6-Gingerol, a Bioactive Compound of Ginger Attenuates Renal Damage in Streptozotocin-Induced Diabetic Rats by Regulating the Oxidative Stress and Inﬂammation

pharmaceutics

Article 6-Gingerol, a Bioactive Compound of Ginger Attenuates Renal Damage in Streptozotocin-Induced Diabetic Rats by Regulating the Oxidative Stress and Inﬂammation

Saleh A. Almatroodi 1 , Abdullah M. Alnuqaydan 2, Ali Yousif Babiker 1, Mashael Abdullah Almogbel 1 , Amjad Ali Khan 3 and Arshad Husain Rahmani 1,*

1 Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia; [email protected] (S.A.A.); [email protected] (A.Y.B.); [email protected] (M.A.A.) 2 Department of Medical Biotechnology, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia; [email protected] 3 Department of Basic Health Sciences, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia; [email protected] * Correspondence: [email protected]; Tel.: +966-16-3800050 (ext. 15477)

Abstract: The aim of present study is to investigate the role of 6-gingerol in ameliorating the renal injury in streptozotocin (STZ)-induced diabetic rats. The diabetes was induced by using a single dose of freshly prepared STZ (55 mg/kg body weight) intraperitoneally which causes the degeneration of pancreatic Langerhans islet β-cells. The diabetic rats were treated with oral gavage of 6-gingerol Citation: Almatroodi, S.A.; (10 mg/kg b.w.). The treatment plan was continued for 8 weeks successively and the body weight Alnuqaydan, A.M.; Babiker, A.Y.; and fasting blood glucose levels were weekly checked. The biochemical parameters like lipid profile, Almogbel, M.A.; Khan, A.A.; Husain kidney profile, antioxidant enzyme levels, lipid peroxidation and anti-inflammatory marker levels Rahmani, A. 6-Gingerol, a Bioactive were investigated after the treatment plant. The pathological condition of kidneys was examined by Compound of Ginger Attenuates haematoxylin-eosin (H&E) staining besides this analysis of NF-κB protein expression by immuno- Renal Damage in histochemistry was performed. Some of the major parameters in diabetes control vs. normal control Streptozotocin-Induced Diabetic Rats were reported as fasting blood glucose (234 ± 10 vs. 102 ± 8 mg/dL), serum creatinine (109.7 ± 7.2 by Regulating the Oxidative Stress and Inflammation. Pharmaceutics vs. 78.9 ± 4.5 µmol/L) and urea (39.9 ± 1.8 vs. 18.6 mg/dL), lipid profile levels were significantly 2021, 13, 317. https://doi.org/ enhanced in diabetic rats. Moreover, diabetic rats were marked with decreased antioxidant enzyme 10.3390/pharmaceutics13030317 levels and increased inflammatory markers. Treatment with 6-gingerol significantly restored the fasting blood glucose level, hyperlipidaemia, Malondialdehyde (MDA) and inflammatory marker Academic Editor: Ariane Leite Rozza levels, NF-κB protein expression and augmented the antioxidant enzyme levels in the kidneys of diabetic rats. The kidney damage was significantly normalized by the treatment of 6-gingerol and it Received: 7 February 2021 provides an evidence that this novel compound plays a significant role in the protection of kidney Accepted: 24 February 2021 damage. These findings demonstrate that 6-gingerol reduces lipid parameters, inflammation and Published: 28 February 2021 oxidative stress in diabetic rats, thereby inhibiting the renal damage. Our results demonstrate that use of 6-gingerol could be a novel therapeutic approach to prevent the kidney damage associated Publisher’s Note: MDPI stays neutral with the diabetes mellitus. with regard to jurisdictional claims in published maps and institutional affil- Keywords: 6-gingerol; diabetes mellitus; inflammation; oxidative stress; renal damage iations.

1. Introduction Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. The prevalence of diabetes mellitus is increasing dramatically worldwide and is This article is an open access article projected to continue with same pace, if proper guidelines are not taken [1]. At present, distributed under the terms and there are 382 million people suffering from diabetes and it is presumed that by the end conditions of the Creative Commons of 2035, this population is may reach up to 592 million, according to the International Attribution (CC BY) license (https:// Diabetes Federation [2]. creativecommons.org/licenses/by/ Diabetic nephropathy, a common late-stage complication of diabetes mellitus patients, 4.0/). usually leads to severe morbidity and mortality. Moreover, this disease is one of the

Pharmaceutics 2021, 13, 317. https://doi.org/10.3390/pharmaceutics13030317 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, 317 2 of 15

most common microvascular complications of diabetes mellitus [3], and is categorized by persistent proteinuria, advanced loss of renal function and morphological changes, such as mesangial expansion, glomerular hypertrophy, glomerular basement membrane thickening, as well as interstitial fibrosis [4]. A novel analysis has confirmed that early blood glucose control plays a major role in the reduction of the incidence of diabetic nephropathy [5]. Additionally, hyperglycaemic damage to mesangial cells is concerned with the progression of diabetic nephropathy [6,7]. The β-cytotoxic drugs such as streptozotocin (STZ) is commonly used to develop diabetes in experimental animals. The STZ induction leads to the necrosis of the pancreatic β-cells and also caused various types of pathogenesis [8]. Usually in rats, diabetes is induced by taking 55 mg/kg b.w. of STZ [9]. The STZ is preferred to develop type I diabetic models as it does not lead to neurotoxicity like alloxan. However, the disadvantage of this drug as a single high dose injection leads to lethality. Numerous drugs such as taurine, melatonin, vitamin C, and vitamin E are used for the treatment and protection of diabetes. Taurine is a ubiquitous sulfur-containing amino acid that is abundant in mammalian tissues. It plays important role in osmoregulation, pharmacological actions and prevention of oxidative tissue damage [10]. Melatonin is a ubiquitous molecule that carries out many functions and it performs different functions as antioxidant and anti-inflammatory activities [11]. Vitamin C (ascorbic acid), plays important role in the inhibition of pathogenesis. This compound protects the cells against lipid peroxidation by scavenging reactive oxygen species (ROS) [12]. Vitamin E, a potent peroxyl radical scavenger, is a chain-breaking antioxidant that prevents the propagation of free radicals [13]. The therapeutic efficiency of such drugs is really limited by some factors such as adverse effects and incapability to regulate blood glucose levels [14–16]. In addition, most of the oral hypoglycemic mediators used to improve diabetic nephropathy are adipogenic [17] and synthetic oral hypoglycemic therapies are narrowly linked with numerous toxicities [18]. Therefore, the search for drugs that can protect against kidney damage and limit the side effects of diabetes is needed. In this regard, natural products or their purified compound from medicinal plants play a vital role in the management of diabetes and its associated complications through the regulation of various biological activities. Therefore, some powerful antioxidants present in these medicinal plants could possibly serve as treatments for diabetes related diseases [19]. 6-gingerol ((S)-5-hydroxy-1-(4-hydroxy-3methoxyphenol)-3-decan-one) is an aromatic polyphenol, an active ingredient of ginger (Zingiber officinale) which belongs to the fam- ily Zingiberaceae. The role of this compound has been investigated as antioxidant, anti- inflammatory and anti-cancer activities [20,21]. Some studies like antihyperglycemic and antioxidant activities have been investigated earlier [22,23]. Remarkably, the previous studies demonstrate that 6-gingerol shows potent insulin secreting, lipid-lowering, antihyperglycemic and antioxidant properties in type-II diabetic animal models [24]. However, some more aspects of its mechanism of action, as anti-glycemic and the reno-protection needs to be examined in detail. To examine the role of 6-gingerol in the management of diabetic complications, in this study streptozotocin (STZ)-induced renal damage animal models were used to identify some molecular mechanisms through the evaluation of oxidative stress, lipid profile, inflammation and histopathological alterations.

