基于LKB1-AMPK通路及16S rDNA测序技术研究“新建曲”发酵炮制前后降脂功效差异及作用机制  

Difference of lipid-lowering efficacy of"Xinjianqu"before and after fermentation and its mechanism based on LKB1-AMPK pathway and 16S rDNA sequencing technology

作　　者：李德华 王瑞生[1,2] 张振凌 朱建光[1,2] 孙梦梅 乔嘉[1,2] LI De-hua;WANG Rui-sheng;ZHANG Zhen-ling;ZHU Jian-guang;SUN Meng-mei;QIAO Jia(Henan University of Chinese Medicine;Henan Research Center for Special Processing Technology of Chinese Medicine;Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases Co-constructed by Henan province&Education Ministry of China)

机构地区：[1]河南中医药大学,河南郑州450046 [2]河南省中药特色炮制技术工程研究中心,河南郑州450046 [3]呼吸疾病中医药防治省部共建协同创新中心,河南郑州450046

出　　处：《中国中药杂志》2023年第8期2146-2159,共14页China Journal of Chinese Materia Medica

基　　金：河南省高等学校重点科研项目(22B360004);国家重点研发计划项目(2018YFC1707202);国家公益性行业专项(00104296);河南中医药大学博士基金项目(RSBSJJ2019-09);2021年河南省本科高校省级大学生创新创业训练计划项目(S202110471027)。

摘　　要：在建立新建曲处方,明确发酵炮制升高新建曲降脂有效成分含量的基础上,进一步比较发酵炮制前后新建曲降脂作用差异,研究新建曲治疗高脂血症的作用机制。随机将70只SD大鼠分为7组,包括正常组、模型组、阳性药辛伐他汀组(0.02 g·kg^(-1))、发酵前新建曲低高剂量组(1.6、8 g·kg^(-1))、新建曲发酵品低高剂量组(1.6、8 g·kg^(-1)),每组10只,连续给予高脂饲料6周,建立高脂血症(hyperlipidemia,HLP)模型。造模成功后,继续给予高脂饲料并灌胃对应药物6周,每天1次,对比新建曲发酵炮制前后对高脂血症大鼠体质量、肝脏系数、小肠推进率的影响。采用酶联免疫吸附测定法(enzyme-linked immunosorbnent assay,ELISA)检测新建曲发酵前后对高脂血症大鼠血清总胆固醇(total cholesterol,TC)、三酰甘油(triglyceride,TG)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)、丙氨酸转氨酶(alanine aminotransferase,ALT)、天冬氨酸转氨酶(aspartate aminotransferase,AST)、尿素氮(blood urea nitrogen,BUN)、肌酐(creatinine,Cr)、胃动素(motilin,MTL)、胃泌素(gastrin,GAS)、Na+-K+-ATP酶(Na+-K+-ATPase,Na+-K+-ATP)、水通道蛋白2(aquaporin 2,AQP-2)水平的影响;通过苏木素-伊红(hemoxylin eosin,HE)染色法和油红O脂肪染色法研究新建曲对HLP大鼠肝脏组织形态的影响;采用免疫组化检测新建曲对肝脏组织中激活腺苷酸活化蛋白激酶(AMPK)、磷酸化AMPK(p-AMPK)、肝激酶B1(LKB1)、3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGCR)蛋白表达的影响;基于16S rDNA高通量测序技术研究新建曲对HLP大鼠肠道菌群结构的调节作用。研究结果显示,与正常组相比,模型组大鼠体质量、肝脏系数显著升高(P<0.01),小肠推进率显著下降(P<0.01),血清中TC、TG、LDL-C、ALT、AST、BUN、Cr、AQP2水平显著升高(P<0.01),血清中HDL-C、MTL、GAS、Na+-K+-AOn the basis of establishing the prescription of Xinjianqu and clarifying the increase of the lipid-lowering active ingredients of Xinjianqu by fermentation,this paper further compared the differences in the lipid-lowering effects of Xinjianqu before and after fermentation,and studied the mechanism of Xinjianqu in the treatment of hyperlipidemia.Seventy SD rats were randomly divided into seven groups,including normal group,model group,positive drug simvastatin group(0.02 g·kg~(-1)),and low-dose and high-dose Xinjianqu groups before and after fermentation(1.6 g·kg~(-1)and 8 g·kg~(-1)),with ten rats in each group.Rats in each group were given high-fat diet continuously for six weeks to establish the model of hyperlipidemia(HLP).After successful modeling,the rats were given high-fat diet and gavaged by the corresponding drugs for six weeks,once a day,to compare the effects of Xinjianqu on the body mass,liver coefficient,and small intestine propulsion rate of rats with HLP before and after fermentation.The effects of Xinjianqu before and after fermentation on total cholesterol(TC),triacylglyceride(TG),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood urea nitrogen(BUN),creatinine(Cr),motilin(MTL),gastrin(GAS),and the Na~+-K~+-ATPase levels were determined by enzyme-linked immunosorbent assay(ELISA).The effects of Xinjianqu on liver morphology of rats with HLP were investigated by hematoxylin-eosin(HE)staining and oil red O fat staining.The effects of Xinjianqu on the protein expression of adenosine 5′-monophosphate(AMP)-activated protein kinase(AMPK),phosphorylated AMPK(p-AMPK),liver kinase B1(LKB1),and 3-hydroxy-3-methylglutarate monoacyl coenzyme A reductase(HMGCR)in liver tissues were investigated by immunohistochemistry.The effects of Xinjianqu on the regulation of intestinal flora structure of rats with HLP were studied based on 16S rDNA high-throughput sequencing technology.The results showed that compa

关 键 词：发酵 炮制 高脂血症 LKB1-AMPK通路 肠道菌群 

分 类 号：R285.5[医药卫生—中药学]