瓜蒌薤白半夏汤预处理对心肌缺血再灌注损伤模型大鼠的心肌保护作用及机制研究

Protective Effect and Mechanism of Pretreatment of Gualou Xiebai Banxia Decoction on Myocardial Ischemia-Reperfusion Injury Model Rats

目的探讨瓜蒌薤白半夏汤对心肌缺血再灌注损伤(MIRI)模型大鼠的心肌保护作用及机制。方法本实验时间为2022年2月至2023年2月。采用随机数字表法将40只大鼠分为假手术组(A组)、模型组(B组)、瓜蒌薤白半夏汤组(C组)、核因子κB(NF-κB)抑制剂组(D组),每组10只。C组、D组大鼠给予瓜蒌薤白半夏汤预处理,A组、B组大鼠给予等量0.9%氯化钠溶液预处理,四组均连续干预7 d;A组大鼠于末次给药后1 h开胸,但不结扎;B组、C组、D组大鼠于末次给药后1 h制备MIRI模型(采用左前降支结扎法制备),其中D组大鼠于再灌注前10 min腹腔注射吡咯烷二硫代甲酸铵。比较四组大鼠心肌梗死面积百分比、心肌组织形态学、血清炎症因子(IL-1β、TNF-α)水平、血清氧化应激指标〔丙二醛(MDA)、超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)〕、心肌组织超微结构及心肌组织Toll样受体4(TLR4)、NF-κB p65蛋白表达水平、mRNA相对表达量。结果B组大鼠心肌梗死面积百分比高于A组,C组和D组大鼠心肌梗死面积百分比低于B组,D组心肌梗死面积百分比高于C组(P<0.05)。HE染色结果显示,C组大鼠心肌组织纤维排列、心肌组织形态改变、横纹结构改变均优于B组;D组大鼠细胞核散落在外侧明显。Masson染色结果显示,B组大鼠心肌细胞排列紊乱,少部分胶原纤维染色阳性;C组和D组大鼠心肌细胞排列较规则,几乎无胶原纤维染色阳性。B组大鼠血清IL-1β、TNF-α、MDA水平及LDH活性高于A组,血清SOD活性低于A组(P<0.05);C组和D组大鼠血清IL-1β、TNF-α、MDA水平及LDH活性低于B组,血清SOD活性高于B组(P<0.05);D组大鼠血清IL-1β、TNF-α、MDA水平及LDH活性低于C组,血清SOD活性高于C组(P<0.05)。透射电镜扫描结果显示,B组大鼠细胞质中存在较多空泡,自噬体较多,C组大鼠细胞质中可见少数自噬泡,D组大鼠细胞质部分呈空泡化。B组大鼠心肌组织TLR4、NF-κB p65蛋白表达水平、mRNA相对表达量高于A组,C组和D组大鼠心肌组织TLR4、NF-κB p65蛋白表达水平、mRNA相对表达量低于B组,D组大鼠心肌组织TLR4、NF-κB p65蛋白表达水平、mRNA相对表达量低于C组(P<0.05)。结论瓜蒌薤白半夏汤预处理可减轻炎症反应、氧化应激及抑制心肌细胞自噬小体产生,进而缩小MIRI模型大鼠心肌梗死面积,保护心肌细胞,其机制可能与瓜蒌薤白半夏汤抑制TLR4/NF-κB信号通路有关。

Objective To investigate the protective effect and mechanism of pretreatment of Gualou Xiebai Banxia decoction on myocardial ischemia-reperfusion injury(MIRI)model rats.Methods This experiment was conducted from February 2022 to February 2023.A total of 40 rats were divided into sham group(A group),model group(B group),Gualou Xiebai Banxia decoction group(C group),and nuclear factor-κB(NF-κB)inhibitor group(D group)using random number table method,10 rats in each group.Rats in groups C and D were pretreated with Gualou Xiebai Banxia decoction,while rats in groups A and B were pretreated with an equal amount of 0.9%sodium chloride solution.All four groups were continuously intervened for 7 days.Rats in group A underwent thoracotomy at 1 hour after the last administration,but were not ligated;while rats in groups B,C,and D were used to prepare MIRI models using the left anterior descending branch ligation method at 1 hour after the last administration.Rats in group D were intraperitoneally injected with ammonium pyrrolidine dithioformate at 10 minutes before reperfusion.The percentage of myocardial infarction area,myocardial tissue morphology,serum inflammatory factor(IL-1βand TNF-α)levels,serum oxidative stress indicators[malondialdehyde(MDA),superoxide dismutase(SOD),lactate dehydrogenase(LDH)],myocardial tissue ultrastructure,Toll like receptor 4(TLR4),NF-κB p65 protein expression level and relative mRNA expression level in myocardial tissue were compared in the four groups of rats.Results The percentage of myocardial infarction area in group B was higher than that in group A,the percentage of myocardial infarction area in groups C and D was lower than that in group B,while the percentage of myocardial infarction area in group D was higher than that in group C(P<0.05).The HE staining results showed that the fiber arrangement,morphological changes,and transverse structural changes of myocardial tissue in group C were superior to those in group B,and the nuclei of group D were scattered and obvious on the outer side.The results of Masson staining showed that the arrangement of myocardial cells in group B was disordered,and a few collagen fibers were positively stained;the arrangement of cells in groups C and D was relatively regular,with almost no positive staining of collagen fibers.Serum levels of IL-1β,TNF-α,MDA and serum LDH activity in group B were higher than those in group A,while the serum SOD activity was lower than that in group A(P<0.05);serum levels of IL-1β,TNF-α,MDA and serum LDH activity in groups C and D were lower than those in group B,while the serum SOD activity was higher than that in group B(P<0.05);serum levels of IL-1β,TNF-α,MDA and serum LDH activity in group D were lower than those in group C,while the serum SOD activity was higher than that in group C(P<0.05).The scanning results of the projection electron microscope showed that there were many vacuoles and autophagosomes in the cytoplasm of group B,a few autophagosomes in the cytoplasm of group C,and partial cytoplasmic vacuolization of group D.The protein expression level and mRNA relative expression level of TLR4 and NF-κB p65 in myocardial tissue of group B were higher than those of group A,the protein expression level and mRNA relative expression level of TLR4 and NF-κB p65 in myocardial tissue of groups C and D were lower than those of group B,while the protein expression level and mRNA relative expression level of TLR4 and NF-κB p65 in myocardial tissue of group D were lower than those of group C(P<0.05).Conclusion Pretreatment of Gualou Xiebai Banxia decoction can reduce inflammatory response,oxidative stress,and inhibit the production of autophagosomes in myocardial cells,thereby reducing the myocardial infarction area and protecting myocardial cells in MIRI model rats.Its mechanism may be related to the inhibition of TLR4/NF-κB signal pathway by Gualou Xiebai Banxia decoction.