2. Materials and Methods 2.1. Chemicals The diabetes inducing drug, Streptozotocin was purchased from Abcam (ab 142155, Cambridge, UK), 6-gingerol (G1045) was purchased Sigma Aldrich, St. Louis, MO, USA. Antioxidant enzymes like superoxide dismutase (SOD) (ab 65354), Glutathione-S- transferase (GST) (ab 65326), catalase (CAT) (ab 83464) and Trichrome Stain Kit (Connective Pharmaceutics 2021, 13, 317 3 of 15

Tissue Stain) (ab150686) were bought from Abcam, Cambridge, UK. ELISA kits for the assay of inﬂammation by C-reactive protein (ab 108827), TNF-α (ab 46070), IL-1β (ab 100768), and IL-6 (ab 100772) were procured from Abcam, Cambridge, UK. Primary antibodies (TNF-α) and Goat Anti-Mouse IgG H&L (HRP) (abcam, Cambridge, UK) used in this study were purchased from Abcam, Cambridge, UK. All supplementary chemicals used in this study were of high purity grade obtained from commercial sources.

2.2. Animals Healthy male Albino Wistar rats weighing between 180–220 g were procured from the animal house of King Saud University, Riyadh, Saudi Arabia. The animals were kept in polypropylene cages at temperature of 22 ± 2 ◦C with a relative humidity of 50–60% and a 12 h light/dark cycle in the animal house at College of Applied Medical Sciences, Qassim University. The animal experiments were carried out as per the guidelines of College of Applied Medical Sciences, Qassim University and approved by the Institutional Animal Ethics Committee (2019-2-2-I-5623), College of Applied Medical Sciences, Qassim University. Throughout the experimental duration, the animals consumed a standard pellet diet and water ad libitum.

2.3. Experimental Design The animals were randomly selected and divided into four groups (group I to group IV) with 8 animals in each group (Table1). All the animals were fasted overnight prior to the start of the experiment. The group I served and normal control and the diabetes was induced in rats (group II, III and IV) by a single intraperitoneal (i.p.) injection of freshly prepared STZ (55 mg/kg b.w.) dissolved in 0.01 M citrate buffer, pH 4.5 [25] except for group I animals which received same buffer as placebo. The blood glucose levels of control and diabetes induced rats were measured through tail vein puncture to check hyperglycaemia. The induction of diabetes was checked after the third day of the STZ injection and animals having fasting blood glucose level more than 200 mg/dL were selected as diabetic and were included for the experiments. Treatments were started on day 4 after STZ injection, and this was considered day 1 for treatment and continued for eight weeks.

Table 1. A total of 32 male albino Wistar rats were randomly divided into 4 groups as.

Group Number Group Short Name Treatment Plan Rats were allowed to have free access to I Normal Control NC a standard pellet diet The rats were intraperitoneally injected II Diabetes Control DC with freshly prepared STZ (55 mg/kg b.w.) in citrate buffer pH 4.5 The diabetic rats were treated with III Diabetes Control + 6-Gingerol DC + 6-Ging 6-gingerol (10 mg/kg b.w.) [26] Diabetes Control + The diabetic rats were treated with IV DC + Gliben Glibenclamide Glibenclamide (5 mg/kg b.w.) [27]

2.4. Measurement of Body Weight and Biochemical Parameters Once weekly, the body weights of all the animals in all four groups were recorded to check the change in their overall weight and the results were analysed accordingly. At the end of the study, blood samples were collected and allowed to clot at room temperature for 30 min and serum was obtained by centrifugation at 3.500 rpm for 10 min. The estimation of total cholestrol, triglycerides, and low density lipoprotein were performed by Crescent Diagnostics, Jeddah, KSA and the result were interpreted accordingly. Additionally, creatinine and blood urea were assayed accordingly using reagent kits purchased from Crescent Diagnostics, Jeddah, KSA. Pharmaceutics 2021, 13, 317 4 of 15

2.5. Measurement of Lipid Peroxidation The lipid peroxidation in the kidney homogenate of experimental group rats was determined by assaying MDA level run in duplicate, and the results obtained were interpreted accordingly.

2.6. Determination of Antioxidant Enzymes Levels Small portion of kidney were harvested from all the rats and stored in phosphate buffer saline. All the tissues were homogenized and centrifuged at 1100× g for 15 min at 4 ◦C. Antioxidant enzymes including SOD, GST, CAT and GSH levels were measured in duplicate, using the available kits, according to manufacturer’s protocol.

2.7. Measurement of Pro-Inﬂammatory Parameters Inﬂammatory markers such as CRP, TNF-α, IL-6 and IL-1β were measured in duplicate, by using ELISA kits from Abcam Company according to the manufactures protocol and results were interpreted accordingly.

2.8. Histopathological Examination of Kidney All the kidney tissue samples were fixed in neutral formalin and paraffin embedded block was made. After thin sectioning (5 µm), the sections were mounted on slides. The hematoxylin and eosin (H&E) staining was performed to examine the architecture of kidney tissues. Histopathological examination was performed by using a light microscope (Olympus, Tokyo, Japan) and the slide pictures were shot accordingly. The histological findings were performed by double-blinded histopathologist. Furthermore, Masson’s trichrome was used to evaluate the morphology of fibrosis as per the manufacturer’s protocol (Abcam, UK).

