Pharmic Activation of PKG2 Alleviates Diabetes-Induced Osteoblast Dysfunction by Suppressing Plcβ1-Ca2+-Mediated Endoplasmic Reticulum Stress

Hindawi Oxidative Medicine and Cellular Longevity Volume 2021, Article ID 5552530, 21 pages https://doi.org/10.1155/2021/5552530

Research Article Pharmic Activation of PKG2 Alleviates Diabetes-Induced Osteoblast Dysfunction by Suppressing PLCβ1-Ca2+-Mediated Endoplasmic Reticulum Stress

Tingting Jia,1,2,3 Ya-nan Wang,1,2,3 Yao Feng,1,2,3 Chenchen Wang,4 Dongjiao Zhang ,1,2,3 and Xin Xu 1,2,3

1Department of Implantology, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, China 2Shandong Key Laboratory of Oral Tissue Regeneration, Jinan, Shandong 250012, China 3Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, Jinan, Shandong 250012, China 4Department of Oral Medicine, Qilu Hospital of Shandong University, Jinan, Shandong 250012, China

Correspondence should be addressed to Dongjiao Zhang; [email protected] and Xin Xu; [email protected]

Received 11 January 2021; Revised 8 April 2021; Accepted 17 May 2021; Published 17 June 2021

Academic Editor: Yong Zhou

Copyright © 2021 Tingting Jia et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

As reported in our previous study, cinaciguat can improve implant osseointegration in type 2 diabetes mellitus (T2DM) rats by reactivating type 2 cGMP-dependent protein kinase (PKG2), but the downstream mechanisms remain unclear. In the present study, we investigated the favorable effect of cinaciguat on primary rat osteoblast, which was cultivated on titanium disc under vitro T2DM conditions (25 mM glucose and 200 μM palmitate), and clarified the therapeutic mechanism by proteomic analysis. The results demonstrated that T2DM medium caused significant downregulation of PKG2 and induced obvious osteoblast dysfunction. And overexpression of PKG2 by lentivirus and cinaciguat could promote cell proliferation, adhesion, and differentiation, leading to decreased osteoblasts injury. Besides, proteomic analysis revealed the interaction between PKG2 and phospholipase Cβ1 (PLCβ1) in the cinaciguat addition group, and we further verified that upregulated PKG2 by cinaciguat could inhibit the activation of PLCβ1, then relieve intracellular calcium overload, and suppress endoplasmic reticulum (ER) stress to ameliorate osteoblast functions under T2DM condition. Collectively, these findings provided the first detailed mechanisms responsible for cinaciguat provided a favorable effect on promoting osseointegration in T2DM and demonstrated a new insight that diabetes mellitus-induced the aberrations in PKG2-PLCβ1-Ca2+-ER stress pathway was one underlying mechanism for poor osseointegration.

1. Introduction diabetes-mediated poor implant osteointegration, such as dysfunction of mitochondria [7] and accumulation of reac- Type 2 diabetes mellitus (T2DM) is a common metabolic dis- tive oxygen species [8] in osteoblasts. Meanwhile, our previ- ease characterized by hyperglycemia and dyslipidemia [1], ous study has demonstrated that the activity of type 2 cGMP- which is associated with osseous complications, such as oste- dependent protein kinase (PKG2) was decreased in the peri- oporosis, fractures, or osteoarthrosis [2–4]. It also acts as a implant bone area, and cinaciguat treatment provided a risk factor for the process of osseointegration, even lead to favorable effect on promoting osseointegration in T2DM rats titanium implant loss or lost due to impaired wound healing [9]. However, detailed cellular mechanisms of these findings [5], and subsequent bone resorption. Multiple studies have are still unclear and need further investigations. indicated that patients with diabetes have an increased risk Cyclic GMP (cGMP) plays a critical role in maintaining for implantation failure than that of nondiabetics [6]. glucose metabolism and bone homeostasis, which is pro- Numerous theories have been proposed for the etiology of duced by soluble guanylyl cyclases (sGCs) and hydrolyzed 2 Oxidative Medicine and Cellular Longevity by phosphodiesterase-5 (PDE5) [10]. Then, cGMP can be potential molecular target for further intensive exploration regarded as secondary messenger to activate two types of regarding the effect of T2DM on implant osteointegration. protein kinases (PKG1 and PKG2) [11]. The expression of PKG1 is limited in the vasculature, and PKG2 is mainly 2. Materials and Methods expressed in specific cell types, such as bone, intestine, brain, and kidney [12]. Increasing literatures have shown that 2.1. Primary Rat Osteoblast Culture and Treatment. Neonatal PKG2 is a key modulator in skeletal homeostasis and bone Sprague-Dawley rats (<24 h old) were selected for osteoblast remodeling. The PKG2-deficient mice developed dwarfism isolation, which were approved by the Animal Ethics Com- that was caused by interfered with endochondral ossification mittee of the School of Stomatology, Shandong University. [13]. And treatment with PKG2-generating agents could Osteoblasts were primarily obtained from the calvaria of rat fi restore osteoblast function and bone formation in diabetes by tissue block method and puri ed using repetitive adhe- ’ fi ’ [14]. In recent years, the impaired cGMP-PKG2 pathway in sion. Cells were cultured in Dulbecco s modi ed Eagle s various tissues has been stated to be associated with the risk medium (DMEM, BI, USA), supplemented with 10% fetal of diabetes and diabetic complications, for instance, cardio- bovine serum (FBS, BI, USA) and 1% penicillin/streptomy- ° fi myopathy [15] and nephropathy [16]. Our research group cin, at 37 C under a humidi ed atmosphere of 5% CO2. also demonstrated that low expression of PKG2 and high The morphology and growth of cells were observed by an fi expression of PDE5 appeared in the peri-implant bone in inverted microscope. Also, the isolated and puri ed osteo- fi diabetic rats [9]. Therefore, drugs are demanded to reactivate blasts were identi ed by alkaline phosphatase staining and the interfered cGMP/PKG2 pathway to exert its protective alizarin red staining. All osteoblasts at cell passages 3-5 were effect in T2DM conditions. seeded on Titanium (Ti) discs (Grade 1; Baoti, Shanxi, China) ff Cinaciguat (BAY58-2667) was selected from 800 ana- in di erent plates. logues as the first NO-independent activator of sGC in To detect the role of PKG2 activation in T2DM-induced ff 2002 [17], which could activate sGC in the low nanomolar osteoblast dysfunction, cells were cultured in di erent range, thereby high-efficiently generate cGMP-producing groups, including normal medium (5.5 mM glucose), control activity and augment PKG2. Several studies have reported medium (adding 20 mM mannitol to the 5 mM glucose its beneficial effects in heart failure [18], pulmonary hyper- medium and palmitate vehicle), T2DM medium (25 mM glu- μ tension [19], and erectile dysfunction [20]. Especially, cinaci- cose and 200 M palmitate to mimic diabetic conditions) guat was considered to be a novel treatment target for [22], T2DM medium with cinaciguat (100 nM, MedChem- μ osteoporosis, which enhances osteocyte survival in OVX Express, USA), or T2DM medium with m-3M3FBS (0.5 M, μ mice [21]. Recent studies have shown that cinaciguat could Santa Cruz, USA), U73122 (2 M, MedChemExpress, USA), μ play protective action in hyperglycemic environments to 4-PBA (200 M, MedChemExpress, USA), Tunicamycin μ improve cardiac dysfunction and prevent bone loss of dia- (0.2 g/ml, MedChemExpress, USA). betic animals. Moreover, we concluded that long-term use 2.2. Cell Proliferation Assessments. EDU assay kit (RiboBio, of cinaciguat could improve poor implant osteointegration China) was used to test the proliferation of osteoblasts based in T2DM rats. This favorable effect of cinaciguat was deliv- on the manufacturer’s instructions. Cells were seeded in Ti ered by normalization of PKG2 expression, but little infor- disc (diameter 10 mm) placed in a 48-well plate (104 cells/- mation is available about the downstream mechanisms. well). EDU detection was monitored 3 days after cells seed- With the development of high-throughput proteomic ing. Different culture medium was replaced by 50 μmol/L sequencing, much progress has been made to elucidate the EDU labelling medium and incubated for an additional 2 h underlying proteins involved in diabetes development. In at 37°C. Then, cells were fixed with paraformaldehyde, the current study, we examined the role of reactivation enhanced permeability with 0.5% TritonX-100, and stained PKG2 on osteoblasts, which were cultured on titanium sheets with Apollo®567 solution and Hoechst 33342 solution. in vitro T2DM models, and hypothesized that osteoblasts Finally, the osteoblasts were observed by fluorescence micro- would exhibit an ameliorative osteogenic ability when cul- scope (Leica, Germany). The percentage of EDU-positive tured with cinaciguat. Furthermore, isobaric tag for relative cells (red fluorescence) among the total cells (blue fluores- and absolute quantitation (iTRAQ) of osteoblasts in T2DM cence) was calculated by ImageJ software. environment with and without cinaciguat treatment was conducted to clarify the underlying molecular mechanism, 2.3. Cell Apoptosis Assessments. The apoptosis of different thus, we found the reduced expression of phospholipase group cells was detected by flow cytometry kit (Liankebio, β β C 1 (PLC 1) may play a vital role in this process. We China). Briefly, osteoblasts were harvested and centrifuged β supposed that the excessive activation of PLC 1 under for 3 min at 1000 rpm, then resuspended in 1x Annexin V diabetic microenvironment caused stimulation of phos- binding solution containing 5 μl Annexin V-FITC and 10 μl phatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and PI for 10 min at room temperature in dark. The apoptosis inositol 1,4,5-trisphosphate (IP3) generation, inducing IP3- rate was acquired by flow cytometer (BD Accuri TM, USA), dependent calcium overload and endoplasmic reticulum and data were analyzed with BD Accuri TMC6 Plus Software. (ER) stress, eventually lead to poor osteointegration. Fortu- nately, reactivation PKG2 by cinaciguat could rescue this 2.4. Cell Adhesion Assessments. Rhodamine-phalloidin stain- adverse process. Overall, our study elaborated a new mecha- ing was employed to observe the osteoblast morphology, nism of PKG2-PLCβ1-Ca2+-ER stress axis, which could be a which was cultured on Ti discs with different treatments. Oxidative Medicine and Cellular Longevity 3

