对香豆酸对重症肌无力大鼠免疫平衡和骨骼肌线粒体生物发生的影响

Effects of p⁃coumaric acid on immune balance and skeletal muscle mitochondrial biogenesis in rats with myasthenia gravis

目的 探究对香豆酸(p-CA)对重症肌无力(MG)大鼠的治疗作用及可能机制。方法 将雌性Lewis大鼠分为5组:对照组、模型组、对香豆酸低剂量组(L-p-CA组)、对香豆酸中剂量组(M-p-CA组)、对香豆酸高剂量组(H-p-CA组),每组12只。对照组大鼠为未建模的大鼠,其他组均通过对大鼠皮下注射乙酰胆碱受体(AChR)α亚基97-116肽段序列(Rα97-116)建立MG大鼠模型。建模后,L-p-CA组、M-p-CA组和H-p-CA组大鼠分别灌胃1 mL的50,100,200 mg/kg对香豆酸溶液。对照组和模型组大鼠分别灌胃1 mL的玉米油。各组大鼠均灌胃4周。采用Lennon分级法进行大鼠临床症状评分,并检测大鼠的低频重复神经电刺激(RNS)衰减率。通过苏木素伊红(HE)染色观察腓肠肌形态。通过ELISA法检测血清AChR抗体(AChR-Ab)、干扰素γ(IFN-γ)和白细胞介素-4(IL-4)水平。按照试剂盒方法检测腓肠肌组织中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)的水平。通过Western blot检测腓肠肌组织中过氧化物酶体增殖物激活受体γ共激活因子-1α(PGC-1α)、磷酸化AMP依赖的蛋白激酶α(p-AMPKα)、AMPKα、核呼吸因子1(NRF1)和线粒体转录因子A(TFAM)的蛋白表达水平。通过免疫荧光染色观察腓肠肌组织中的TFAM表达。结果 与对照组比较,模型组大鼠的Lennon评分和RNS衰减率均显著升高(P<0.05),血清AChR-Ab和IFN-γ水平显著升高(P<0.05);腓肠肌出现损伤,腓肠肌中SOD、GSH-Px水平、以及PGC-1α、p-AMPKα、NRF1和TFAM蛋白表达水平均显著降低(P<0.05),TFAM相对荧光强度显著降低(P<0.05),MDA水平显著升高(P<0.05)。与模型组比较,L-p-CA组、M-p-CA组和H-p-CA组大鼠的Lennon评分和RNS衰减率显著降低(P<0.05),血清AChR-Ab和IFN-γ水平显著降低(P<0.05);腓肠肌形态明显改善,腓肠肌中SOD、GSH-Px水平、以及PGC-1α、p-AMPKα、NRF1和TFAM蛋白表达水平均显著升高(P<0.05),TFAM相对荧光强度显著升高(P<0.05),MDA水平显著降低(P<0.05)。与L-p-CA组和Mp-CA组比较,H-p-CA组大鼠的Lennon评分和RNS衰减率显著降低(P<0.05),血清AChR-Ab和IFN-γ水平显著降低(P<0.05);腓肠肌形态明显改善,腓肠肌中SOD、GSH-Px水平、以及PGC-1α、p-AMPKα、NRF1和TFAM蛋白表达水平均显著升高(P<0.05),TFAM相对荧光强度显著升高(P<0.05),MDA水平显著降低(P<0.05)。结论 对香豆酸对MG大鼠具有良好的治疗效果,其机制可能与改善免疫平衡和激活AMPK/PGC-1α信号通路介导的线粒体生物发生有关。

Objective To explore the therapeutic effect of p-coumaric acid(p-CA)on myasthenia gravis(MG)rats and its possible mechanism.Methods The female Lewis rats were randomly divided into 5 groups:control group,model group,low dose p-coumaric acid group(L-p-CA),medium dose p-coumaric acid group(M-p-CA)and high dose p-coumaric acid group(H-p-CA),with 12 rats in each group.The rats in control group were unmodeled,and the rats in the other groups were subcutaneously injected with acetylcholine receptor(AChR)alpha subunit 97-116 peptide sequence(Rα97-116)to induce MG models.After modeling,the rats in L-p-CA group,M-p-CA group and H-p-CA group were intragastrically fed with 1 mL of 50,100,200 mg/kg p-coumaric acid solution respectively,while the rats in control group and MG group were intragastrically fed with 1 mL corn oil.The rats in each group were treated for 4 weeks.The clinical symptoms of rats were scored by Lennon grading method,and the attenuation rate of low frequency repetitive nerve stimulation(RNS)was measured.The morphology of gastrocnemius muscle was observed by HE staining.The serum levels of AChR antibody(AChR-Ab),interferon gamma(IFN-γ)and interleukin-4(IL-4)were detected by ELISA method.The levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)in gastrocnemius muscle were detected by different kits.The protein expressions of peroxisome proliferator activated receptorγcoactivator-1α(PGC-1α),phosphorylated AMP dependent protein kinaseα(p-AMPKα),AMPKα,nuclear respiratory factor 1(NRF1)and mitochondrial transcription factor A(TFAM)in gas-trocnemius muscle were detected by Western blot.The expression of TFAM in gastrocnemius muscle was observed by immunofluores-cence staining.Results Compared with control group,Lennon score and RNS attenuation rate were significantly increased in MG group(P<0.05),serum AChR-Ab and IFN-γlevels were significantly increased(P<0.05),the injury of gastrocnemius muscle was found,SOD and GSH-Px levels,PGC-1α,p-AMPKα,NRF1 and TFAM protein expressions in gastrocnemius muscle were significantly decreased(P<0.05),TFAM relative fluorescence intensity was significantly decreased(P<0.05),and MDA level was significantly increased(P<0.05).Compared with MG group,Lennon score and RNS attenuation rate were significantly decreased in L-p-CA group,M-p-CA group and H-p-CA group(P<0.05),serum AChR-Ab and IFN-γlevels were significantly decreased(P<0.05),the gastrocne-mius muscle injury was significantly improved,SOD and GSH-Px levels,PGC-1α,p-AMPKα,NRF1 and TFAM protein expressions in gastrocnemius muscle were significantly increased(P<0.05),TFAM relative fluorescence intensity was significantly increased(P<0.05),and MDA level was decreased(P<0.05).Compared with L-p-CA group and M-p-CA group,Lennon score and RNS attenuation rate were significantly decreased in H-p-CA group(P<0.05),serum AChR-Ab and IFN-γlevels were significantly decreased(P<0.05),the gastrocnemius muscle injury was significantly improved,SOD and GSH-Px levels,PGC-1α,p-AMPKα,NRF1 and TFAM protein expressions in gastrocnemius muscle were significantly increased(P<0.05),TFAM relative fluorescence intensity was significantly increased(P<0.05),and MDA level was decreased(P<0.05).Conclusion The p-coumaric acid plays a good therapeutic effect on MG rats by improving the immune balance and activating the mitochondrial biogenesis mediated by AMPK/PGC-1αsignal pathway.