2.9. Immunohistochemical Evaluation of TNF-alpha All the kidney tissues were fixed, dehydrated, paraffin embedded, and thin sections were cut. The tissue sections were deparaffinised with xylene, rehydrated with graded alcohol, washed in water and incubated to antigen retrieval in 0.1 M citrate buffer (pH 6.0). After blocking, all the tissue sections were incubated with TNF-α antibody (1:250 dilution) for overnight. After washing with PBS (pH 7.0), the sections were treated with secondary antibody (no dilution as per the manufacturers’ instructions) for 45 min. Finally, these samples were stained with Diaminobenzedine, washed with phosphate buffer salin and mounted. The stained sections were examined through Olympus (Tokyo, Tokyo, Japan) microscope and the results were interpreted accordingly. The expression pattern of TNF-α was evaluated manually based on the cytoplasmic expression. The quantification of cytoplasmic positivity of TNF-α was evaluated by a pathologist, blinded to the different animal groups. A total of 500 cells from the five different selected regions were counted. The positive stained cells were expressed as the percentage of total cells counted in each case. If less than 5% of cells showed positivity for TNF- α, were considered as negative. If equal or more than 5% of the cells were positive for TNF- α, were considered as positive. Based on the staining intensity or expression pattern, the stained sections were graded as 0 for no staining/expression, 1 was represented for weak but detectable (~25%) staining, 2 was represented for moderate staining (25–50%) and 3 was represented for intense or high staining (>75%).

2.10. Statistical Analysis All the values are presented as the means ± standard deviation. For comparisons in multiple groups, one-way analysis of variance (ANOVA) was used. The probability, p < 0.05 was considered as statistically signiﬁcant for a two-tailed test. Pharmaceutics 2021, 13, x 5 of 15

All the values are presented as the means ± standard deviation. For comparisons in multiple groups, one-way analysis of variance (ANOVA) was used. The probability, p < 0.05 was considered as statistically significant for a two-tailed test.

3. Results 3.1. Effect of 6-Gingerol on Body Weight and Blood Glucose Levels

Pharmaceutics 2021, 13, 317 The body weight of all experimental rat groups was recorded weekly during5 ofthe 15 8- week long experimental period. In comparison with the control rats, there was a significant reduction in the body weight of diabetic rats. After continuous treatment with 6-gingerol to group III animals, this procedure attenuated the weight loss of the diabetic rats. 3.As Results compared with the diabetes control rats (group II), the surge in body weight was nearly 3.1.26.22% Effect ofin 6-Gingerol6-gingerol-treated on Body Weightdiabetic and rats Blood (Figure Glucose 1). Levels TheThe body rats weightwere allowed of all experimental to fast for 12 rat h to groups evaluate was their recorded fasting weekly blood duringglucose. the The 8-weekfastinglong blood experimental glucose level period. in group In comparison II animals was with significantly the control rats,higher there as compared was a sig- to niﬁcantanimals reduction present inin thecontrol body group weight (p of< 0.05). diabetic As rats.compared After continuousto the STZ group, treatment the withfasting 6-gingerol to group III animals, this procedure attenuated the weight loss of the diabetic blood glucose levels in the STZ plus 6-gingerol group were significantly decreased (p < rats. As compared with the diabetes control rats (group II), the surge in body weight was 0.05). nearly 26.22% in 6-gingerol-treated diabetic rats (Figure1).

350 300 Initial body weight Final body weight # 300 250 *

250 200 * 200 # 150 150 100 Body Body weight (g)

100 Glucose level (mg/dL) 50 50

0 0 NC DC DC+6-Ging DC+Gliben NC DC DC+6-Gin DC+Gliben Animal groups Animal groups

(a) (b)

FigureFigure 1. (1.a )(a The) The initial initial and and the the ﬁnal final body body weight weight of of different different groupsgroups ofof ratsrats after 8 weeks weeks of of experimental experimental design. design. The The rats ratswere were equally equally divided divided into into 4 groups, 4 groups, each each group group (n (=n 8).= 8).Data Data areare represented represented as mean as mean ± standard± standard error error of the of mean the mean (SEM). (SEM).Groups: Groups: Normal Normal control control (NC); (NC); disease disease control control (DC), (DC),i.e., STZ- i.e.,treated STZ-treated group; group; treatment treatment group group (DC + (DC 6-gingerol); + 6-gingerol); positive

positivecontrol control (PC) animal (PC) animal treated treated with STZ with + glibenclamide; STZ + glibenclamide; * p < 0.05 * (significantp < 0.05 (significant difference difference of final b.w. of between final b.w. DC between vs. NC) # p < 0.05 (significant difference of final b.w. between DC vs. DC + 6-gingerol). (b): The values of serum glucose in different DC vs. NC) # p < 0.05 (significant difference of final b.w. between DC vs. DC + 6-gingerol). (b): The values of serum groups of rats after 8 weeks of treatment. The rats were equally divided into 4 groups, each group (n = 8). Data are repre- glucose in different groups of rats after 8 weeks of treatment. The rats were equally divided into 4 groups, each group sented as mean ± standard error of the mean (SEM). Groups: Normal control (NC); disease control (DC), i.e., STZ-treated (n group;= 8). Data treatment are represented group (DC as+ 6-ginger mean ±ol);standard positive error control of (PC) the mean animal (SEM). treated Groups: with STZ Normal + glibenclamide; control (NC); * p < disease 0.05 (sig- controlnificant (DC), differencei.e., STZ-treated of final b.w.group; between treatment DC vs. group NC) (DC # p < + 0.05 6-gingerol); (significant positive difference control of (PC)final animalb.w. between treated DC with vs. STZ DC + 6- glibenclamide;gingerol). * p < 0.05 (significant difference of final b.w. between DC vs. NC) # p < 0.05 (significant difference of final b.w. between DC vs. DC + 6-gingerol). 3.2. Effect of 6-Gingerol on Lipid Profile The rats were allowed to fast for 12 h to evaluate their fasting blood glucose. The As presented in Figure 2, The STZ-induced diabetic rats had markedly elevated level fasting blood glucose level in group II animals was significantly higher as compared to of TG (238.7 mg/dL vs. 130.9 mg/dL) and TC (147.37 mg/dL vs. 95.34 mg/dL) and LDL-C animals present in control group (p < 0.05). As compared to the STZ group, the fasting blood (179.9 mg/dL vs. 112.8 mg/dL) levels as compared with those of the control group. Ani- glucose levels in the STZ plus 6-gingerol group were significantly decreased (p < 0.05). mals treated with 6-gingerol (150 mg/kg) showed significantly attenuated the TC (123.45 3.2.mg/dL), Effect of TG 6-Gingerol (181.3 mg/dL), on Lipid LDL-C Profile (138.23 mg/dL) as compared with STZ treated animal group (p < 0.05). As presented in Figure2, The STZ-induced diabetic rats had markedly elevated level of TG (238.7 mg/dL vs. 130.9 mg/dL) and TC (147.37 mg/dL vs. 95.34 mg/dL) and LDL-C (179.9 mg/dL vs. 112.8 mg/dL) levels as compared with those of the control group. Animals treated with 6-gingerol (150 mg/kg) showed significantly attenuated the TC (123.45 mg/dL), TG (181.3 mg/dL), LDL-C (138.23 mg/dL) as compared with STZ treated animal group (p < 0.05).