After fixed with 4% formaldehyde, cells were washed with (Millipore, USA), and intensities were calculated by ImageJ PBS and 0.5% Triton X-100, then stained with Rhodamine- software. phalloidin and Dapi for observation by fluorescence microscope (Leica, Germany). 2.8. Transmission Electron Microscopy (TEM) Investigations. TEM investigations were conducted to confirm the dilation 2.5. Osteogenic Differentiation Assessments. Alkaline phos- of endoplasmic reticulum. Osteoblasts were harvested and fi phatase (ALP) activity assay, ALP staining, and alizarin red xed in 3% glutaraldehyde at room temperature. After ff fi staining were used to measure the cell osteogenetic ability washed by 0.1 M phosphate bu er (PBS), cells were xed under different conditions. At day 7 after induction, the again by 1% osmic acid, then, the samples were subjected to ALP activity assay was performed following the instructions gradient ethanol dehydration, acetone penetration, embed- ° of the ALP activity assay kit (Nanjing Jiancheng, China), ding, and polymerized in a drying oven for 12 h at 37 C, ° ° and the absorbance at 520 nm was measured by microplate 12 h at 45 C, and 48 h at 60 C. Subsequently, ultrathin sections spectrophotometer. For staining, cells were washed thrice (50-70 nm) were sliced and stained with 2% uranyl acetate and with phosphate-buffered saline (PBS) and fixed in 4% para- lead citrate. Photos were acquired using transmission electron formaldehyde, then stained with NBT/BCIP staining kit microscope (FEI F200C, USA). (Beyotime, China) or Alizarin Red (Solarbio, China), and 2.9. siRNA Knockdown and Lentivirus-Mediated PKG2 finally washed twice with ddH O. 2 Overexpression of Osteoblasts. Targeted small interfering RNA (siRNA) was designed to knock down PKG2 gene 2.6. Cell Immunostaining Staining. Immunofluorescence expression by Genepharma (Shanghai, China). Osteoblasts staining was used to examine the expression levels and were transfected using Micropoly-transfecter (Jiangsu, China), nuclear-cytoplasm distribution of PKG2 or PDE5. The cells and knockdown efficiency was detected by total RNAs were seeded on Ti disc in 12-well plate and treated with dif- and proteins, which were extracted at 48 h or 72 h after ferent stimuli. Three days after treatment, osteoblasts were transfection. washed with PBS, fixed with paraformaldehyde, blocked with For overexpression, lentivirus packaging osteoblasts were 1% BSA, and incubated with primary antibodies to PKG2 transfected with LV-Prkg2 and LV-Con purchased from (Santa Cruz, sc-393126, 1 : 200, USA) and PDE5 (Abcam, Genechem (Shanghai, China). After infection, cells were sta- ab14672,1 : 500, UK) overnight at 4°C in sequence. Subse- bly selected with puromycin (Solarbio, China). quently, the cells were incubated with fluorescent secondary antibodies (goat anti-rabbit IgG, Abcam, ab150084, 1 : 400, 2.10. Proteomic Analysis. Osteoblasts from T2DM medium UK; goat anti-mouse IgG, Abcam, ab150117, 1 : 400, UK) in and T2DM medium with cinaciguat were prepared as sam- the dark, and the nucleus was stained by DAPI (Sigma, ples for proteomic analysis with three biological replicates fl USA). The positive cells were visualized by uorescence for each group. Six groups of protein were separated, and fl microscopy (Leica, Germany), and the uorescent quantita- the concentrations were determined with the BCA method, tive analysis was conducted by Image J software. then SDS-polyacrylamide gels electrophoresis was performed for protein quantification. After proteolysis with Filter-Aided 2.7. Protein Isolation and Western Blot. Proteins were Sample Preparation (FASP), the protein samples were sepa- ff extracted from osteoblasts by RIPA lysis bu er (Solarbio, rately labeled by TMT kit (Thermo Fisher Scientific, USA) China) on ice, and the supernatant lysate was collected after according the manufacturer’s instructions. Subsequently, centrifugation at 12,000 rpm for 15 min. Then, the concen- the labeled peptides of each group were mixed and fraction- tration was measured using BCA Protein Assay Kit (Solarbio, ated by 1260 infinity II high-performance liquid chromatog- ° China). After protein denaturation for 5 min at 100 C, the raphy (Agilent, Germany), and mass spectrometry (MS) proteins were separated on SDS-polyacrylamide gels and analysis was performed. fl transferred to polyvinylidene di uoride (PVDF) membranes For bioinformatic analysis, the GO and KEGG pathway by a wet transfer apparatus. Bands were blocked in 5% dried enrichment analyses were performed to identify candidate ° fi milk for 1 h and incubated overnight at 4 C with rst anti- biomarkers by the Fisher’s Exact Test. For cluster analysis, bodies against COLI (Cell Signaling Technology, CST, the quantitative information of the target protein was nor- 84336, 1 : 1000, USA), ALP (Abcam, ab108337, 1 : 1000, malized firstly, then, we used Matplotlib software to classify UK), RUNX2 (CST, 8486, 1 : 1000, USA), PKG2 (Santa Cruz, the two dimensions of sample and protein expression at the sc-393126,1 : 500,USA), PDE5 (Abcam, ab14672,1 : 1000, same time and generated hierarchical clustering heat map. β UK), PLC 1 (Abcam, ab182368, 1 : 1000, UK), IP3R (Santa The Search Tool for the Retrieval of InteractingGenes/Pro- Cruz, sc-377518,1 : 500,USA), PERK (Bioss, bs-2469R, 1 : 500, teins (STRING) database and Cytoscape software were used China), phospho-PERK (Bioss, bs-3330R, 1 : 500, China), to generate a protein-protein interaction (PPI) network. GRP78 (Bioss, bs-1219R, 1 : 500, China), ATF4 (Bioss, bs- 1531R, 1 : 500, China), CHOP (CST, 2895, 1 : 1000, USA), β- 2.11. Determination of Intracellular Ca2+ Concentration. The actin (Abways, AB0035, 1 : 5000 China), and GAPDH concentration of intracellular Ca2+ was measured in osteo- (Proteintech, 10494-1-AP, 1 : 5000, China). The next day, the blasts using a fluorescent Ca2+ indicator Calbryte™ 520 AM membranes were washed and incubated with anti-rabbit or (AAT Bioquest, USA). After washing by Hanks and Hepes anti-mouse HRP-conjugated secondary antibodies. The pro- Buffer (HHBS), 200 μLof2μM Calbryte™ 520 AM was tein bands were visualized under Amersham Imager 600 added to the wells and incubated for 45 min at 37°C and then 4 Oxidative Medicine and Cellular Longevity incubate the