基于骨骼肌线粒体动力学探讨健脾清化方对高脂饮食诱导的肥胖小鼠胰岛素敏感性的影响

The Effect of Jianpi Qinghua Prescription(健脾清化方)on Insulin Sensitivity in Obese Mice Induced by High-Fat Diet Based on Mitochondrial Dynamics of Skeletal Muscle

目的:观察健脾清化方对高脂饮食诱导的肥胖模型小鼠(DIO)胰岛素敏感性的影响,并基于骨骼肌线粒体动力学讨论其作用机制。方法:将30只小鼠随机分为正常组(NOR组,n=6)和模型对照组(n=24),NOR组小鼠予正常饲料喂养。MOD组小鼠利用高脂饲料诱导建立C57BL/6J肥胖模型。将18只造模成功小鼠随机分为模型组(MOD组)、健脾清化方组(JPQH组)、二甲双胍组(MET组),每组6只,分别予生理盐水、健脾清化方(1.3g/mL)、二甲双胍(30 mg/mL)进行干预。干预6周后,称量小鼠体质量,检测空腹胰岛素(FINS)、胰岛素敏感性、葡萄糖耐量,反转录聚合酶链式反应法(RT-PCR)检测小鼠骨骼肌有丝分裂融合蛋白1(Mfn1)mRNA、有丝分裂融合蛋白2(Mfn2)mRNA、视神经萎缩蛋白1(Opa1)mRNA、动力相关蛋白1(Drp1)mRNA、线粒体分裂蛋白1(Fis1)mRNA表达水平,蛋白质免疫印迹法(Western blotting)检测Mfn1、Mfn2、Opa1、Fis1、Drp1、肌球蛋白重链(MYH)蛋白表达水平。结果:干预1周后,JPQH组小鼠体质量低于MOD组(P<0.05);干预2、3、4、5、6周后,JPQH组小鼠体质量与MOD组比较,差异无统计学意义(P>0.05);干预2、3、4、5、6周后,MET组小鼠体质量低于MOD组(P<0.05);干预1周后,MET组小鼠体质量与MOD组比较,差异无统计学意义(P>0.05)。JPQH组小鼠FINS水平低于MOD组(P<0.05),与MET组比较,差异无统计学意义(P>0.05)。IPITT试验中,JPQH组小鼠各时间点血糖水平均低于MOD组(P<0.05);JPQH组小鼠0 min血糖水平高于MET组(P<0.05);JPQH组小鼠30、60、90、120 min血糖水平与MET组比较,差异无统计学意义(P>0.05);JPQH组曲线下面积AUC低于MOD组(P<0.05),与MET组比较,差异无统计学意义(P>0.05)。OGTT试验中,JPQH组小鼠0 min血糖水平与MOD组比较,差异无统计学意义(P>0.05),JPQH组小鼠30、60、90、120 min血糖水平均低于MOD组(P<0.05),与MET组比较,差异无统计学意义(P>0.05);JPQH组曲线下AUC低于MOD组(P<0.05),与MET组比较,差异无统计学意义(P>0.05)。JPQH组小鼠Mfn1 mRNA相对表达量低于MOD组(P<0.05),高于MET组(P<0.05);JPQH组小鼠Mfn2 mRNA相对表达量高于MOD组、MET组(P<0.05);JPQH组小鼠Opa1 mRNA相对表达量低于MOD组(P<0.05),高于MET组(P<0.05);JPQH组小鼠Fis1 mRNA相对表达量高于MOD组(P<0.05),低于MET组(P<0.05);JPQH组小鼠Drp1 mRNA相对表达量与MOD组比较,差异无统计学意义(P>0.05)。JPQH组小鼠Mfn1、Mfn2蛋白相对表达量高于MOD组、MET组(P<0.05);JPQH组小鼠Opa1、Fis1蛋白相对表达量低于MOD组、MET组(P<0.05);JPQH组小鼠Drp1蛋白相对表达量与MOD组比较,差异无统计学意义(P>0.05);JPQH组小鼠MYH蛋白相对表达量低于MOD组(P<0.05),与MET组比较,差异无统计学意义(P>0.05)。结论:健脾清化方可以降低高脂饮食诱导的肥胖小鼠体质量,改善小鼠的胰岛素敏感性,降低胰岛素抵抗;健脾清化方可能通过影响骨骼肌线粒体动力学进而改善骨骼肌胰岛素抵抗,以稳定血糖。