3.3. Effect of 6-Gingerol on Serum Creatinine, and Blood Urea Level Following the induction of hyperglycemia via STZ, elevated levels of serum creatinine (109.7 µmol/L vs. 78.9 mol/L), and blood urea (39.9 mg/dL vs. 18.6 mg/dL) were observed in the STZ group as compared to the control group. However, 6-gingerol treatment signiﬁ- cantly reduced renal dysfunction biomarkers such as serum creatinine (89.39 µmol/L), and blood urea (26.7 mg/dL) as compared to STZ treated animals. These results indicate that 6-gingerol protects rental function in STZ induced diabetes (Figure3). Pharmaceutics 2021, 13, x 6 of 15

Pharmaceutics 2021, 13, x 6 of 15 Pharmaceutics 2021, 13, 317 6 of 15

Figure 2. The lipid profile in different groups of rats after 8 weeks of continues treatment. The rats were equally divided into 4 groups, each group (n = 8). Data are presented as mean ± standard error of the mean (SEM). Groups: Normal control (NC); disease control (DC), i.e., STZ-treated group; treatment group (DC + 6-gingerol); positive control (PC) animal treated with STZ + glibenclamide; * p < 0.05 (significant difference of final b.w. between DC vs. NC) # p <0.05 (significant difference of final b.w. between DC vs. DC + 6-gingerol).

3.3. Effect of 6-Gingerol on Serum Creatinine, and Blood Urea Level Following the induction of hyperglycemia via STZ, elevated levels of serum creatinine (109.7 μmol/L vs. 78.9 mol/L), and blood urea (39.9 mg/dL vs. 18.6 mg/dL) were ob- Figure 2. The lipid profileserved in different in the groups STZ ofgroup rats after as compared 8 weeks of to continues the control treatment. group. The However, rats were equally6-gingerol divided treatment into 4 groups, each group (n = 8). Data are presented as mean ± standard error of the mean (SEM). Groups: Normal control Figure 2. The lipid profile in differentsignificantly groups reduced of rats renalafter 8dysfunction weeks of continues biomarkers treatment. such asThe serum rats werecreatinine equally (89.39 divided (NC); disease control (DC), i.e., STZ-treated group; treatment group (DC + 6-gingerol); positive control (PC) animal treated into 4 groups, each group (n = 8).μmol/L), Data are and presented blood urea as mean(26.7 mg/dL) ± standard as comp errorared of the to meanSTZ treated (SEM). animals. Groups: These Normal results control with STZ + glibenclamide;indicate * p < 0.05 that (significant 6-gingerol difference protects of final rental b.w. between function DC in vs. STZ NC) induced # p < 0.05 (significantdiabetes (Figure difference 3). (NC); diseaseof final control b.w. between (DC), i.e., DC vs.STZ-treated DC + 6-gingerol). group; treatment group (DC + 6-gingerol); positive control (PC) animal treated with STZ + glibenclamide; * p < 0.05 (significant difference of final b.w. between DC vs. NC) # p <0.05 (significant difference of final b.w. between DC vs. DC + 6-gingerol).

3.3. Effect of 6-Gingerol on Serum Creatinine, and Blood Urea Level Following the induction of hyperglycemia via STZ, elevated levels of serum creatinine (109.7 μmol/L vs. 78.9 mol/L), and blood urea (39.9 mg/dL vs. 18.6 mg/dL) were observed in the STZ group as compared to the control group. However, 6-gingerol treatment significantly reduced renal dysfunction biomarkers such as serum creatinine (89.39 μmol/L), and blood urea (26.7 mg/dL) as compared to STZ treated animals. These results indicate that 6-gingerol protects rental function in STZ induced diabetes (Figure 3).

Figure 3. The kidney function test profile (creatinine and urea) in different groups of rats after Figure8 weeks 3. The of treatment.kidney function The rats test were profile equally (creatinine divided an intod urea) 4 groups, in different each group groups (n of= 8).rats Data after are 8 weekspresented of treatment. as mean The± standard rats were error equa oflly the divided mean into (SEM). 4 groups, Groups: each Normal group control (n = 8). (NC); Data diseaseare pre- sentedcontrol as (DC),mean i.e., ± standard STZ-treated error group; of the treatment mean (SEM). group Groups: (DC + 6-gingerol); Normal control positive (NC); control disease (PC) animal control (DC),treated i.e., withSTZ-treated STZ + glibenclamide; group; treatment * p < 0.05group (significant (DC + 6-gingerol); difference ofpositive final b.w. control between (PC) DC animal vs. NC) treated# p < 0.05with (significant STZ + glibenclamide; difference of * final p < 0.05 b.w. (significant between DC difference vs. DC + 6-gingerol).of final b.w. between DC vs. NC) # p < 0.05 (significant difference of final b.w. between DC vs. DC+6-gingerol). 3.4. Effect of 6-Gingerol on Oxidative Stress 3.4. EffectThe of protective 6-Gingerol role on ofOxidative 6-gingerol Stress on the redox status of the normal and diabetic rats was measured via assessment of the levels of MDA, and antioxidants enzyme (CAT, SOD, The protective role of 6-gingerol on the redox status of the normal and diabetic rats GST and GSH) levels. STZ-induced diabetic rats showed a significant reduction in CAT was(22.2 measured U/mg vs.via assessment 48.5 U/mg), of SOD the levels (34.3 of U/mg MDA, vs. and 73.3 antioxidants U/mg), GST enzyme (87.9 U/mg(CAT, SOD, vs. GST142.6 and U/mg GSH)) and levels. GSH STZ-induced (25.9 µmol/g diabetic vs. 46.6 ratsµmol/g) showed level a significant as compared reduction to the control in CAT (22.2group. U/mg Treatment vs. 48.5 withU/mg), 6-gingerol SOD (34.3 meaningfully U/mg vs. improved73.3 U/mg), the GST altered (87.9 oxidative U/mg vs. stress 142.6 U/mg)levels and in STZ GSH induced (25.9 diabeticμmol/g animalsvs. 46.6 resultingμmol/g) inlevel increased as compared antioxidant to the levels control (Figure group.4 ). TreatmentMoreover, with STZ-induced 6-gingerol diabetic meaningfully rats showed impr aoved significant the altered surge oxidative in kidney stress MDA levels levels in STZ induced diabetic animals resulting in increased antioxidant levels (Figure 4). Moreo- Figure(151.3 3. The nmol/g kidney vs. function 110.7 nmol/g) test profile in comparison (creatinine with an thed normalurea) in control different group. groups Treatment of rats after 8 ver,with STZ-induced 6-gingerol significantly diabetic rats decreased showed MDA a significant levels in surge the kidney in kidney of diabetic MDA rats. levels (151.3 weeks of treatment. The rats were equally divided into 4 groups, each group (n = 8). Data are presented as mean ± standard error of the mean (SEM). Groups: Normal control (NC); disease control (DC), i.e., STZ-treated group; treatment group (DC + 6-gingerol); positive control (PC) animal treated with STZ + glibenclamide; * p < 0.05 (significant difference of final b.w. between DC vs. NC) # p < 0.05 (significant difference of final b.w. between DC vs. DC+6-gingerol).