plate at room temperature for another 15 min. 3.3. Overexpression of PKG2 Relieved Osteoblast Dysfunction Replace the dye working solution with HHBS, and Images under T2DM Condition. To determine the effects of PKG2 were acquired in the FITC channel. on diabetes-induced osteoblast dysfunction, lentiviral vector was used to overexpress PKG2. The overexpression efficiency 2.12. Statistical Analysis. All data were expressed as mean ± was examined by western blot (Figure 3(a)). Compared with standard deviations (SD) at least three independent experi- the negative control (OENC) group, an obvious increase in ments. Statistically significant differences (p value of less than cell proliferation was discovered in the T2DM-treated osteo- 0.05) were analyzed using two-tailed Student’s t-test and blast after overexpression of PKG2 (Figures 3(b) and 3(c)). one-way analysis of variance (ANOVA) with SPSS 16.0 Also, the cytoskeleton of cells inclined to be extended and software. dense on Ti surfaces in the PKG2-higher expression group, while the T2DM and OENC groups acquired wizened and 3. Results sparse cytoskeletal structure (Figure 3(d)). Furthermore, T2DM-induced decrease of differentiation capability in oste- 3.1. Impaired Proliferation, Adhesion, and Differentiation oblast was recovered by overexpression of PKG2. ALP activ- Capability of Osteoblast in T2DM Medium. In this study, ity and mineralization capacity (Figures 3(e) and 3(f)) were we first identified the purity of the rat primary osteoblast. clearly increased in osteoblast, and the protein expression After three sample purifications, the isolated cells exhibited level of COL1, ALP, and RUNX2(Figure 3(g)) was upregu- typical morphology of osteoblasts with good proliferation lated because of transfected with lentiviruses PKG2. Taken ability (Figure 1(a)). ALP staining showed positive results, together, overexpression of PKG2 in vitro reduced pathway and alizarin red staining presented calcium nodules with dif- dysfunction and promoted osteogenesis. ferent sizes (Figure 1(b)), which indicated the cells possessed osteogenetic property. 3.4. Cinaciguat Alleviates T2DM-Induced Osteogenesis Injury Meanwhile, we mimicked diabetic conditions in vitro by by Upregulating PKG2. Our previous study has confirmed using 25 mM glucose and 200 μM palmitate, as previously that pharmacologic activation of the cGMP/PKG2 signaling reported [22]. To investigate whether T2DM medium had a by cinaciguat could improve implant osseointegration in profound effect on many aspects of osteoblasts, we explored T2DM rats [9], but its effects on osteoblast biological behav- its proliferation, adhesion, and differentiation capability. ior on titanium surface in vitro remain unclear. First, western EDU test displayed that T2DM-treated osteoblasts had blot and immunofluorescence confirmed the disturbed path- reduced proliferation ability relative to normal group, as way was ameliorated by cinaciguat, which mainly manifested revealed by less EDU-positive cells (Figures 1(c) and 1(d)). as high-expression of PKG2 in T2DM-cinacguat group T2DM medium also induced more cell apoptosis, as quanti- (Figures 4(a)–4(c)). Second, our work showed that PKG2 fied by evaluating Annexin V-FITC and PI staining was necessary for the proproliferative and prosurvival effects (Figures 1(f) and 1(g)). Cell adhesion was directly qualitative of cinaciguat in osteoblasts under T2DM condition. Cultur- analysis by Rhodamine-phalloidin staining 48 h after stimu- ing osteoblasts in T2DM medium with cinaciguat recovered lation on Ti surfaces. We could find that osteoblasts in the EDU incorporation (Figure 4(e)) and cytoskeletal structure normal and control group appeared more stretched and (Figure 4(i)) and also reduced cell apoptosis to 10.8% larger than cells in the T2DM group (Figure 1(e)). After (Figures 4(g) and 4(h)). The efficiency of knockdown PKG2 treating with osteogenic induction medium for 7 days and by siRNA was examined by western blot (Figure 4(d)). As 28 days, the ALP activity and Alizarin red staining were obvi- expected, the favorable effects were faded down when knock- ously reduced upon T2DM medium addition (Figures 1(h) down of PKG2 with siRNA. Finally, adding cinaciguat to the and 1(i)). Consistently, T2DM medium led to lower expres- T2DM medium obviously increased ALP production at 7 sion of osteogenesis-related protein (including COL1, ALP, days (Figures 4(j) and 4(k)). Cinaciguat also induced an and RUNX2) detected by western blotting compared with increase in expression of the osteoblastic differentiation the normal group (Figure 1(j)). And the differences were marker proteins COL1, ALP, and RUNX2 (Figure 4(l)). Sim- not from elevated osmolarity and vehicle, because the control ilarly, cinaciguat was ineffective to enhance osteogenesis in group neither affected cell viability nor cell differentiation. prkg2-deficient osteoblasts. These results demonstrated that cinaciguat could reverse the negative effects of diabetes on 3.2. Downregulation of PKG2 Expression in Osteoblast osteoblast in vitro, which was mediated by PKG2. Culturing with T2DM Medium. Existing evidences have demonstrated that the cGMP/PKG2 pathway could be dete- 3.5. Quantitative Proteomic Analysis of Differentially- riorated under diabetic conditions, and this phenomenon Expressed Proteins (DEPs) Regulated by Cinaciguat in was also proved by Figure 2. Western blot showed that the Osteoblasts under T2DM Condition. Further, we explored downregulated expression of PKG2 and upregulated expres- the potent molecular mechanisms that how upregulated sion of PDE5 were observed in the T2DM group. Immuno- PKG2 by cinaciguat affected the fate of osteoblasts in the fluorescence displayed a further verification to western blot, T2DM medium. Protein components between the T2DM primarily manifested as a stronger red fluorescence (PDE5) group and T2DM + cinaciguat group were compared by pro- and weaker green fluorescence (PKG2) under T2DM condi- teomic analysis. The identification of DEPs was based on two tion. The above results demonstrated that PKG2 was sup- primary principles, p value < 0.05 and an absolute fold pressed by diabetes. change ≥ 1:2. Thus, among a total of 6437 proteins quantified, Oxidative Medicine and Cellular Longevity 5

