TRPV1激动剂辣椒素在大鼠肝缺血再灌注损伤中的作用及机制

Effect of TRPV1 agonist capsaicin on hepatic ischemia-reperfusion injury in rats and its mechanism

目的研究瞬时感受器电位香草酸受体-1 (TRPV1)激动剂辣椒素对大鼠肝缺血再灌注损伤(IR)的保护作用及机制。方法将实验大鼠30只按双盲法随机均分为假手术组(Sham组)、缺血再灌注损伤损伤组(IR组)和辣椒素预处理组(CPS组),每组10只。采用夹闭左、中叶肝蒂建立70%大鼠IR模型,术前给予药物治疗。再灌注2 h后,取静脉血利用ELISA检测血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)和乳酸脱氢酶(LDH)的浓度;取肝脏组织利用TUNEL法检测缺血肝脏细胞凋亡指数(AI),Western blotting检测肝脏瞬时感受器电位香草酸受体-1 (TRPV1)、磷酸化瞬时感受器电位香草酸受体-1 (p-TRPV1)、B淋巴细胞瘤-2 (BCL-2)、细胞外信号调节激酶(ERK)、磷酸化细胞外信号调节激酶(p-ERK)、Bcl-2相关X蛋白(Bax)和半胱氨酸天冬氨酸蛋白酶3 (Caspase-3)表达水平;并利用苏木精—伊红染色(HE)染色观察肝脏病理损伤。结果 IR组大鼠血清中AST、ALT和LDH表达水平分别为(158.14±21.15) U/L、(327.78±36.43) U/L和(577.94±68.31) U/L,明显高于CPS组的(66.19±6.58) U/L、(155.36±15.81)U/L和(254.76±38.69) U/L,差异均有统计学意义(P<0.05);IR组大鼠的损伤评分为(3.50±0.50)分,明显高于CPS组的(1.50±0.20)分;AI为(80.00±15.00)个,明显多于CPS组的(35.00±7.00)个,差异均有统计学意义(P<0.05);IR组大鼠的肝组织中p-TRPV1、BCL-2和Caspase-3灰度比值分别为0.35±0.09、0.14±0.04和0.27±0.05,明显低于CPS组的0.57±0.12、0.48±0.06和0.56±0.16,差异均有统计学意义(P<0.05);而IR组大鼠的肝组织Bax和p-ERK灰度比值分别为0.42±0.09和1.34±0.27,明显高于CPS组的0.15±0.06和0.85±0.17,差异均有统计学意义(P<0.05);IR组大鼠的肝组织TRPV1和ERK灰度比值分别为0.69±0.20和1.27±0.20,CPS组大鼠的肝组织TRPV1和ERK灰度比值分别为0.76±0.21和1.16±0.31,差异均无统计学意义(P>0.05)。结论 TRPV1激动剂辣椒素具有良好的抗IR作用,其作用机制可能与抑制ERK途径介导的细胞凋亡相关。

Objective To study the protective effect and mechanism of TRPV1 agonist capsaicin on hepatic ischemia-reperfusion injury(IR)in rats and its mechanism.Methods Thirty experimental rats were randomly divided into the sham operation group(Sham group),the ischemia-reperfusion injury(ischemia-reperfusion,IR)group,and the capsaicin preconditioning group(capsaicin,CPS),with 10 rats in each group,by douple blind methods.The IR model of70%rats was established by clamping the left and middle lobe liver pedicle,and the drug was given before the operation.After reperfusion for 2 h,the serum levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),and lactate dehydrogenase(LDH)was measured by ELISA.TUNEL was used to detect the apoptosis index(apoptotic index,AI)of the liver.Western blotting was used to detect the expression level of the transient receptor potential vanilloid 1(TRPV1),phosphorylated TRPV1(p-TRPV1),B lymphoma-2(BCL-2),extracellular signal-regulated kinase(ERK),phosphorylated ERK(p-ERK),Bcl-2-related X protein(Bax),and Caspase-3 in the liver.HE was used to examine the pathological injury of liver.Results The serum expression levels of AST,ALT,and LDH in IR group were(158.14±21.15)U/L,(327.78±36.43)U/L,(577.94±68.31)U/L,significantly higher than(66.19±6.58)U/L,(155.36±15.81)U/L,(254.76±38.69)U/L in CPS group(P<0.05).The hepatic injury score in IR group was 3.50±0.10,significantly higher than 1.50±0.20 in CPS group(P<0.05).The AI in IR group was 80.00±15.00,significantly higher than 35.00±7.00 in CPS group(P<0.05).In addition,the gray scale ratios of p-TRPV1,BCL-2,and Caspase-3 in IR group were 0.35±0.09,0.14±0.04,and 0.27±0.05,significantly lower than 0.57±0.12,0.48±0.06 and 0.56±0.16 in CPS group with statistically significant differences(P<0.05);The gray scale ratios of p-ERK and Bax in IR group were 0.42±0.09 and 1.34±0.27,significantly higher than 0.15±0.06 and 0.85±0.17 in CPS group(P<0.05).The gray scale ratios of TRPV1 and ERK in IR group were 0.69±0.20 and 1.27±0.20,versus 0.76±0.21 and 1.16±0.31 in CPS group(P>0.05).Conclusion TRPV1 agonist capsaicin has good anti-IR effect,and its mechanism may be related to suppressing ERK mediating apoptotic signal.