3.4. Effect of 6-Gingerol on Oxidative Stress The protective role of 6-gingerol on the redox status of the normal and diabetic rats was measured via assessment of the levels of MDA, and antioxidants enzyme (CAT, SOD, GST and GSH) levels. STZ-induced diabetic rats showed a significant reduction in CAT (22.2 U/mg vs. 48.5 U/mg), SOD (34.3 U/mg vs. 73.3 U/mg), GST (87.9 U/mg vs. 142.6 U/mg) and GSH (25.9 μmol/g vs. 46.6 μmol/g) level as compared to the control group. Treatment with 6-gingerol meaningfully improved the altered oxidative stress levels in STZ induced diabetic animals resulting in increased antioxidant levels (Figure 4). Moreo- ver, STZ-induced diabetic rats showed a significant surge in kidney MDA levels (151.3

Pharmaceutics 2021, 13, x 7 of 15

nmol/g vs. 110.7 nmol/g) in comparison with the normal control group. Treatment with 6- gingerol significantly decreased MDA levels in the kidney of diabetic rats. Pharmaceutics 2021, 13, 317 7 of 15

180 160 * 140 # 120

100

MDA (nmol/g) MDA 60

0 NC DC DC+6-Ging DC+Gliben Animal Groups

(a)

(b)

Figure 4. (a, b):Figure The lipid 4. (a, peroxidation b): The lipid peroxidation (MDA) and (MDA) antioxidant and antioxidant enzyme enzyme (CAT, (CAT,SOD, SOD, GST GSTand and GSH) GSH) level in differentlevel groups in different of rats groups after 8 of weeks rats after of treatment. 8 weeks of The treatment. rats were The equally rats were divided equally into divided 4 into groups, each group4 groups, (sample each groupsize = (sample 8). Data size are = 8).show Datan areas shownmean as± standard mean ± standard error of error the of mean the mean (SEM). (SEM). Groups: NormalGroups: control Normal (NC); controldisease (NC); control disease (DC) control, i.e., (DC),STZ-treated i.e., STZ-treated group; group;treatment treatment group group (DC (DC+ 6-gingerol); positive+ 6-gingerol); control positive (PC) anim controlal treated (PC) animal with treated STZ + with glibenclamide; STZ + glibenclamide; * p < 0.05 * p(significant< 0.05 (significant difference of finaldifference b.w. between of final b.w. DC between vs. NC) DC # vs.p < NC)0.05 # (significantp < 0.05 (significant differenc differencee of final of final b.w. b.w. between between DC DC vs. DC + 6-gingerol).vs. DC + 6-gingerol).

Pharmaceutics 2021, 13, x 8 of 15

Pharmaceutics 2021, 13, 317 8 of 15 3.5. 6-Gingerol Decrease the Levels of CRP, IL-6, IL-1β and TNF-α The diabetic rats showed noticeable inflammatory responses in the kidney, were significantly enhanced in the diabetic rats TNF-α (54.64 pg/mL vs. 37.49 pg/mL), IL-6 (108.64 3.5. 6-Gingerol Decrease the Levels of CRP, IL-6, IL-1β and TNF-α pg/mL vs. 74.49 pg/mL), IL-1β (25.3 pg/mL vs. 18.8 pg/mL), CRP (0.97 ng/mL vs. 0.59 The diabetic rats showed noticeable inflammatory responses in the kidney, were ng/mL)significantly as compared enhanced with in thethose diabetic of the rats normal TNF-α rats(54.64 (p < pg/mL 0.05). vs.Treatment 37.49 pg/mL), with 6-gingerol IL-6 significantly(108.64 pg/mL decreased vs. 74.49 the pg/mL), levels IL-1of inflammatoryβ (25.3 pg/mL markers vs. 18.8 pg/mL),in the kidneys CRP (0.97 of ng/mLdiabetic rats as vs.compared 0.59 ng/mL) to STZ as treated compared only with (Figure those 5). of the normal rats (p < 0.05). Treatment with 6-gingerol significantly decreased the levels of inflammatory markers in the kidneys of diabetic rats as compared to STZ treated only (Figure5).

60 * 120 * 50 # 100 #

40 80

30 60 IL-6 (pg/ml) TNF-α (pg/ml) 20 40

10 20

0 0 NC DC DC+6-Ging DC+Gliben NC DC DC+6-Ging DC+Gliben Animal Groups Animal Groups 30 * 1.2 25 # 1 *

20 0.8 #

15 0.6 IL-1β (pg/ml)

10 CRP(ng/ml) 0.4

5 0.2

0 0 NC DC DC+6-Ging DC+Gliben NC DC DC+6-Ging DC+Gliben Animal Groups Animal Groups

Figure 5. The inﬂammatory markers level in different groups of rats after 8 weeks of treatment.

