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Free PDF Download European Review for Medical and Pharmacological Sciences 2019; 23: 3088-3095 The effects of TRPM2, TRPM6, TRPM7 and TRPM8 gene expression in hepatic ischemia reperfusion injury T. BILECIK1, F. KARATEKE2, H. ELKAN3, H. GOKCE4 1Department of Surgery, Istinye University, Faculty of Medicine, VM Mersin Medical Park Hospital, Mersin, Turkey 2Department of Surgery, VM Mersin Medical Park Hospital, Mersin, Turkey 3Department of Surgery, Sanlıurfa Training and Research Hospital, Sanliurfa, Turkey 4Department of Pathology, Inonu University, Faculty of Medicine, Malatya, Turkey Abstract. – OBJECTIVE: Mammalian transient Introduction receptor potential melastatin (TRPM) channels are a form of calcium channels and they trans- Ischemia is the lack of oxygen and nutrients port calcium and magnesium ions. TRPM has and the cause of mechanical obstruction in sev- eight subclasses including TRPM1-8. TRPM2, 1 TRPM6, TRPM7, TRPM8 are expressed especial- eral tissues . Hepatic ischemia and reperfusion is ly in the liver cell. Therefore, we aim to investi- a serious complication and cause of cell death in gate the effects of TRPM2, TRPM6, TRPM7, and liver tissue2. The resulting ischemic liver tissue TRPM8 gene expression and histopathologic injury increases free intracellular calcium. Intra- changes after treatment of verapamil in the he- cellular calcium has been defined as an important patic ischemia-reperfusion rat model. secondary molecular messenger ion, suggesting MATERIALS AND METHODS: Animals were randomly assigned to one or the other of the calcium’s effective role in cell injury during isch- following groups including sham (n=8) group, emia-reperfusion, when elevated from normal verapamil (calcium entry blocker) (n=8) group, concentrations. The high calcium concentration I/R group (n=8) and I/R- verapamil (n=8) group. leads to depressed compensative vasodilation TRPM 2, 6, 7, 8 gene expression level was as- following a period of vasoconstriction. All these sessed by Real Time-quantitative Polymerase reasons, including the lack of oxygen and nutri- Chain Reaction (RT-qPCR) and histopathologic ents start the pathway of apoptosis and necrosis3. changes were determined by the hematoxylin Verapamil, which is a member of the dihydropyri- and eosin (HE) examination. 4 RESULTS: The expression level of TRPM 2, 6, dine family, is a calcium entry blocker . Two stud- 7, and 8 genes was significantly higher in isch- ies have reported that the addition of verapamil emia-reperfusion (I/R), verapamil, IR-verapamil in perfusate and the iv/ip pretreatment showed groups compared to sham group. The p-values beneficial effects in ischemia-reperfusion inju- were 0.0024, < 0.0001, 0.0002, 0.006 for TRPM2, ry prevention on animal models. Moreover, ver- TRPM6, TRPM7, and TRPM8, respectively. Se- apamil has a vital effect on calcium homeostasis vere necrotic, degenerative differentiations and severe hemorrhagic areas were observed in he- and could reduce ischemia and reperfusion in the 5,6 patocytes from IR group. Also, moderate necrotic injured liver . The calcium channel blockers are and degenerative differentiations and moderate also used to improve the post ischemic myocar- hemorrhagic areas were observed in hepato- dial function7 and the survival of ischemic skin cytes from IR-verapamil group. flaps8. CONCLUSIONS: This is the first study report- The mammalian transient receptor potential ing an association between the expression level of TRPM 2, 6, 7, 8 in a hepatic ischemia-reperfu- (TRP) channels are a form of calcium channels sion rat model. Moreover, TRPM 2, 6, 7, 8 affect and they transport calcium, magnesium and trace hepatic ischemia-reperfusion. metal ions9. Therefore, TRPs cause changes in cal- cium, magnesium and trace metal ions concentra- Key Words Ischemia, Reperfusion, TRP, TRPM, Verapamil, Cal- tions. These contributions are pivotal for several cium entry blocker. physiological processes including pheromone sig- naling, temperature sensation, cell proliferation, 3088 Corresponding Author: Faruk Karateke, MD; e-mail: [email protected] TRPM calcium channels affect hepatic ischemia-reperfusion and muscle contraction10. Almost every cell type Ischemia-reperfusion group (I/R): none of the expresses TRPs which are localized in the plasma rats were treated with any substance. Ischemia membrane9. TRP superfamily includes TRPC (ca- was applied to the left hepatic artery and the por- nonical), TRPV (vanilloid), TRPM (melastatin), tal vein by a clamp for 60 min after the laparot- TRPML (mucolipin), TRPP (polycystin), TRPA omy. Within 60 min after reperfusion, relaparot- (ankyrin transmembrane protein) and TRPN (nom omy was performed on all group members, and PC-like)11. The family of TRPMs has eight sub- liver and blood of the subjects were isolated (n=8). classes including TRPM1-8 and they are expressed Ischemia-reperfusion/calcium entry blocker in different cells. TRPM2, TRPM6, TRPM7, (I/R-verapamil): as pretreatment, verapamil (1.25 TRPM8 are expressed especially in the liver cell12. mg/kg) was administered orally to the rats 30 min Moreover, TRPM7 has been found to be involved