200 �m 200 �m (a)

200 �m 200 �m (b) Normal Control T2DM EDU

20 ⁎ ⁎ 15 Hoechst 10

5 Merge EDU-positive percentage (%) 0 Normal Control T2DM (c) (d) Normal Control T2DM RP Dapi Merge

(e)

Figure 1: Continued. 6 Oxidative Medicine and Cellular Longevity

Gate: P1 Gate: P1 Gate: P1 107.2 107.2 107.2 Q1-UL Q1-UR Q1-UL Q1-UR Q1-UL Q1-UR 0.2% 1.4% 0.4% 1.6% 0.3% 5.0% 106 106 106 105 105 105 104 104 104

3 3 3

PerCP-A 10 PerCP-A 10 PerCP-A 10

102 102 102

101 Q1-LL Q1-LR 101 Q1-LL Q1-LR 101 Q1-LL Q1-LR 95.9% 2.5% 95.7% 2.3% 73.8% 20.9% 100 100 100 100 101 102 103 104 105 106 107.2 100 101 102 103 104 105 106 107.2 100 101 102 103 104 105 106 107.2 FITC-A FITC-A FITC-A

Normal Control T2DM (f)

⁎ ⁎ 30 ⁎ 180 ⁎ 150

20 120

10 60 Apoptosis index (%)

ALP activity (U/gprot) 30

0 0 Normal Control T2DM Normal Control T2DM (g) (h) Normal Control T2DM Normal Control T2DM

COL1

ALP

RUNX2

GAPDH

(i) (j)

Figure 1: Impaired proliferation, adhesion, and differentiation capability of osteoblast in T2DM medium. (a) The morphology of osteoblast, left: primary culture, right: passages P3. Scale bar = 200 μm. (b) The identification of osteoblast, left: ALP staining, right: Alizarin red Staining. Scale bar = 200 μm. (c) EDU staining was performed to test the osteoblasts undergoing DNA replication. Red, EDU positive nucleus. Blue, all nucleus. Scale bar = 100 μm. (d) The quantification analysis of EDU-positive cells. (e) Cytoskeleton (red) was stained by rhodamine- phalloidin, and nucleus (blue) was stained by DAPI. Scale bars = 100 μm. (f) Flow cytometer analysis was conducted to detect cells apoptosis. (g) The quantification analysis of apoptosis rate. (h) ALP activity assay after 7 days induction. (i) ALP staining (upper) and Alizarin red staining (lower) after 7 days or 28 days induction. (j) The protein levels of COL1, ALP, and RUNX2 were detected by western blot after 7 days induction. Data were presented as means ± SD from at least three independent experiments. ∗: p <0:05. RP: rhodamine-phalloidin.

130 proteins were upregulated, and 91 proteins were downreg- 25 KEGG pathway terms were enriched with statistical signif- ulated after adding cinaciguat, as demonstrated by the heat icance (Figure S1 A-D). map and volcano plot (Figures 5(a) and 5(b)). GO enrichment Furthermore, PPI analysis was constructed by STRING analysis showed the biological processes, molecular functions, platform, and the network was visualized in cytoscape, which and cellular components associated with DEPs, and a total of provided a 22-node protein module with PKG2 as the central Oxidative Medicine and Cellular Longevity 7

PKG2 PDE5 Dapi Merge Normal

100 �m 100 �m 100 �m 100 �m Control

100 �m 100 �m 100 �m 100 �m T2DM

100 �m 100 �m 100 �m 100 �m (a) Normal Control T2DM

50 ⁎ ⁎ ⁎ PKG2 ⁎ 40

30 PDE5

10 �-Actin Mean fuorescence intensity fuorescence Mean 0 PKG2 PDE5 Normal Control T2DM (b) (c)

Figure 2: Downregulation of PKG2 expression in osteoblast culturing with T2DM medium. (a) Immunofluorescent double staining was performed to examine the expression of PKG2 and PDE5 after 3 days of T2DM stimulation. Scale bars = 100 μm. (b) Quantitative analysis of mean fluorescence intensity of PKG2 and PDE5. (c) The protein levels of PKG2 and PDE5 were detected by western blot after 3 days of T2DM stimulation. Data were presented as means ± SD from at least three independent experiments. ∗: p <0:05. hub protein, and 5 of those (Prkg1, Atp2b4, Pln, Vasp, and stress, Ca2+ excessive release through the IP3R results in the Plcβ1) had a stronger relationship than the others, which exhaustion of Ca2+ in the endoplasmic reticulum and the accu- connected with thicker lines in Figure 5(c). Based on compre- mulation of Ca2+ in the cytoplasm [25], which disorders Ca2+ hensive literature retrieval of the node proteins, we were able homeostasis triggering ER stress and accelerating cell damage. to recognize a putative regulatory relationship between Based on the above, we suspected that upregulated PKG2 by PKG2 and PLCβ1, whose expression was greatly increased cinaciguat could inhibit the activation of PLCβ1, relieve in the T2DM medium. Specifically, PLCβ1 activity is inhib- intracellular calcium overload, and then suppress ER stress ited by PKG2 via phosphorylation of regulator of G protein to ameliorate osteoblast functions under T2DM condition. signaling 4 (RGS4) [23]. Further, the activation of PLCβ1 could lead to the hydrolysis of PIP2, generating two second 3.6. Cinaciguat Inhibited the Activation of PLCβ1/IP3R via messengers: diacylglycerol (DAG) and IP3 [24]. The latter PKG2 under T2DM Condition. To verify the protein mass could bind of IP3 receptor (IP3R), which is the Ca2+-release spectrometry results, the protein expression level of PLCβ1 channel on the endoplasmic reticulum. When exposure to was detected by western blotting (Figure 5(d)). As expected, 8 Oxidative Medicine and Cellular Longevity

Normal T2DM T2DM+OENC T2DM+OEPKG2 EDU

100 �m 100 �m 100 �m 100 �m

OENC OEPKG2 Hoechst

100 �m 100 �m 100 �m 100 �m PKG2

�-Actin Merge

100 �m 100 �m 100 �m 100 �m (a) (b) 0.20 ⁎ ⁎ 0.15

0.10

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EDU-positive percentage (%) percentage EDU-positive 0.00 T2DM Normal T2DM+OENC T2DM+OEPKG2 (c) Normal T2DM T2DM+OENC T2DM+OEPKG2 RP

100 �m 100 �m 100 �m 100 �m ⁎ 150 ⁎ Dapi

100

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100 �m 100 �m 100 �m 100 �m 0 T2DM Normal T2DM+OENC T2DM+OEPKG2 (d) (e)

Figure 3: Continued. Oxidative Medicine and Cellular Longevity 9

Normal T2DM T2DM+OENC T2DM+OEPKG2

T2DM Normal T2DM OENC OEPKG2

COL1

ALP

RUNX2

GAPDH

(f) (g)

Figure 3: Overexpression of PKG2 relieved osteoblast dysfunction under T2DM condition. (a) The overexpression efficiency of PKG2 was verified by western blot. (b) EDU staining was performed to test the osteoblasts undergoing DNA replication. Red, EDU positive nucleus. Blue, all nucleus. Scale bar = 100 μm. (c) The quantification analysis of EDU-positive cells. (d) Cytoskeleton (red) was stained by rhodamine-phalloidin, and nucleus (blue) was stained by DAPI. Scale bars = 100 μm. (e) ALP activity assay after 7 days induction. (f) ALP staining (upper) and Alizarin red staining (lower) after 7 days or 28 days induction. (g) The protein levels of COL1, ALP, and RUNX2 were detected by western blot after 7 days induction. Data were presented as means ± SD from at least three independent experiments. ∗: p <0:05. OENC: overexpression negative control; OEPKG2: overexpression of PKG2; RP: rhodamine-phalloidin.

