Study On The Cold-sensitive TRPM8 Channel As A Potential Drug Target For Inhibiting Myocardial Ischemia Reperfusion

Objective: To investigate whether the 25 ℃ cold-sensitive transient receptor potential melastatin 8(TRPM8)channel can reduce myocardial ischemia-reperfusion injury and become a potential drug target for myocardial protection,and explore its underlying mechanism.Methods: Forty male SD rats(200g-220g)were randomly divided into four groups,including a 25 ℃ hypothermic ischemia / reperfusion group(25℃+I/R),a 37 ℃ normothermic ischemia / reperfusion group(37℃+I/R),a 25 ℃ hypothermic reperfusion plus TRPM8 antagonist BCTC group(25℃+BCTC+I/R)and 37 ℃ normothermic reperfusion plus TRPM8 agonist Icilin group(37℃+Icilin+I/R),10 in each group.The purpose of setting up 25℃ +IR and 25℃ +BCTC+IR groups is to observe whether 25℃ hypothermic reperfusion can play a role in myocardial protection,and whether the protective effect plays a role by activating TRPM8;the purpose of setting up 37℃ +IR and the 37℃ +Icilin+IR group is to observe whether the drug-activated TRPM8 can also play a role in myocardial protection when the body temperature is 37℃ reperfusion.The Langendorff apparatus was used for perfusion of isolated hearts.The myocardial ischemia/reperfusion injury was induced by global ischemia for 30 minutes,and then reperfusion for 120 minutes.Western blot and real-time quantitative PCR technique were used to detect the expression of TRPM8 in myocardial tissue and the protein expression of Bcl-2,Bax,phosphorylated caspase-3,Rho A,and ROCK2 in myocardial tissue;UV-visible spectrophotometry was used to detect lactate dehydrogenase(LDH)depletion in cardiac coronary effluent,superoxide dismutase(SOD)activity and malondialdehyde(MDA)content in heart tissue;TTC staining method was used to detect the infarct area of the heart;Lab Chart physiological signal recorder monitors was used to record the left ventricular development pressure(LVDP),left ventricular end-diastolic pressure(LVEDP),and maximum left ventricular pressure rise / fall rate(+ dp / dtmax /-dp / dtmax);HE staining method was used to observe the morphological changes of myocardium,and TUNEL staining method was used to detect myocardial cell apoptosis.Results:(1)Both TRPM8 protein and m RNA are expressed in SD rat myocardium;(2)Compared with normothermic reperfusion at 37 ℃,the area of myocardial infarction decreased significantly at 25 ℃ hypothermic reperfusion(P <0.01),LDH depletion in cardiac coronary effluent decreased significantly(P <0.01),and cardiac LVDP increased significantly(P <0.01),LVEDP decreased significantly(P <0.05),and ±dp/dtmaxincreased significantly(P <0.01);(3)Compared with normothermic reperfusion at 37 ℃,the content of MDA in myocardial tissue was significantly reduced(P <0.05),and the SOD activity was significantly increased(P <0.05).The apoptosis index was also significantly reduced(P <0.01);(4)Compared with normothermic reperfusion at 37 ℃,the expressions of Bax and phosphorylated caspase-3 in myocardial tissues were significantly decreased at25 ℃ hypothermic reperfusion(P <0.01),while the expression of Bcl-2 was significantly increased(P <0.05),and the expressions of Rho A and ROCK2 were significantly decreased(P <0.01).When the TRPM8 antagonist BCTC(8 μmol/L)was added at 25 ℃ hypothermic reperfusion,the changes of the above indexes were like those in normothermic reperfusion at 37 ℃,while the TRPM8 agonist Icilin(2 μmol/L)was added at 37 ℃ normothermic reperfusion,caused the similar changes of above indexes as hypothermic reperfusion at 25 ℃.Conclusion: During myocardial ischemia / reperfusion process,activation of cardiac TRPM8 during the onset of rperfusion can reduce myocardial oxidative stress and inhibit myocardial cell apoptosis by inhibiting the Rho A/ROCK2 signaling pathway,and thus provide myocardial protection,suggesting that the cold-sensitive TRPM8 channel can be a potential drug intervention target to reduce myocardial ischemia-reperfusion injury.