通腑泄热法中药通过线粒体DNA/Toll样受体9/微小RNA-223通路抑制肝脏缺血再灌注损伤

Inhibitory effect of"Tongfuxiere"method on hepatic ischemia reperfusion injury via mitochondria,deoxyribonucleic acid/toll-like receptor 9/micro ribonucleic acid-223 pathway

目的探讨通腑泄热法中药通过线粒体DNA(mtDNA)/Toll样受体9(TLR9)/微小RNA(miR)-223通路抑制肝脏缺血再灌注损伤(HIRI)的作用。方法随机数字法将80只健康无特定病原体(SPF)级SD大鼠分成通路正常组(NP组)、通路激活组(AP组)、通路正常中药组(NPT组)、通路激活中药组(APT组)4组,"通路激活"指在大鼠HIRI建模麻醉后采用TLR9激活剂50μg/只腹腔注射激活mtDNA/TLR9/miR-223通路,"通路正常"则腹腔注射对应剂量生理盐水;"中药"指手术处理前3 d以通腑泄热法中药按照5 ml/只/次剂量,每日2次给大鼠灌胃。每组20只,均建立肝脏HIRI模型,以分组要求施加不同干预后分别于再灌注后24、48 h两个时间点采集血清及肝组织标本,并进行相关生化指标、病理、蛋白质印迹法(Western blot)及聚合酶链反应(PCR)检测。采用多因素方差分析。结果"通路激活"及"中药干预"均是术后血清谷丙转氨酶(ALT)、谷草转氨酶(AST)和乳酸脱氢酶(LDH)变化的影响因素。通路激活可促进术后血清中ALT、AST、LDH升高,AP组大鼠术后24 h血清ALT、AST、LDH高于NP组[ALT:(962.34±42.35)U/L比(747.33±40.04)U/L,F=11.446,P<0.05;AST:(1421.58±93.25)U/L比(1041.87±48.60)U/L,F=5.893,P<0.05;LDH:(2312.99±95.77)U/L比(1974.23±75.80)U/L,F=5.019,P<0.05];而中药干预可抑制术后血清中ALT、AST、LDH升高,NPT组大鼠术后24 h血清ALT、AST、LDH低于NP组[ALT:(561.31±40.49)U/L比(747.33±40.04)U/L,F=688.807,P<0.05;AST:(792.38±44.03)U/L比(1041.87±48.60)U/L,F=838.255,P<0.05;LDH:(1702.75±62.32)U/L比(1974.23±75.80)U/L,F=514.608,P<0.05];通路激活及中药干预的交互作用呈现出抑制作用,APT组大鼠术后24 h血清ALT、AST、LDH低于NP组[ALT:(437.88±47.88)U/L比(747.33±40.04)U/L,F=156.296,P<0.05;AST:(510.02±55.54)U/L比(1041.87±48.60)U/L,F=272.576,P<0.05;LDH:(1473.69±71.94)U/L比(1974.23±75.80)U/L,F=134.480,P<0.05];且术后48 h,NP组、AP组、NPT组、APT组4组大鼠血清ALT、AST、LDH均较术后24 h降低。术后24 h 4组大鼠肝细胞病理均呈现出不同程度坏死,以AP组坏死最严重,且术后48 h各组肝细胞坏死程度较术后24 h轻。术后24 h,各组大鼠肝组织中核因子-κB(NF-κB)及ICAM1的表达以NP组表达最高,APT组最低,而术后48 h各组NF-κB及ICAM1表达趋势同术后24 h,且各自较术后24 h表达有所下降。术后24 h,相对于NP组,AP组及APT组中miR-223、TLR9的表达明显升高,NPT组的表达明显降低;而NPT组、AP组及APT组中mtDNA的表达均明显降低,以APT组降低最显著,术后48 h各组三者表达量均较术后24 h降低,以APT组降低最显著。结论通腑泻热法中药可以通过mtDNA/TLR9/miR-223通路改善肝脏缺血再灌注损伤。

Objective To investigate whether liver ischemia reperfusion injury(HIRI)can be inhibited by Chinese traditional medicine"Tongfuxiere"method through the mitochondria,deoxyribonucleic acid(mtDNA)/toll-like receptor 9(TLR9)/micro ribonucleic acid(miR)-223 pathways.Methods A total of 80 healthy specific pathogen free(SPF)SD rats were randomly divided into 4 groups:the pathway-normal group(NP group),the pathway-activated group(AP group),the pathway-normal TCM group(NPT group)and the pathway-activated TCM group(APT group),with 20 rats each group.The HIRI experiment was performed on all rats.Serum and liver tissue samples were then collected at two time points:24 h and 48 h after reperfusion,after applying different interventions according to group requirements.These specimens were used for biochemical,pathological,Western blotting,and PCR examinations.Results were analyzed by SPSS 25.0 statistical software.Measurement data meeting normal distribution were expressed as mean±standard deviation(SD),using multi-way ANOVA.P<0.05 was used as a statistically significant difference.Results"Pathway activation"and"TCM intervention"were all the factors influencing the postoperative changes in serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),and lactate dehydrogenase(LDH).Pathway activation could promote the elevation of ALT,AST,and LDH in the postoperative serum.Serum ALT,AST and LDH were higher at 24 h after surgery in the AP group than in the NP group[ALT:(962.34±42.35)vs.(747.33±40.04)U/L,F=11.446,P<0.05;AST:(1421.58±93.25)vs.(1041.87±48.60)U/L,F=5.893,P<0.05;LDH:(2312.99±95.77)vs.(1974.23±75.80)U/L,F=5.019,P<0.05].TCM intervention could inhibit the elevation of serum ALT,AST,and LDH postoperatively.Serum ALT,AST and LDH at 24 h after surgery in the NPT group were lower than in the NP group[ALT:(561.31±40.49)vs.(747.33±40.04)U/L,F=688.807,P<0.05;AST:(792.38±44.03)vs.(1041.87±48.60)U/L,F=838.255,P<0.05;LDH:(1702.75±62.32)vs.(1974.23±75.80)U/L,F=514.608,P<0.05].The interaction effects of pathway activation and TCM intervention showed inhibitory effects.Serum ALT,AST and LDH at 24 h after surgery in the APT group were lower than in the NP group[ALT:(437.88±47.88)vs.(747.33±40.04)U/L,F=156.296,P<0.05;AST:(510.02±55.54)vs.(1041.87±48.60)U/L,F=272.576,P<0.05;LDH:(1473.69±71.94)vs.(1974.23±75.80)U/L,F=134.480,P<0.05].Moreover,at 48 h,the serum ALT,AST and LDH decreased in all groups as compared with those at 24 h,showing a significant difference.And the difference in the AP group was the most prominent.The necrosis of hepatocytes was observed in all rats at 24 h after surgery,and necrosis was the most severe in the AP group,and hepatocyte necrosis was milder in each group at 48 h than at 24 h.At 24 h after surgery,the expression of nuclear factor-κB(NF-κB)and ICAM1 in rat liver tissues of each group was highest in the NP group and lowest in the AP group.The expression of NF-κB and ICAM1 in all groups showed a similar trend at 48 h after surgery,and their respective expression decreased compared with 24 h later.At 24 h after surgery,miR-223 and TLR9 expression was significantly higher in the AP group and APT group than in the NPT group.The expression of mt-DNA in the AP group,NPT group and APT group was significantly reduced,and the expression of each group decreased at 48 h after surgery as compared with that at 24 h after surgery,with the AP group being the most significantly reduced.Conclusion The"Tongfuxiere"methods can improve LIRI through the mtDNA/TLR9/miR-223 pathway.