FigureThe rats5. The were inflammatory equally divided markers into 4 groups,level in eachdifferent group groups (sample of size rats = after 8). Data 8 weeks are summarized of treatment. Theas rats mean were± standard equally errordivided of the into mean 4 groups, (SEM). each Groups: group Normal (sample control size (NC); = 8). disease Data are control summarized (DC), as meani.e., ± STZ-treated standard errorgroup; of treatment the mean group (SEM). (DC Groups: + 6-gingerol); Normal positive control control (NC); (PC) disease animal control treated with(DC), i.e., STZ-treatedSTZ + glibenclamide; group; treatment * p < 0.05 group (signiﬁcant (DC + difference6-gingerol of); ﬁnalpositive b.w. control between (PC) DC vs.animal NC) #treatedp < 0.05 with

STZ(significant + glibenclamide; difference * ofp < final 0.05 b.w. (significant between DCdifference vs. DC +of 6-gingerol). final b.w. between DC vs. NC) # p < 0.05 (significant difference of final b.w. between DC vs. DC + 6-gingerol). 3.6. Effect of 6-Gingerol on Kidney Architecture 3.6. EffectThe of kidney 6-Gingerol architecture on Kidney was Architecture examined through hematoxylin-eosin (H&E), and Mas- son’s trichrome staining. Control group rats tissue presented normal cell architecture The kidney architecture was examined through hematoxylin-eosin (H&E), and Mas- within the kidney with normal glomeruli and renal tubules. As presented in Figure6, son’sdiabetic trichrome group ratsstaining. displayed Control infiltration group of lymphocytes,rats tissue presented thickened glomerularnormal cell basement architecture withinmembranes, the kidney and congestion with normal as compared glomeruli to normaland renal control tubules. rats. However, As presented administration in Figure 6, diabeticof 6-gingerol group rats for 8 displayed weeks markedly infiltration diminished of lymphocytes, the deformities thickened in both glomerular the glomerular basement membranes,and tubular and architecture congestion in ratsas compared and displayed to no mildrmal inflammation, control rats. However, and congestion. administration The of 6-gingerolkidney damage for 8 wasweeks significantly markedly normalized diminished by the the deformities treatment of in 6-gingerol both the glomerular providing and tubularevidence architecture that 6-gingerol in rats plays and adisplayed significant m functionild inflammation, in the protection and congestion. of kidney damage. The kidney damageMasson’s was trichrome significantly staining normal was performedized by the on treatment all experimental of 6-gingerol group tissue providing to evaluate evidence the distribution of collagen fibers as a previously described method [28]. Control group that 6-gingerol plays a significant function in the protection of kidney damage. Masson’s rats tissue showed normal distribution of collagen. As presented in Figure7, STZ treated trichromerats displayed staining high was degree performed of collagen on deposition.all experimental Treatment group with tissue 6-gingerol to evaluate significantly the distri- butiondecreased of collagen the collagen fibers deposition as a previously when compared described with method the diabetic [28]. Control controlgroup. group rats tissue showed normal distribution of collagen. As presented in Figure 7, STZ treated rats displayed high degree of collagen deposition. Treatment with 6-gingerol significantly decreased the collagen deposition when compared with the diabetic control group.

Pharmaceutics 2021, 13Pharmaceutics, 317 2021, 13, x 9 of 15 9 of 15 Pharmaceutics 2021, 13, x 9 of 15

Figure 6. Effect of gingerol in histopathological changes of kidney tissues. (a): Typical architecture of kidney in control animals. (b): STZ only treated kidney tissues showed infiltration of lymphocytes, thickened glomerular basement mem- FigureFigure 6. 6. EffectEffect of of gingerol gingerol in in histopathological histopathological changes changes of of kidney kidney tissues. tissues. ( (aa):): Typical Typical architecture architecture of of kidney kidney in in control control branes and congestion. (c): Administration of 6-gingerol markedly mitigated the irregularities in the glomerular and tub- animals.animals. ( (bb):): STZ STZ only only treated treated kidney kidney tissues tissues showed showed infiltration inﬁltration of of lymphocytes, lymphocytes, thickened thickened glomerular glomerular basement basement mem- mem- ular architecture and displayed mild congestion, (d): positive control (diabetic rats treated with glibenclamide) showing branesbranes and and congestion. ( c): Administration ofof 6-gingerol6-gingerol markedly markedly mitigated mitigated the the irregularities irregularities in in the the glomerular glomerular and and tubular tub- normal architecture (e): Animal group treated with gingerol only showed normal architecture of kidney. (Scale bar = 100 ulararchitecture architecture and displayedand displayed mild mild congestion, congestion, (d): positive (d): positive control control (diabetic (diabetic rats treated rats treated with glibenclamide) with glibenclamide) showing showing normal μm) normalarchitecture architecture (e): Animal (e): Animal group treated group withtreated gingerol with ging onlyerol showed only showed normal architecturenormal architec of kidney.ture of (Scalekidney. bar (Scale = 100 barµm). = 100 μm)

Figure 7. Masson trichrome staining. Masson’s trichrome staining was showing that (a): Normal Figure 7. Masson trichrome staining. Masson’s trichrome staining was showing that (a): Normal kidney architecture show- kidney architecture showing no collagen deposition; (b): signiﬁcant increase in collagen deposition in ing no collagen deposition; (b): significant increase in collagen deposition in animals treated with STZ was noticed; (c): animals treated with STZ was noticed; (c): Following treatment with gingerol and STZ treated group Figure 7. Masson trichromeFollowing staining. treatment Masson’s with gingerol trichrome and stainingSTZ treated was groupshowing collagen that (a ):deposition Normal kidney was reduced architecture as compared show- to the disease ing no collagen deposition;control group, (collagenb): significant(d): positive deposition increase control was in (diabetic reduced collagen rats as deposition compared treated with in to animals theglibenclamide) disease treated control withnot group,showingSTZ was (d any ):noticed; positive collagen (c): control deposition, (e): Following treatmentGingerol-treated with gingerol(diabetic group and rats STZ only treated treated did with not group show glibenclamide) collagendeposition deposition not of showingcollagen. was any(Scale reduced collagen bar = as 100 deposition, compared μm) (toe): the Gingerol-treated disease control group, (d): positive groupcontrol only (diabetic did not rats show treated deposition with glibenclamide) of collagen. (Scale not showing bar = 100 anyµm). collagen deposition, (e): Gingerol-treated group only did not show deposition3.7. Effects of ofcollagen. 6-Gingerol (Scale on bar TNF- = 100α Expression μm) 3.7. Effects of 6-Gingerol on TNF-α Expression The expression of TNF-α protein was not noticed in kidneys of the control group The expression of TNF-α protein was not noticed in kidneys of the control group 3.7. Effects of 6-Gingerolwhile it onshowed TNF-α intense Expression expression in the diabetic control group as compared to the nor- while it showed intense expression in the diabetic control group as compared to the normal The expressionmal control of TNF- groupα protein (p < 0.01). was Moreover,not noticed diabetic in kidneys groups of treated the control with 6-gingerol,group exhibited control group (p < 0.01). Moreover, diabetic groups treated with 6-gingerol, exhibited low while it showedlow intense expression expression in the in diabetic the diabetic group control plus the group 6-gingerol as compared group as tocompared the nor- to the diabetic expression in the diabetic group plus the 6-gingerol group as compared to the diabetic mal control groupcontrol (p < 0.01).group Moreover, (p < 0.05). diabeticAs a result groups of treatment treated withof diabetic 6-gingerol, rats with exhibited 6-gingerol, showed control group (p < 0.05). As a result of treatment of diabetic rats with 6-gingerol, showed low expression ina significant the diabetic decrease group plus TNF- theα protein 6-gingerol expression group asas comparedcompared towith the the diabetic diabetic group (Fig- a signiﬁcant decrease TNF-α protein expression as compared with the diabetic group control group (pure < 0.05). 8A and As 8B). a result of treatment of diabetic rats with 6-gingerol, showed (Figure8A,B). a significant decrease TNF-α protein expression as compared with the diabetic group (Fig- ure 8A and 8B).