T2DM treatment led to higher expression of PLCβ1 com- ATF4, and CHOP expression was higher in T2DM + m- pared to the control group, while cinaciguat decreased the 3M3FBS group than in T2DM, which was reversed by expression of PLCβ1, which was consistent with proteomic exposing to cinaciguat and in siNC group. Additionally, the analysis. Consequently, cinaciguat reduced the expression alterations of subcellular ER structure were detected by of IP3R compared with the T2DM group. The drug was TEM. Gross ER dilation and distortions were observed in impotent in prkg2-deficient cells, which represented the cells in response to T2DM, and the dilation was exacerbated reducing effect was mediated by PKG2. after the addition of m-3M3FBS. Fortunately, the negative phenomena were largely repressed by U73122 treatment and 3.7. Cinaciguat Relieved Intracellular Calcium Overload and reactivation of PKG2 by cinaciguat. ER Stress via PKG2 by Suppressing PLCβ1. To further under- stand the interaction between PKG2 and PLCβ1, m-3M3FBS 3.8. Cinaciguat Showed Osteoprotective Effects via PKG2 by and U73122 were used to activate and inhibit PLCβ1, respec- Suppressing ER Stress under T2DM Condition. To detect tively. The relevant effect of two drugs was verified by western whether ER stress contributed to T2DM-related dysfunction blot (Figure S2). Meanwhile, the concentration of intracellular in osteoblasts, we analyzed the impact of 4-phenylbutyric Ca2+was detected in osteoblast by Calbryte™ 520 AM, and the acid (4-PBA), an inhibitor of ER stress, and tunicamycin, fluorescence intensity (Figures 6(a) and 6(b)) indicated that an activator of ER stress, on the proliferation, apoptosis, both cinaciguat and U73122 treatment could inhibit the and differentiation of cells. Western blot displayed that 4- accumulation of Ca2+ in the cytoplasm induced by T2DM PBA and tunicamycin could inhibit and activate the pathway medium. The addition of m-3M3FBS achieved more of ER stress (Figure S3). Both cinaciguat and 4-PBA cytoplasm Ca2+ than the T2DM group, demonstrating that ameliorated DM-induced decreased proliferation (Figures 7(a) the activation of PLCβ1 by m-3M3FBS aggravated calcium and 7(b)), excessive apoptosis (Figures 7(c) and 7(d)), overload, while cinaciguat could mitigate this adverse effect. disordered adhesion (Figure 7(e)), and impaired osteogenesis Also, cinaciguat was impotent to rescue the above adverse (Figures 7(f)–7(h)). Moreover, tunicamycin treatment alone effect when knockdown of PKG2. Mounting evidences have under T2DM condition could aggravate these dysfunctions in shown that intracellular calcium increase could induce ER osteoblasts. In addition, the inhibitory effects of tunicamycin stress [26, 27]. In our study, ER stress response was assessed on proliferation and differentiation were countered by the by TEM investigations (Figure 6(c)) and detecting the addition of cinaciguat, while the effects were disappeared in protein expression of ER stress markers (Figure 6(d)). T2DM the siPKG2 group. stimulation increased the expression of phosphorylated PERK, GRP78, ATF4, and CHOP, while cinaciguat and 4. Discussion U73122 inhibited the activation of the above proteins. To verify whether cinaciguat could restrain the activation of the Type 2 diabetes mellitus is closely related to impaired ER stress through PLCβ1, we added m-3M3FBS into implant osteointegration, and increased risk of implantation osteoblasts. Western blot showed that p-PERK, GRP78, failure, which is due to the function of osteoblasts, is 10 Oxidative Medicine and Cellular Longevity

T2DM Normal T2DM DMSO Cina

PKG2

GAPDH

(a)

PKG2 Dapi Merge Normal

100 �m 100 �m 100 �m T2DM

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100 �m 100 �m 100 �m T2DM+Cina

100 �m 100 �m 100 �m (b)

50 ⁎ ⁎ ⁎ 40 siNC siPKG2

30 PKG2

10 �-Actin Mean fuorescence intensity fuorescence Mean 0 T2DM Normal T2DM+Cina T2DM+DMSO (c) (d)

Figure 4: Continued. xdtv eiieadClua Longevity Cellular and Medicine Oxidative

PerCP-A 10 10 10 10 10 10 10 10

7.2 Merge Hoechst EDU 0 1 2 3 4 5 6 10 0.1% Q1-UL 0 96.4% Q1-LL 10 1 10 Normal Normal 2 10 Gate: P1 FITC-A 3 10 4 10 5 2.6% Q1-LR 1.0% Q1-UR 10 6 10 100 �m 100 �m 100 �m 7.2

PerCP-A 10 10 10 10 10 10 10 10 7.2 0 1 2 3 4 5 6 10 0.8% Q1-UL 0 75.1% Q1-LL 10 1 10 T2DM 2 T2DM 10 Gate: P1 FITC-A 3 10 4 10 5 19.8% Q1-LR 4.2% Q1-UR 10 6 100 �m 100 �m 100 �m 10 EDU-positive percentage (%) 7.2 0.00 0.05 0.10 0.15 0.20

PerCP-A 10 10 10 10 10 10 10 10 7.2 0 1 2 3 4 5 6 10 0.9% Q1-UL 0 76.1% Q1-LL 10 1 10 2 Normal DMSO DMSO 10 Gate: P1 FITC-A ⁎ Figure 3 10 4 10 5 17.3% Q1-LR

5.6% Q1-UR T2DM 10 6 :Continued. 4: 100 �m 100 �m 100 �m 10 (g) (e) (f) 7.2

PerCP-A 10 ⁎ 10 10 10 10 10 10 10

7.2 T2DM+DMSO 0 1 2 3 4 5 6 10 0.5% Q1-UL 0 90.0% Q1-LL ⁎ 10 1 10

2 T2DM+Cina Cina 10 Gate: P1 FITC-A Cina 3 10 4 10

5 T2DM+Cina+siNC 7.9% Q1-LR ⁎ 1.5% Q1-UR 10 100 �m 100 �m 100 �m 6 10 T2DM 7.2 T2DM

PerCP-A 10

10 10 10 10 10 10 10 T2DM+Cina+siPKG2 7.2 0 1 2 3 4 5 6 10 0.6% Q1-UL 0 90.2% Q1-LL 10 1 Cina+siNC 10 Cina+siNC 2 10 Gate: P1 FITC-A 3 10 4 10 5 8.0% Q1-LR 1.2% Q1-UR 10 100 �m 100 �m 100 �m 6 10 7.2

PerCP-A 10 10 10 10 10 10 10 10 7.2 0 1 2 3 4 5 6 10 0.6% Q1-UL 0 80.0% Q1-LL 10 1 Cina+siPKG2 Cina+siPKG2 10 2 10 Gate: P1 FITC-A 3 10 4 10 5 14.5% Q1-LR 5.0% Q1-UR 10 100 �m 100 �m 6 10 7. 2 11 12

Merge Dapi RP Normal 100 �m 100 �m 100 �m T2DM

ALP activity (U/gprot) Apoptosis index (%) 150 200 100 10 20 30 50 0 100 �m 100 �m 100 �m 0

Normal Normal DMSO ⁎ Figure ⁎

T2DM T2DM :Continued. 4: 100 �m 100 �m 100 �m (h) (i) (j) ⁎ ⁎ T2DM+DMSO T2DM+DMSO ⁎ ⁎

T2DM+Cina Cina T2DM+Cina ⁎ T2DM+Cina+siNC T2DM+Cina+siNC ⁎ 100 �m 100 �m 100 �m T2DM T2DM+Cina+siPKG2 T2DM+Cina+siPKG2 Longevity Cellular and Medicine Oxidative iasN Cina+siPKG2 Cina+siNC 100 100 100 �m �m �m 100 100 100 �m �m �m Oxidative Medicine and Cellular Longevity 13

