基于AMPK信号通路的附子在不同机体状态下对神经—内分泌—免疫网络的生物学效应表达机制研究

Research on the mechanism of the expression of biological effect of Fuzi on neuroendocrine immune network in different organismal status based on AMP-Activated protein kinase signaling pathway

目的基于腺苷酸激活蛋白激酶(AMP-activiated protein kinase,AMPK)信号通路,研究附子在寒热不同机体状态下对神经—内分泌—免疫(neural-endocrine-immune,NEI)网络的生物效应表达机制。方法84只雄性KM小鼠随机分为空白组、虚热模型组、虚寒模型组、虚热附子低剂量(10 g/kg)组、虚热附子高剂量(20 g/kg)组、虚寒附子低剂量(10 g/kg)组、虚寒附子高剂量(20 g/kg)组,每组12只。采用醋酸地塞米松建立虚热模型、氢化可的松琥珀酸钠建立虚寒模型,各组灌胃相应药物,连续14日。采用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测环腺苷酸(cyclic adenosine monophosphate,cAMP)、环鸟苷酸(cyclic guanosine monophosphatec,cGMP)、去甲肾上腺素(norepinephrine,NE)、多巴胺(dopamine,DA)、5-羟色胺(5-hydroxytryptamine,5-HT)、三碘甲状腺原氨酸(triiodothyronine,T3)、甲状腺素(thyroxine,T4)、补体C3(complement 3,C3)、C4、免疫球蛋白G(immunoglobin G,IgG)、IgM;通过反转录—荧光定量PCR法(reverse transcript quantitative PCR,RT-qPCR)检测心脏组织脂肪酸合成酶(fatty acid synthase,FASN)、6-磷酸果糖激酶-2/果糖-2,6-二磷酸酶3(6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3,PFKFB3)、硬脂酰辅酶A去饱和酶1(stearoyl coenzyme A desaturase 1,SCD1)、胰岛素受体底物2(insulin receptor substrate,IRS2)的mRNA表达。结果与虚寒模型组比较,附子高剂量能够显著升高虚寒证小鼠的环核苷酸cAMP、cAMP/cGMP含量(P<0.01),神经递质NE、DA、5-HT含量(P<0.01),内分泌激素T3、T4含量(P<0.01),免疫因子C3、C4、IgG、IgM含量(P<0.01),显著降低虚寒证小鼠cGMP含量(P<0.01),FASN、PFKFB3、SCD1、IRS2的mRNA表达(P<0.01,P<0.05,P<0.01,P<0.01);与虚热模型比较,附子对虚热证小鼠的FASN、SCD1、PFKFB3的mRNA表达则无统计学意义。结论辛热中药附子作用于寒热不同机体状态下通过调控AMPK信号通路上FASN、PFKFB3、SCD1基因mRNA表达进而影响NEI网络的生物学效应表达不同,并且无论是虚寒状态还是虚热状态,附子均能显著抑制IRS2的mRNA表达,可能是其“效—毒”效应表达不同的机制。

Objective Research on the mechanism of the expression of biological effect of Fuzi on neuroendocrine immune network in different organismal status(cold and heat)based on AMP-activated protein kinase(AMPK)signaling pathway.Methods KM male mice were randomly divided into control group,deficiency-heat model group,deficiency-cold model group,deficiency-heat+low dose Fuzi group(10 g/kg),deficiency-heat+high dose Fuzi group(20 g/kg),deficiency-cold+low dose Fuzi group(10 g/kg),deficiency-cold+high dose Fuzi group(20 g/kg).The deficiency-heat model was induced by dexamethasone acetate and the deficiency-cold model was induced by hydrocortisone sodium phosphate for 14 days.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of cAMP,cGMP NE,DA,5-HT and the levels of T3,T4,C3,C4,IgG,IgM were detected.The mRNA expressions of heart tissue of FASN,PFKFB3,SCD1 and IRS2 were detected by RT-qPCR.Results Compared with the deficiency-cold model group,high dose Fuzi can significantly increase the levels of neurotransmitters NE,DA,5-HT levels(P<0.01,P<0.01,P<0.01),the endocrine hormones T3,T4(P<0.01,P<0.01)and the immune facters C3,C4,IgG,IgM levels(P<0.01),and significantly decrease the cGMP level(P<0.01),the mRNA expressions of FASN,PFKFB3,SCD1,IRS2 of mice with deficiency-cold syndrome(P<0.01,P<0.05,P<0.01,P<0.01).Compared with the deficiency-heat model group,there are no significant differences in the mRNA expressions of FASN,PFKFB3,SCD1of mice by Fuzi.Conclusion Fuzi,a pungent-hut traditional Chinese medicine,can regulate the mRNA expressions of FASN,PFKFB3,SCD1 in different organismal status(cold and heat)mice via the AMPK signaling pathway lead to the different biological effect on neuroendocrine immune(NEI)network.Meanwhile,no matter in deficiency-heat or deficiency-cold status,Fuzi can decrease the mRNA expressions of IRS2 significantly,which maybe the mechanism of different expressions of‘effect-toxicity’of it.