Pharmaceutics 2021, 13, 317 10 of 15

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3 ** 2.5

2 * 1.5

0.5 Immunohistochemical score 0 NC DC DC+6-Gin DC+Gliben Gin only Animal groups

Figure 8. (A) (a): The expression of TNF-α protein was not noticed in kidney of normal control group while (b): it showed Figure 8. [A] (a): The expression of TNF-α protein was not noticed in kidney of normal control group while (b): it showed intenseintense expression in in diabetic diabetic control control group. group. Moreover, Moreover, (c (c):): diabetic diabetic groups groups treated treated with with 6-gingerol, 6-gingerol, exhibited exhibited weak weak ex- expressionpression in indiabetic diabetic group group plus plus 6-gingerol 6-gingerol group. group. (d): positive (d): positive control control (diabetic (diabetic rats tr ratseated treated with glibenclamide) with glibenclamide) not show- not showinging any expression, any expression, (e): Gingerol-treated (e): Gingerol-treated group group only only did no didt show not show any anyexpression. expression. (Scale (Scale bar bar= 100 = 100μm).µ m).[B]: (representsB): represents the thegraphical graphical representation representation of TNF- of TNF-α expression.α expression. The The expression expression pattern pattern was was high high in the in thediabetic diabetic control control as compared as compared to the to thenormal normal control control group group (** p (** < p0.01).< 0.01). The Thediabetic diabetic group group treated treated with with 6-ginger 6-gingerolol showed showed low lowexpression expression as compared as compared to the to thediabetic diabetic control control (* p < (* 0.05).p < 0.05). The expression The expression pattern pattern of TNF- of TNF-α in theα in positive the positive control control and 6-gingerol and 6-gingerol treated treated only was only statis- was statisticallytically insignificant insigniﬁcant as comp as comparedared to the to control the control group group (p > 0.05). (p > 0.05).

4. Discussion The prevalence ofof diabetesdiabetes mellitusmellitus has has been been increasing increasing alarmingly alarmingly worldwide worldwide and and is projectedis projected to to continue continue in thein the future future at theat th samee same pace pace if proper if proper measuresa measuresa are are not not taken taken [1]. Animal[1]. Animal models models are are commonly commonly used used to to evaluate evaluate the the role role of of natural naturalcompounds compounds inin thethe management of of diabetes diabetes and and it itss associated associated complications. complications. STZ STZ is commonly is commonly used used as dia- as betesdiabetes inducing inducing agent agent in experimental in experimental animals animals due due to its to capability its capability of specific of speciﬁc necrosis necrosis induction of pancreatic β-cells that consequences in loss of insulin secretion [29], leading to

Pharmaceutics 2021, 13, 317 11 of 15

induction of pancreatic β-cells that consequences in loss of insulin secretion [29], leading to hyperglycaemia and diabetic complications [30]. A single dose injection of streptozotocin in rats has been reported to caused hyperglycemia as well as renal fibrosis [31]. Here in this study, we appraised the effects of 6-gingerol on STZ-induced renal damage rats through the evaluation of oxidative stress, inflammation, histopathological and biochemical alterations among different treatment groups. 6-gingerol has been found to inhibit the pathogenesis including renal damage through the modulation of various biological activities. It has been identified that gingerol through its antioxidant [32] and anti-inflammatory activities stimulates GLP-1 mediated insulin secretion pathway and upregulates Rab27a/Slp4-a that controls insulin granule exocytosis in pancreatic β-cells. Besides this, it facilitates glucose disposal in skeletal muscle by up-regulating glycogen synthase 1 and increases GLUT4 membrane presentation [20,33]. Gingerol promotes nephroprotective effects by the reduction of oxidative stress, inflammatory processes, and renal dysfunction [34]. In addition, gingerol has been found to down-regulate blood glucose level, creatinine and blood urea nitrogen level. It also regulates pro-inflammatory cytokines, nuclear factor kappa B (N-κB) activation, renal p38MAPK, and TGF-β [35]. Gingerol plays a significant role in improving the condition of renal tissue by alteration in p38MAPK and NF-κB activity, and control inflammatory reaction and oxidative stress [35]. The high level of fasting blood glucose is characterized as diabetes mellitus [35] and is establishes risk factors for coronary heart disease [36]. The fasting blood glucose concentration in the STZ group animals was significantly higher as compared to the control group (p < 0.05). In comparison to the STZ group, the fasting blood sugar levels in the STZ plus 6-gingerol group were significantly decreased (p < 0.05) (Figure1b). The proper insulin level does not only affect the blood glucose level but is also associated with cholesterol regulation including its synthesis and absorption [37]. In the current study, STZ-induced diabetic rats showed a markedly elevated level of TGs, TC and LDL-C levels as compared with those of the control group. Animals treated with 6-gingerol (150 mg/kg) showed significantly attenuated lipid profiles compared with STZ treated animal group (Figure2). Moreover, 6-gingerol treatment significantly reduced the renal dysfunction biomarkers such as serum creatinine, and blood urea as compared to STZ treated animals. These results demonstrate that 6-gingerol protects rental function in STZ induced diabetes (Figure3). Some previous reports also support that gingerol significantly down-regulated the blood glucose level, creatinine and blood urea nitrogen level in a dose-dependent fashion [35]. Moreover, gingerol role as antioxidant and anti-inflammatory has been proven [38]. The imbalance between oxidation and anti-oxidant systems, and excessive reactive oxygen species (ROS) generation are the key pathogenic factors in renal disease [39]. It has been explained that hyperglycemia may promote the glomerular mesangial cells and tubular epithelial cells to produce excess ROS, which damage tissue proteins, causes a large number of lipid peroxides and additional worsening of renal oxidative damage [40]. Furthermore, it has been described that hyperglycemia resulted in oxidative injury via shooting up oxidative stress, resulting in overproduction of ROS [41]. Moreover, kidney tissues are more sensitive to ROS linked with oxidative damage because of higher rate of oxygen consumption. In addition, excess ROS could induce an enrichment of MDA content, a good indicator of lipid peroxidation [42]. In the current study, treatment with 6-gingerol appreciably ameliorated the altered oxidative stress levels in STZ induced diabetic animals resulting in increased antioxidant levels. Moreover, STZ-induced diabetic rats showed a significant upsurge in kidney MDA levels. Treatment with 6-gingerol significantly decreased MDA levels in the kidneys of diabetic animals (Figure4a). The previous study has reported that chronic hyperglycemia resulted in a significant increase in malondialdehyde in the kidney of rats. Z. officinale extract efficiently attenuated oxidative stress, and enhanced antioxidant defence in diabetic kidneys [43]. Another study has also reported that diabetic animals treated with gentamicin in an enriched solution of gingerol Pharmaceutics 2021, 13, 317 12 of 15