T2DM Normal T2DM DMSO Cina Cina+siNC Cina+siPKG2

(k) T2DM NormalT2DM DMSO Cina Cina+siNC Cina+siPKG2

COL1

ALP

RUNX2

GAPDH

(l)

Figure 4: Cinaciguat alleviates T2DM-induced osteogenesis injury by upregulating PKG2. (a) The protein level of PKG2 was detected by western blot after 7 days of adding cinaciguat. (b) Immunofluorescent staining was performed to examine the expression of PKG2 after 3 days of adding cinaciguat. Scale bars = 100 μm. (c) Quantitative analysis of mean fluorescence intensity of PKG2. (d) The knockdown efficiency of PKG2 was verified by western blot. (e) EDU staining was performed to test the osteoblasts undergoing DNA replication. Red, EDU positive nucleus. Blue, all nucleus. Scale bar = 100 μm. (f) The quantification analysis of EDU-positive cells. (g) Flow cytometer analysis was conducted to detect cells apoptosis. (h) The quantification analysis of apoptosis rate. (i) Cytoskeleton (red) was stained by rhodamine-phalloidin, and nucleus (blue) was stained by DAPI. Scale bars = 100 μm. (j) ALP activity assay after 7 days induction. (k) ALP staining after 7 days induction. (l) The protein levels of COL1, ALP, and RUNX2 were detected by western blot after 7 days induction. Data were presented as means ± SD from at least three independent experiments. ∗: p <0:05. Cina: cinaciguat; RP: rhodamine-phalloidin. suppressed under T2DM condition. So, it is very necessary to together with anomalous cell adhesion and poor cell differen- choose an ideal T2DM-simulated vitro model to explore the tiation. All of the pathological changes were alleviative in the relationship between characteristics of osteoblasts and path- normal and control groups. These results offered evidence ological microenvironment. There are many methods of imi- that diabetes-induced dysfunction of osteoblasts on Ti sur- tating DM conditions in vitro [28], and the most used models face was established with success. are the addition of high glucose (HG) in culturing conditions. PKG2 is the main target of cGMP in osteoblasts and However, the effects of HG alone on osteoblasts have led to a osteocytes [33], whose deficiency could cause skeletal abnor- controversy, with either inconsistent or contradictory results. malities in humans [34, 35]. Further, we have previously con- Some researchers revealed that hyperglycemia decreased pro- firmed that the poor implant osseointegration observed in liferation, differentiation of osteoblasts [29, 30]. In contrast, a T2DM rats is partly caused by impaired cGMP/PKG2 path- few researchers indicated that hyperglycemia promoted min- way in osteoblasts, while the precise mechanism is still eralization and increased the expression of osteogenesis- unclear. In this study, we focused on the expression of related genes in osteoblasts [31]. Compared to type 1 diabetes PKG2 in vitro and discovered that it was downregulated in mellitus (T1DM), T2DM is a more complicated metabolic diabetic-stimulated osteoblasts, which was proved by western disease, characterized by not only hyperglycemia but also blot and immunofluorescence. Similar to phenomenon dyslipidemia [1, 32]. Therefore, we tried to clarify the influ- appeared in osteoblasts, several studies have indicated the ence of both high glucose (D-glucose, 25 mM) and high lipid reduced PKG expression in cardiomyocytes [15, 36] and (palmitate, 200 μM) on osteoblasts, helping to more closely cytotrophoblast cells [37] under diabetic conditions. In order imitate diabetes condition and subsequent impaired implant to determine if promoting PKG2 expression is sufficient to osteointegration. In this study, we first detected essential protect osteoblasts from diabetes-induced dysfunction, we osteogenesis characteristics of osteoblasts on Ti using increased its activity by transfecting lentivirus and adding T2DM-simulated vitro model, as evidenced by decreased cinaciguat. The overexpression of PKG2 by lentivirus pro- EDU-positive cells as well as increased cell apoptosis, moted cell proliferation, adhesion, and differentiation, leading 14 Oxidative Medicine and Cellular Longevity

Col15a1 Lgals3bp

b21apsH

Smoc1 Tsc22d1 Abi3bp

Smpd4 Supt7l

Dach1 Bgn Aspn

Mfap4 Fhl1 Pdzrn3 Phex Gjb2 Cilp Pln Akap14 Ppm1l Cep192 Tor3a Rpl39 Tmem126a Mfap5 Nexn Col14a1 Lrrc15 Zmat1 Sept4 Tgfi Col4a1 Afap1l2 Scml2 Rassf4 Dcn Comp Fmod Cnn1 Vwf LOC500846 Tbs1 Vsnl1 Pqlc3 LOC100910678 Nav2 Hmcn1 Rbfa Cd14 Plg Pym1 Bbs2 Trmt1l Penk Anks3 Zfp512b Pgm5 Krt2 Sf1 Tnfsf18Pls3 Bcl3 Uckl1 Pdlim1 Tubb4a Lmcd1 Atp2b4 Cavin3 Ehd2 Cntn2 Dpysl4 Cryab Cav2 Slfn13 Svep1 Krt5 Prkg1 Plcd1 Fam160a2 C1qtnf3 Nid1 Gstm3 Cst3 Med11 Gtpbp1 Sptlc2 Rapgef4 Tinagl1 Eps8l2 Psmd10 Fn1 Dnhd1 Ctif Col4a2 Arrb1 Usp12 Ca3 Lmod1 Gstm2 Zfp69 Prelp Vasp Lum Ogfod2

DM_Cina1

DM_Cina2 Tagln 2 DM_Cina3 Col5a1

DM1

DM2 Loxl4 DM3 1 Spp1 Lpin3 Fgb 0 Adam23 Ncf4 Aldob C3 B9d1 −1 Tubb1 Kat7 Cul9 Tril Acox2 −2 Ndfp2 F13a1 Iftm2 Ogn Hmgb2 Pex6 Sqstm1 Gpr4 Apod Fga Cyr61 Prkg2 COX2 Ptgis DM_Cina3 Akr1b8 Pcdh19 Mettl7b DM_Cina2 Tti2 Pdpn DM_Cina1 Dpp4 Fcgbp Fgg DM3 RGD1311703 ND2 Ecm1 DM2 Lyrm7 ND5 DM1 Fgfr1 Glyatl3 Itm2a Usp16 Ythdf3 Adh7 Sdc4 Slc39a8 Nqo1 Gnao1 Gpm6b Krt9 Mest C4b Tgfr2 Sema3a Sfrp2 Lix1l Adam12 Crabp2 Aldh3a1 Cobll1 Anxa5 Tmem26 Ccdc126 Ints9 Eif4a1 Ugcg Rgn Cyp1b1 Ldlr Cox6c2 Itfg1 Cntn1 AlcamNrp2 Hcn4 Laptm4a Lpar6 Dcun1d5 Fam92a Tnfrsf19 RGD1310352 Ptn Egf6 Serpine2 Gap43 Atg2b iftm3 Ncoa4 Pdia4 A1m Slc24a1 Igfp7 Gpc3 Adh1 Tmem248 Mme

Synb Pld3

Slc7a8 Cpe Tax1bp1 Ifngr1 Itih3

Lgr5 2plpA

Plcb1

Slc1a3 Vom2r30

Tmem59

Tmem178a (a)

Figure 5: Continued. Oxidative Medicine and Cellular Longevity 15

5 Tgfr2 Hspa12b Sema3a Sf1 4 Prkg1

Gnao1 Plcb1 Scml2 3 Arrb1 P- value

10 Prkg2 Akap14 Prkg2 Atp2b4 Lpin3

− log 2 Spp1 Plcb1 Rapgef4 Phex Vasp 1 Pln

Adh1 0 Aldh3a1 Adh7 −2 −0.26 0 0.26 2 Rgn

log2 (fold change) (b) (c) T2DM Normal T2DM Cina Cina+siNC Cina+siPKG2

IP3R

PLC�1

GAPDH

(d)