Objective:To observe the effect of Jianpi Qinghua Prescription on insulin sensitivity in high-fat diet-induced obesity model mice(DIO),and to discuss its mechanism of action based on mitochondrial dynamics of skeletal muscle.Methods:Totally 30 mice were randomly divided into normal group(NOR group,n=6)and model control group(n=24),and the mice in the NOR group were fed with normal diet.The mice in the model control group were established a C57BL/6J obesity model by high-fat diet.The 18 successfully modeled mice were randomly divided into model group(MOD group),Jianpi Qinghua Prescription group(JPQH group)and meformin group(MET group),with 6 mice in each group.And the three groups were treated with normal saline,Jianpi Qinghua Prescription(1.3 g/mL)and metformin(30 mg/mL),respectively.After 6 weeks of drug intervention,body weight was weighed.Fasting insulin(FINS),insulin sensitivity,and glucose tolerance were measured,and reverse transcription polymerase chain reaction(Rt-PCR)was used to detect mitotic fusion protein 1(Mfnl)mRNA,mitotic fusion protein 2(Mfn2)mRNA,optic atrophy protein 1(Opal)mRNA,dynamin related protein 1(Drpl)mRNA,and mitochondrial fission protein 1(Fisl)mRNA in mouse skeletal muscle.The expression levels of Mfnl,Mfn2,Opal,Fisl,Drpl and myosin heavy chain(MYH)proteins were detected by Western blotting.Results:After a week of intervention,the JPQH group showed lower body weight than MOD group(P<0.05).After 2,3,4,5 and 6 weeks of intervention,there was no significant difference in body weight between JPQH group and MOD group(P>0.05).After 2,3,4,5 and 6 weeks of intervention,the MET group showed lower body weight than MOD group(P<0.05),and after 1 week of intervention,there was no significant difference in body weight between MAT group and MOD group(P>0.05).The JPQH group showed lower FINS level than MOD group(P<0.05),while there was no significant`difference in FINS level between JPQH group and MET group(P>0.05).In the IPITT experiment,the JPQH group showed lower blood glucose levels than MOD group(P<0.05);The JPQH group showed higher O-min blood glucose level than MET group(P<0.05),while there was no significant diference in blood glucose levels at 30,60,90 and 120 min between JPQH group and MET group(P>0.05).The JPQH group showed lower AUC than MOD group(P<0.05),while there was no significant difference between JPQH group and MET group(P>0.05).In the OGTT test,there was no significant difference in the O min blood glucose level between JPQH group and MOD group(P>0.05);The JPQH group showed lower 30,60,90,120 min blood glucose level than MOD group(P<0.05),while there was no significant difference between JPQH group and MET group(P>0.05);The JPQH group showed lower AUC than MOD group(P<0.05),while there was no statistical difference between JPQH group and MET group(P>0.05).The JPQH group showed lower relative expression of Mfnl mRNA than MOD group(P<0.05),while higher than MET group(P<0.05).The JPQH group showed higher relative expression of Mfn2 mRNA than MOD group and MET group(P<0.05).The JPQH group showed lower relative expression of Opal mRNA than MOD group(P<0.05),while higher than MET group(P<0.05).The JPQH group showed higher relative expression of Fisl mRNA than MOD group(P<0.05),while lower than MET group(R<0.05).There was no significant difference in the relative expression of Drpl mRNA between JPQH group and MOD group(P>0.05).The JPQH group showed higher relative expression levels of Mfnl and Mfn2 proteins than MOD group and MET group(P<0.05).The JPQH group showed lower relative expression levels of Opal and Fisl proteins than MOD group and MET group(P<0.05).There was no significant difference in the relative expression of Drpl protein between JPQH group and MOD group(P>0.05);The JPQH group showed lower relative expression of MYH protein than MOD group(P<0.05),while there was no significant difference between JPQH group and MET group(P>0.05).Conclusion:Jianpi Qinghua Prescription can reduce the body weight,improve insulin sensitiv ity and reduce insulin resistance in obese mice induced by high-fat diet.Jianpi Qinghua Prescription may improve skeletal muscle insulin resistance by affecting mitochondrial dynamics in skeletal muscle to stabilize blood glucose fluctuations.