groups showed amelioration in renal function parameters and reduced lipid peroxidation and nitrosative stress, in addition to an increment in the levels of GSH and SOD activity [34] and 6-gingerol ameliorates gentamicin induced renal cortex oxidative stress [44] The previous studies based on ginger reported the reno-protective effect through the regulation of lipid peroxidation and maintenance of histopathological changes. The effect of ginger (Zingiber zerumbet) exerts an ameliorative effect on renal damage in diabetic rats. Moreover, the histological evaluation demonstrated amelioration of diabetes induced glomerular pathological changes after the treatment with ginger. The finding of this study advocates that the reno-protective effect of Z. zerumbet may be similar to the action of metformin and upregulation of the renal nephrin and podocin [45]. The effects of ginger powder on nephropathy induced by diabetes through the measurement of antioxidant capacity and lipid peroxidation were evaluated. The finding of the study revealed that MDL levels in diabetic rats treated with ginger were significantly lower than any other treatment group. This study exhibited that ginger decreases lipid peroxidation and increases in antioxidant capacity and reduction of renal nephropathy [46]. Inflammation plays an important part in the pathogenesis of diabetes [47,48]. Over- expression of inflammatory cytokines provokes the development of diabetes mellitus. In diabetic nephropathy, the production of pro-inflammatory cytokines enhances the changes in glomerular filtration rate and endothelial cell permeability and provokes the production of ROS and other free radicals [49]. Natural products or active compound of medicinal plants shows role in the inhibition of pathogenesis through inhibition of inflammatory process [50–53]. In the current study, diabetic rats showed significantly increased inflammatory markers, compared with those of the healthy rats. While treatment with 6-gingerol significantly decreased the levels of the inflammatory markers in the kidney of diabetic rats as compared to STZ treated only (Figure5). Taken together, the above results indicated that 6-gingerol treatment ameliorated diabetes-induced inflammatory reaction in kidneys of diabetic rats. Moreover, a previous study reported that gingerol plays a significant role in improving the condition of renal tissue by alteration in p38MAPK and NF-κB activity, and control inflammatory reaction and oxidative stress [38]. In this study, the histopathological investigation of the sections of kidneys from the STZ treated group of rats showed huge pathological changes such as edema, inflammation, fibrosis, and lymphocytic infiltrate in comparison to the normal structure observed in the normal control group. These findings are in parallel with previous studies and it has been reported that severe destruction as well as glomerular sclerosis occurs in the kidneys of diabetic animals [54]. The administration of 6-gingerol markedly decreased the abnormalities in both the glomerular and tubular architecture in rats and displayed normal renal glomeruli and mild degenerative changes and minimal fibrosis. The kidney damage was significantly normalized by the treatment of 6-gingerol providing evidence that 6-gingerol plays a role in the protection of kidney damage. Additionally, a previous study reported that the treatment with 200 mg/kg of the Z. officinale hydroalcoholic extract, the expansion of the mesangial was slightly attenuated and a dose of 400 mg/kg of the same extract ameliorated enlargement of the mesangial in glomeruli [55]. Immunohistochemical study revealed no expression of TNF-α protein in the normal control group while it showed high expression in the diabetic control group. Moreover, diabetic groups treated by 6-gingerol displayed low expression of TNF-α protein. Ad- ministration of 6-gingerol markedly decreased the expression levels of TNF-α protein and suggests that it can ameliorate renal damages in diabetic rats, probably through the downregulation of TNF-α protein. A study based on Ursolic acid, a commonly found pentacyclic triterpenoid compound was used to study its effects on some renal disease parameters. The renal structural irregularities and the rise of TNF-α, MCP-1 and IL-1β expression level were diminished by the administration of Ursolic acid [56]. Furthermore, the TUNEL positivity of all treatment groups were examined and no TUNEL positive tissues were seen in any experimental group. Pharmaceutics 2021, 13, 317 13 of 15

In conclusion, the novelty of our results imply that 6-gingerol attenuated weight loss of diabetic rats and decreased the blood glucose level. Moreover, 6-gingerol shows reno- protective effects in diabetic rats via the regulation of urea and creatinine level, inhibition of oxidative stress, hyperglycemic and inflammatory markers like CRP, IL-6, IL-1β and TNF-α. Moreover, 6-gingerol ameliorates renal fibrosis and pathological changes via the decrease in expression of TNF-α protein in streptozotocin-induced diabetes. The pharmacokinetic and bioavailability studies are required to check its hypoglycemic actions and specific dosage in clinical practice.

Author Contributions: Conceptualization, interpretation of data for the work, designed the study and wrote the manuscript S.A.A. and A.H.R.; Formal analysis, assisted with the experiments A.Y.B.; Investigation, acquisition A.M.A.; Methodology, contributed to the development of the Diabetic animal model, A.Y.B., A.A.K. and A.H.R.; Project administration, Writing—review and editing M.A.A.; Resources, M.A.A.; Methodology, Writing—original draft, S.A.A. and A.H.R.; Writing— review and editing, data curation S.A.A., A.M.A. and A.A.K. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded and supported by Deanship of Scientific Research, Qassim Univer- sity, and Grants No. CAMS1-2019-2-2-I-5623. Institutional Review Board Statement: The animals were maintained at animal facility of the College of Applied Medical Sciences (CAMS) accordance with the guidelines of the Qassim University on Animal Care. The animal experiments were carried out as per the guidelines of CAMS, Qassim University and approved by the Institutional Animal Ethics Committee (2019-2-2-I-5623), CAMS, Qassim University. Informed Consent Statement: Not applicable. Data Availability Statement: The data used to support the findings of this study are included within the article. Conflicts of Interest: All authors declare that they have no conflict of interest.

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