before anesthesia. Ischemia was applied to the left in delayed neuronal death after ischemia13. hepatic artery and the portal vein by a clamp for 60 There is no study about the association be- min after the laparotomy. Within 60 min after reper- tween the application of calcium entry blocker fusion, relaparotomy was performed on all group aiming at preventing ischemia-reperfusion liver members, and liver and blood were isolated (n=8). injury and TRPM calcium channels. To under- stand the roles of TRPM calcium channels in liver Surgical Procedures tissue, it is important to explain the pathogenesis Rats were anesthetized with intraperitoneal in- of ischemia-reperfusion. Therefore, we aim to in- jections of xylazine (12 mg/kg) and ketamine (80 vestigate the effects of TRPM2, TRPM6, TRPM7, mg/kg), and placed in a supine position on a tem- and TRPM8 gene expression and histopathologic perature-controlled heating table, maintaining the changes after treatment of the calcium entry block- body temperature in the range of 36.5-37.5°C. Rats er in the hepatic ischemia-reperfusion rat model. were allowed to breathe spontaneously during sur- gery. For the preparation of the liver, abdominal skin was shaved and sterilized with 70% of ethyl alcohol. Materials and Methods After midline laparotomy and subcostal incisions, the liver was carefully mobilized from all the liga- In vivo Experiment mentous attachments. The left hepatic artery and the The experiments were carried out on 32 male portal vein were clamped for 60 min using an atrau- Wistar rats (average body weight 225±25 g) matic vascular clamp for complete ischemia of the housed in an environmentally controlled room median and left hepatic lobes. Then, the edges of the (24°C to 26°C temperature, 50% to 60% moisture abdominal incision were approximated to each other rate) with a 12:12 h light: dark cycle, and kept on and covered by a piece of gauze soaked with warm commercial rat chow and tap water ad libitum. isotonic saline to prevent the undue loss of body flu- The experimental protocol was in accordance ids. After the removal of the clamp, the median and with EU directive 2010/63 for the protection of left hepatic lobes were removed and the abdomen animals used for scientific purposes. This investi- was properly irrigated with isotonic saline. During gation was also approved by the local Committee IR periods, the abdomen was covered with a plas- on the Ethics of Animal Experiments of the Mus- tic wrap to minimize fluid loss via evaporation. At tafa Kemal University, Hatay, Turkey (2014/5-9). the end of ischemia, the abdomen was closed with continuous stitches using Vicryl (Ethicon Endo-Sur- Experimental Design gery, Inc., Cincinnati, Ohio, USA). 3/0 sutures and The animals were randomly assigned to one the animals were returned to their cages. After 60 or other of the following groups including sham, min of reperfusion, animals were anesthetized with verapamil (calcium entry blocker), I/R group and intraperitoneal injection of ketamine and xylazine I/R- verapamil groups. (80 and 12 mg/kg, respectively) and were sacrificed Sham Group: rats were subjected to glucose by exsanguination via right ventricular puncture, pretreatment and surgical procedures, except for then blood and histological samples were taken. the induction of liver ischemia, but including liver resection (n=8). Hepatic Histopathologic Evaluation Verapamil (Calcium entry blocker) group: rats After ischemia-reperfusion, the liver specimens were treated with verapamil as a pretreatment were removed and placed in 10 % of formalin. Then, (1.25 mg/kg) and to none of them ischemia-reper- tissues were embedded in paraffin after alcohol and fusion (n=8) was applied. xylene according to the standard protocol. Five mi- 3089 T. Bilecik, F. Karateke, H. Elkan, H. Gokce Table I. Primers used for TRPM gene expressions. Primers β-Actin Left 5’-CCC GCG AGT ACA ACC TTC T-3’ β-Actin Right 5’-CGT CAT CCA TGG CGA ACT-3’ TRPM2 Left 5’-AAT TTG CTC ATC GCC ATG TT-3’ TRPM2 Right 5’-GAT CTG GTC TGT GTG CTC CTG-3’ TRPM6 Left 5’-GCA AGA ACT GGC TTT CCG TG-3’ TRPM6 Right 5’-ATC CGG GTC CTC TTG CAT CT-3’ TRPM7 Left 5’-AGA CGC TTT CCG ATA GAT GG-3’ TRPM7 Right 5’-CTA TCC AGG ATT TCT GGG ACA T-3’ TRPM8 Left 5’-GCC CAG TGA TGT GGA CAG TA-3’ TRPM8 Right 5’-GGA CTC ATT TCC CGA GAA GG-3’ crometer thick sections were stained with hematox- Statistical Analysis ylin and eosin (HE) for light microscopy studies in Data were analyzed by using GraphPad Prism a double-blind manner. The hepatic damage was 5 program (GraphPad Software Inc., La Jolla, evaluated with a histopathologic scoring system. CA, USA). Data were expressed as mean values The assessment was expressed as the sum of the in- ± standard error of the mean (SEM). For normally dividual score grades from 0 (no findings), 1 (mild), distributed data, the one-way analysis of variance 2 (moderate), to 3 (severe) for each of the following (ANOVA) with Bonferroni’s multiple comparison six parameters: cytoplasmic color fading, vacuoliza- post-hoc test was used to test significant differ- tion, nuclear condensation, nuclear fragmentation, ences.
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