补阳还五汤通过cAMP/PKA/PPARγ通路调控脂质代谢抗大鼠脑缺血再灌注损伤的作用

The Effect of Buyang Huanwu Decoction on Cerebral Ischemia-Reperfusion Injury in Rats by Regulating Lipid Metabolism via cAMP/PKA/PPARγPathway

目的探究补阳还五汤通过cAMP/PKA/PPARγ通路调控脂质代谢抗大鼠脑缺血再灌注损伤的作用机理。方法将60只大鼠随机分为假手术组、模型组、丁苯酞组及补阳还五汤低、中、高剂量组。采用短暂性大脑中动脉栓塞法制备脑缺血再灌注模型,补阳还五汤低、中、高剂量组予不同剂量补阳还五汤灌胃(6.413、12.825、25.65 g·kg^(-1)),丁苯酞(NBP)组予丁苯酞灌胃(54 mg·kg^(-1)),假手术组和模型组予等体积蒸馏水灌胃,连续14 d。对大鼠进行神经行为学评分,HE染色观察大脑病理变化,试剂盒检测缺血侧磷酸胆碱(PC)、磷脂酰乙醇胺(PE)、甘油二酯(DAG)、游离脂肪酸(FFA)含量,RT-qPCR和WesternBlot法检测环磷酸腺苷(cAMP)、蛋白激酶A(PKA)、过氧化物酶体增殖物激活受体(PPARγ)中mRNA及蛋白表达。结果与假手术组比较,模型组神经功能缺损评分明显升高(P<0.01);缺血侧皮质出现病理形态损伤;PC、PE含量降低,DAG、FFA含量升高(P<0.01);cAMP的mRNA升高(P<0.05)。与模型组比较,补阳还五汤低剂量组神经功能缺损评分降低(P<0.05),补阳还五汤中、高剂量组和丁苯酞组神经功能缺损评分明显降低(P<0.01);各给药组细胞排列相对规整,细胞间隙减小,正常细胞增多;补阳还五汤中、高剂量组和丁苯酞组PC、PE明显升高,DAG、FFA明显降低(P<0.01);补阳还五汤低剂量组PC升高,FFA、DAG明显降低(P<0.05,P<0.01);补阳还五汤低剂量组PPARγ的mRNA表达升高(P<0.05),补阳还五汤中、高剂量组和丁苯酞组cAMP、PKA、PPARγ的mRNA及蛋白表达升高(P<0.05,P<0.01)。结论补阳还五汤对脑缺血再灌注损伤大鼠具有神经保护作用,其机制可能与调控cAMP/PKA/PPARγ信号通路关键因子的表达,调节脂质代谢有关。

Objective To explore the mechanism of Buyang Huanwu Decoction against cerebral ischemia-reperfusion injury in rats by regulating lipid metabolism through the cAMP/PKA/PPARγpathway.Methods 60 rats were randomly divided into sham operation group(Sham),model group(Model),Buyang Huanwu Decoction low-dose group(BHD-L),Buyang Huanwu Decoction medium-dose group(BHD-M),Buyang Huanwu Decoction highdose group(BHD-H)and Butylphthalide group(NBP).The cerebral ischemia-reperfusion model was prepared by transient middle cerebral artery embolization.The BHD low-,medium-and high-groups were given different doses·667·Traditional Chinese Drug Research&Clinical Pharmacology,2024 May,Vol.35 No.5 of Buyang Huanwu Decoction(6.413,12.825,25.65 g·kg^(-1))by intragastric administration.The NBP group was administered with Butylphthalide(54 mg·kg^(-1)).The sham operation group and the model group were administered with an equal volume of distilled water,all given for 14 days.The rats were subjected to neurobehavioral scoring.HE staining was used to observe brain pathological changes,and the kit was used to detect the levels of phosphocholine(PC),phosphatidylethanolamine(PE),diacylglycerol(DAG),and free fatty acid(FFA)on the ischemic side.RT-qPCR and Western Blot were applied to detect the mRNA and protein expressions of cyclic adenosine monophosphate(cAMP),protein kinase A(PKA),and peroxisome proliferator-activated receptorγ(PPARγ).Results Compared with the sham group,the neurological deficit score was significantly increased(P<0.01),pathomorphological damage in ischemic cortex was found,the contents of PC and PE were reduced,the contents of DAG and FFA were increased(P<0.01),and cAMP mRNA expression increased(P<0.05)in the model group.Compared with the model group,the neurological deficit score of the BHD-L group was decreased(P<0.05),and the neurological deficit score of the BHD-M,BHD-H and NBP groups was significantly decreased(P<0.01),the cells in each treatment group were regularly arranged,the intercellular spaces were reduced,and the normal cells were increased.PC and PE were significantly increased,DAG and FFA were significantly decreased(P<0.01)in the BHD-M,BHD-H and NBP groups.PC was increased,FFA and DAG were decreased in the BHD-L group(P<0.05,P<0.01).The mRNA level of PPARγwas increased in the BHD-L group(P<0.05),and the mRNA and protein levels of cAMP,PKA,and PPARγwere increased in the other treatment groups(P<0.05,P<0.01).Conclusion Buyang Huanwu Decoction has a neuroprotective effect on cerebral ischemia-reperfusion injury rats,and its mechanism may be related to regulating the expression of key factors in the cAMP/PKA/PPARγsignaling pathway and lipid metabolism.