Figure 5: Quantitative proteomics analysis of differentially-expressed proteins regulated by cinaciguat in osteoblasts under T2DM condition. (a) Hierarchical clustering of the differentially expressed proteins. (b) Volcano plots of protein expression profiles. Red/green symbols classified the upregulated/downregulated proteins. (c) Protein-protein interaction was constructed by STRING platform, and the network was visualized in cytoscape. The thick lines represent strong association among proteins. (d) The protein levels of PLCβ1 and IP3R were detected by western blotting to verify the protein mass spectrometry result. Cina: cinaciguat. to decreased osteoblasts injury. The same protective effect also proteins, offering a more accurate picture of the pathways happened to the cinaciguat group. We started by measuring regulated by cinaciguat under T2DM condition. This deep the recovery of PKG2 expression, producing about two-fold proteomic coverage then identified a link between the down- increased PKG2 protein in the T2DM + Cina group versus regulation of PLCβ1 and upregulation of PKG2 in adding the the T2DM group, which demonstrated that PKG2 activity cinaciguat group. PLCβ1 is the major PLC isoform located was elevated in a close to normal physiological range. As on the nuclear envelope [38]. Data of the proteomics and expected, the PKG2 gain of function gave rise to cell- western blot displayed that PLCβ1 was highly expressed in increased in proliferation, cell-inerratic in adhesion, and cell- the T2DM group and inhibited in T2DM + cina. Moreover, resistive in apoptosis, explaining ameliorative differentiation the inhibitory effect was mediated by PKG2, which is consis- ability of osteoblasts. Also, we further afforded evidence that tent with PPI analysis. Current evidence suggests that exces- cinaciguat improved diabetic-induced dysfunction of osteo- sive activation of PLCβ1 under diabetic conditions means to blasts mediated by PKG2, because when PKG2 was silenced, generate much more IP3, so as to activate IP3R [39], the Ca2+ the opposite results were observed. channel on the ER. Wozniak et al. indicated that PLCβ1 The above results and our previous study demonstrated could encourage Ca2+ release through IP3R from the ER, that cinaciguat could attenuate T2DM-induced osteoblast resulting in a high cytoplasmic Ca2+ concentration [40]. injury and poor osseointegration. However, the precise Although Ca2+ is a crucial second messenger that regulates mechanisms leading to the protective effects of cinaciguat many processes of cellular biology, the excessive accumulation remain to be elucidated. In this study, we conducted proteo- of intracellular Ca2+ could trigger ER stress, which eventually mic analysis to enable the detection of some low abundance causes damage to cells and leads to many diseases [27, 41]. 16 Oxidative Medicine and Cellular Longevity

Normal T2DM T2DM+Cina T2DM+U73122 ⁎ ⁎ 50 ⁎ ⁎ ⁎ ⁎ 40

100 �m 100 �m 100 �m 100 �m 30 T2DM+m-3M T2DM+m-3M+Cina T2DM+m-3M+Cina+siNC T2DM+m-3M+Cina+siPKG2

10 Mean fuorescence intensity fuorescence Mean

100 �m 100 �m 100 �m 100 �m 0 T2DM Normal T2DM+Cina T2DM+m-3M T2DM+U73122 T2DM+m-3M+Cina T2DM+m-3M+Cina+siNC T2DM+m-3M+Cina+siPKG2 (a) (b)

Normal T2DM T2DM+Cina T2DM+U73122 T2DM m-3M

Normal T2DM Cina U73122 m-3M Cina Cina+siNC Cina+siPKG2 p-PERK

PERK

T2DM+m-3M T2DM+m-3M+Cina T2DM+m-3M +Cina+siNC T2DM+m-3M+Cina+siPKG2 GRP78

ATF4

CHOP

�-Actin

Figure 6: Cinaciguat relieved intracellular calcium overload and ER stress via PKG2 by suppressing PLCβ1. (a) Images of intracellular Ca2+ ﬂuorescence stained with Calbryte™ 520 AM. Scale bars = 100 μm. (b) Quantitative analysis of mean ﬂuorescence intensity of intracellular Ca2+. (c) Transmission electron microscopy images showed ER morphology after 3 days stimulation. ER is denoted by red arrows. Scale bars = 2 μm. (d) The protein levels of p-PERK, GRP78, ATF4, and CHOP were detected by western blotting. Data were presented as means ± SD from at least three independent experiments. ∗: p <0:05. Cina: cinaciguat; m-3M: m-3M3FBS.

Thus, we hypothesized that ER stress may be involved in dia- vation [42]. When undesirable stimulation or damage triggers betic injury, inducing osteoblast dysfunction and poor calcium overload, GRP78 dissociates from PERK and subse- osseointegration, and cinaciguat could ameliorate the adverse quently leads to ER stress. The process promotes the transla- effects by regulating ER stress via the PLCβ1 inhibition. To tion of ATF4 and activation of CHOP [43]. Our results verify the hypothesis, we exposed osteoblasts to cinaciguat, indicated that T2DM treatment remarkably activated the ER U73122 (PLC inhibitor), and m-3M3FBS (PLC activator) stress-related proteins of osteoblast, which agreed with previ- under T2DM condition. Calcium fluorescence was used to ous findings that palmitic acid could induce the high expres- evaluate the concentrations of Ca2+ in cytoplasm, and the sion of GRP78 and CHOP in osteoblast-like Saos-2 cell [44]. result indicated that both cinaciguat and U73122 could Furthermore, we discovered that m-3M3FBS-induced the decrease the cytoplasmic Ca2+ induced by diabetic condition. worse ER stress in osteoblasts was suppressed by cinaciguat When PLCβ1 activated by m-3M3FBS, there was a higher mediated by PKG2. These observations suggested that diabetes cytoplasmic Ca2+ than the T2DM group and then was sup- generates ER stress in osteoblasts that is dependent on Ca2+ pressed by cinaciguat. Moreover, the depressor effect of cinaci- homeostasis; moreover, cinaciguat relieved intracellular cal- guat had gone off while PKG2 silencing. Normally, chaperone cium overload and ER stress via PKG2 by suppressing PLCβ1, GRP78 binds to ER stress sensor, PERK, and inhibits its acti- providing a novel pharmaceutical target for cinaciguat. xdtv eiieadClua Longevity Cellular and Medicine Oxidative

Merge Hoechst EDU

PerCP-A 10 PerCP-A 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 7.2 7.2 0 1 2 3 4 5 6 0 1 2 3 4 5 6 omlTD ia4-PBA Cina T2DM Normal 10 10 0 0 0.2% Q1-UL 0.2% Q1-UL 66.1% Q1-LL 95.6% Q1-LL 10 10 1 1 10 10 2 2 Normal T2DM+Tm 10 10 FITC-A FITC-A Gate: P1 Gate: P1 3 3 10 10 4 4 10 10 5 5 1.2% Q1-UR 3.0% Q1-LR Q1-LR 10 10 25.5% 8.1% 6 6 10 10 7.2 7.2 10 PerCP-A PerCP-A 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 7.2 7.2 0 1 2 3 4 5 6 0 1 2 3 4 5 6 10 10 0 0 0.3% Q1-UL 80.7% Q1-LL 0.1% Q1-UL 74.8% Q1-LL 10 10 EDU-positive percentage (%) 1 1 10 15 20 0 5 10 10 T2DM+Tm+Cina 2 2 T2DM 10 10 Gate: P1 FITC-A FITC-A Gate: P1 3 3 Normal 10 10 ⁎ 4 4 Figure 10 10 5 5 T2DM 8.1% Q1-UR ⁎ 10.6% Q1-LR 17.0% Q1-LR 9.4% Q1-UR 10 10 ⁎ 6 6 10 10 T2DM+Cina 7.2 ⁎ 7.2 (c) :Continued. 7: (b) (a)

PerCP-A 10 PerCP-A 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 7.2 7.2 0 1 2 3 4 5 6 0 1 2 3 4 5 6 10

10 T2DM+4-PBA 0 0.1% Q1-UL 0 83.9% Q1-LL 0.3% Q1-UL 90.2% Q1-LL 10 10 T2DM+Tm+Cina+siNC 1 1

T2DM+Tm ⁎ Tm 10 10 2 2 T2DM+Cina 10 10 Gate: P1 Gate: P1 FITC-A FITC-A

3 T2DM+Tm+Cina 3 10 10 4 4 ⁎ T2DM 10 10 T2DM+Tm+Cina+siNC 5 5 6.3% Q1-UR 6.4% Q1-UR 3.2% Q1-LR 9.6% Q1-LR 10 10 6 iaCn+iCCina+siPKG2 Cina+siNC Cina 6 T2DM+Tm+Cina+siPKG2 10 10 7.2 7.2 PerCP-A 10 10 10 10 10 10 10 10 10 PerCP-A 7.2 10 10 10 10 10 10 10 7.2 0 1 2 3 4 5 6 0 1 2 3 4 5 6 10 10 0 0.3% Q1-UL 68.8% Q1-LL 0 0.4% Q1-UL 93.1% Q1-LL T2DM+Tm+Cina+siPKG2 10 10 1 1 10 10 2 2 T2DM+4-PBA Tm 10 10 Gate: P1 Gate: P1 FITC-A FITC-A 3 3 10 10 4 4 10 10 5 5 3.5% Q1-UR 2.9% Q1-LR 12.3% Q1-UR 18.6% Q1-LR 10 10 6 6 10 10 7.2 7.2 17 18 Merge Dapi RP omlTD ia4-PBA Cina T2DM Normal

ALP activity (U/gprot) Apoptosis index (%) 100 150 10 20 30 40 50 0 0

Normal Normal ⁎ ⁎ Figure T2DM

T2DM ⁎ ⁎ ⁎ ⁎ T2DM+Cina ⁎ (e) :Continued. 7: T2DM+Cina ⁎ (d) (f) T2DM+4-PBA T2DM+4-PBA Tm T2DM+Tm T2DM+Tm ⁎ ⁎

T2DM+Tm+Cina T2DM+Tm+Cina T2DM ⁎

T2DM+Tm+Cina+siNC ⁎ T2DM+Tm+Cina+siNC iaCn+iCCina+siPKG2 Cina+siNC Cina xdtv eiieadClua Longevity Cellular and Medicine Oxidative T2DM+Tm+Cina+siPKG2 T2DM+Tm+Cina+siPKG2 Tm Oxidative Medicine and Cellular Longevity 19

Normal T2DM T2DM+Cina T2DM+4-PBA

T2DM+Tm T2DM+Tm+Cina T2DM+Tm+Cina+siNC T2DM+Tm+Cina+siPKG2

(g) T2DM Tm Normal T2DM Cina 4-PBA Tm Cina Cina+siNC Cina+siPKG2 COL1

ALP RUNX2

GAPDH (h)

Figure 7: Cinaciguat showed osteoprotective effects via PKG2 by suppressing ER stress under T2DM condition. (a) EDU staining was performed to test the osteoblasts undergoing DNA replication. Red, EDU positive nucleus. Blue, all nucleus. Scale bar = 100 μm. (b) The quantification analysis of EDU-positive cells. (c) Flow cytometer analysis was conducted to detect cell apoptosis. (d) The quantification analysis of apoptosis rate. (e) Cytoskeleton (red) was stained by rhodamine-phalloidin, and nucleus (blue) was stained by DAPI. Scale bars = 100 μm. (f) ALP activity assay after 7 days stimulation. (g) ALP staining after 7 days stimulation. (h) The protein levels of COL1, ALP, and RUNX2 were detected by western blot after 7 days stimulation. Data were presented as means ± SD from at least three independent experiments. ∗: p <0:05. RP: rhodamine-phalloidin; Cina: cinaciguat; Tm: tunicamycin; 4-PBA: 4-phenylbutyric aci.

Then, we explored the interaction between osteoblast dysfunction and ER stress. Tunicamycin, a N-linked glyco- Osteoblasts Type 2 diabetic mellitus Cinaciguat sylation inhibitor, could induce ER stress, and 4-PBA could PLC� promote protein folding and secretion to suppress ER stress. PLC� β β Both of them were added in medium to examine the anti-ER PKG2 stress effects of cinaciguat. Our data showed that 4-PBA PKG2 could restrain T2DM-induced decrease in proliferation, dis- ff 2+ order adhesion, and poor di erentiation in osteoblasts, espe- Ca overload Ca2+ balance cially in excessive apoptosis. Similarly, Guan et al. also provided that palmitic acid could activate ER stress to induce IP3R IP3R 2+ apoptosis in cardiomyocytes, and 4-PBA could reduce apo- Ca Ca2+ ptosis by inhibition of ER stress [45], which further expanded our results that T2DM induced osteoblast dysfunction via ER ER stress ER recovery stress. The similar effects were achieved with the application Inducing dysfunction of cinaciguat. With the addition of tunicamycin, osteoblasts Improving bio-function suffered the most damage in all groups, and the adverse effect of tunicamycin was inhibited by cinaciguat. However, there was more serious osteoblast injuries in the siPKG2 group Poor implant osseointegration Better implant osseointegration than in the siNC group, which indicate that cinaciguat played anti-ER stress role through PKG2. The above findings Figure 8: Schematic illustration of the detailed mechanisms showed that the excessive activation of ER stress was involved responsible for cinaciguat provided a favorable effect on promoting in osteoblast dysfunction under diabetic environment, and osseointegration in T2DM. 20 Oxidative Medicine and Cellular Longevity cinaciguat played an osteoprotective role via suppressing the [2] N. Napoli, M. Chandran, D. D. Pierroz et al., “Mechanisms of ER stress activation. diabetes mellitus-induced bone fragility,” Nature Reviews Endocrinology, vol. 13, no. 4, pp. 208–219, 2017. 5. Conclusions [3] I. Kanazawa, “Interaction between bone and glucose metabolism,” Endocrine Journal, vol. 64, no. 11, pp. 1043–1053, 2017. In summary, our T2DM-simulated vitro model could impair [4] N. S. Asfandiyarova, A. A. Nizov, T. I. Nekhaeva, N. A. the biological characters of osteoblasts on Ti disc, and cinaci- Sakaeva, T. E. Filatova, and N. S. Zhuravleva, “Osteoarthrosis guat could alleviate this injury by reactivation PKG2. in patients with type 2 diabetes mellitus,” Therapeutic Archive, Moreover, integrated proteomic analysis identified a link vol. 85, no. 5, pp. 44–47, 2013. between PKG2 and PLCβ1 in adding cinaciguat group. Then, [5] F. Javed and G. E. Romanos, “Chronic hyperglycemia as a risk we basically demonstrated that cinaciguat inhibited the PLCβ1 factor in implant therapy,” Periodontology 2000, vol. 81, no. 1, activation to attenuate the intracellular Ca2+ overload-ER pp. 57–63, 2019. stress pathway and protected the osteoblasts from diabetes. [6] S. Annibali, N. Pranno, M. P. Cristalli, G. La Monaca, and These results provide the first detailed mechanisms responsi- A. Polimeni, “Survival analysis of implant in patients with dia- ble for cinaciguat treatment provided a favorable effect on betes mellitus,” Implant Dentistry, vol. 25, no. 5, pp. 663–674, promoting osseointegration in T2DM (Figure 8). Further- 2016. more, the present study provides a new insight that diabetes [7] X. F. Hu, L. Wang, Y. Z. 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