温补肾阳中药对虚证模型小鼠肾上腺皮质激素合成的调节作用

Chinese medicines of warming and tonifying kidney yang regulates synthesis of adrenocortical hormone in mice with deficiency syndrome

目的:研究温补肾阳中药及其配伍对虚证模型小鼠肾上腺皮质激素合成的影响。方法:120只雄性ICR小鼠随机分成10组,即对照组、模型组、淫羊藿(5.3 mg·kg^-1·d^-1)组、仙茅(4.3 mg·kg^-1·d^-1)组、巴戟天(4.3 mg·kg^-1·d^-1)组、杜仲(5.3 mg·kg^-1·d^-1)组、补骨脂(5.3 mg·kg^-1·d^-1)组、菟丝子(6.0 mg·kg^-1·d^-1)组、肉苁蓉(5.3 mg·kg^-1·d^-1)组以及配伍(各单味中药等比例混合,4.7 mg·kg^-1·d^-1)组,每组12只。除对照组外,其他各组小鼠灌胃给予0.66 mg·kg^-1·d^-1氢化可的松溶液,连续14 d,以复制药源性虚证模型。造模同时,各中药干预组及配伍组小鼠灌胃给予相应药液,连续14 d。末次给药后,称量小鼠体质量;采集血清和双侧肾上腺组织。ELISA检测血清皮质酮含量;PCR检测肾上腺相关基因的表达,包括类固醇激素合成酶(Cyp11a1、Cyp21a1、Cyp11b1、Hsd3b1)、胆固醇转运分子(Star、StarD3、Npc1、Npc2)、胆固醇摄取分子(Ldlr、Scarb1)、胆固醇合成限速酶(Hmgcr)、胆固醇储存分子(Lipe、Acat1)、胆固醇流出分子(Abca1、Abcg1)、胆固醇稳态分子(Srebf2、Nr1h3);Western blot检测肾上腺CYP11A1、CYP21A2、HSD11B1、StAR、NPC1、NPC2、LDLR、SRBI、HSL、ACAT1的蛋白表达。结果:(1)与模型组相比,各中药干预组及配伍组小鼠的体质量和血清皮质酮含量无明显变化(P>0.05)。(2)与对照组相比,模型组小鼠肾上腺Cyp21a1 mRNA表达和CYP11A1蛋白表达显著降低(P<0.01);与模型组相比,淫羊藿组和仙茅组Cyp21a1 mRNA表达明显上调(P<0.05),各中药组及配伍组CYP11A1蛋白表达呈上调趋势。(3)与对照组相比,模型组小鼠肾上腺Star和Npc1的mRNA表达及StAR和NPC1蛋白表达显著降低(P<0.05,P<0.01),NPC2蛋白表达显著升高(P<0.01);与模型组相比,淫羊藿组、仙茅组、杜仲组、菟丝子组及配伍组StAR蛋白表达明显升高(P<0.05,P<0.01),各中药组NPC1蛋白表达量增多,以补骨脂作用最为显著(P<0.05),除杜仲组外其他中药干预组及配伍组NPC2蛋白表达明显下调(P<0.01)。(4)与对照组相比,模型组小鼠肾上腺Ldlr、Scarb1的mRNA表达以及LDLR、SRBI的蛋白表达均显著降低(P<0.05,P<0.01);与模型组相比,仙茅组、补骨脂组、菟丝子组、肉苁蓉组及配伍组Ldlr mRNA表达明显下调(P<0.01),杜仲组、菟丝子组及配伍组SRBI蛋白表达显著升高(P<0.05,P<0.01)。(5)模型组小鼠肾上腺Hmgcr mRNA表达及HSL蛋白表达较对照组显著降低(P<0.01);与模型组相比,仙茅组、巴戟天组、杜仲组、补骨脂组、肉苁蓉组Hmgcr mRNA表达明显降低(P<0.05,P<0.01),淫羊藿组、补骨脂组、菟丝子组、肉苁蓉组及配伍组HSL蛋白表达明显降低(P<0.05,P<0.01)。(6)模型组小鼠肾上腺Abcg1 mRNA表达较对照组显著升高(P<0.01);与模型组比较,除肉苁蓉组外,其他各中药干预组及配伍组的Abcg1 mRNA表达均显著降低(P<0.05,P<0.01)。(7)模型组小鼠肾上腺Srebf2 mRNA表达较对照组显著降低(P<0.01);与模型组相比,补骨脂组、肉苁蓉组及配伍组Srebf2 mRNA表达显著降低(P<0.05,P<0.01),且补骨脂组、菟丝子组、肉苁蓉组及配伍组Nr1h3 mRNA表达显著降低(P<0.05)。结论:温补肾阳中药可调节氢化可的松诱导的虚证模型小鼠肾上腺皮质激素的合成能力,其中以淫羊藿、仙茅、杜仲、菟丝子的作用较为显著。

Objective:To study the effects of Chinese medicines of warming and tonifying kidney yang and their compatibility on the synthesis of adrenocortical hormone in mice with deficiency syndrome.Methods:One hundred and twenty male ICR mice were randomly divided into ten groups,including control group,model group,Epimedii Folium(5.3 mg·kg^-1·d^-1)group,Curculiginis Rhizoma(4.3 mg·kg^-1·d^-1)group,Morindae Officinalis Radix(4.3 mg·kg^-1·d^-1)group,Eucommiae Cortex(5.3 mg·kg^-1·d^-1)group,Psoraleae Fructus(5.3 mg·kg^-1·d^-1)group,Cuscutae Semen(6.0 mg·kg^-1·d^-1)group,Cistanches Herba(5.3 mg·kg^-1·d^-1)group,and compatibility(above Chinese medicines mixed in equal proportion,4.7 mg·kg^-1·d^-1)group,12 mice in each group.Except the control group,mice in the other groups were treated with 0.66 mg·kg^-1·d^-1 hydrocortisone solution by intragastric administration for 14 days to replicate the drug-induced deficiency syndrome model.Meanwhile the mice in all Chinese medicine treatment groups and the compatibility group were treated with the corresponding decoction by intragastric administration for 14 days.After the last administration,the body mass of mice was weighed,and the serum and bilateral adrenal tissues were collected.The serum content of corticosterone was detected by ELISA.The expressions of related genes in adrenal gland were detected by PCR,including steroid hormone synthases(Cyp11 a1,Cyp21 a1,Cyp11 b1,Hsd3 b1),cholesterol transport molecules(Star,StarD3,Npc1,Npc2),cholesterol uptake molecules(Ldlr,Scarb1),cholesterol synthesis rate-limiting enzyme(Hmgcr),cholesterol storage molecules(Lipe,Acat1),cholesterol outflow molecules(Abca1,Abcg1),and cholesterol homeostasis molecules(Srebf2,Nr1 h3).The protein expressions of CYP11 A1,CYP21 A2,HSD11 B1,StAR,NPC1,NPC2,LDLR,SRBI,HSL and ACAT1 in adrenal gland were detected by Western blot.Results:(1)Compared with the model group,there were no significant changes in body mass and serum corticosterone content in all Chinese medicine treatment groups and the compatibility group(P>0.05).(2)Compared with the control group,the expression of Cyp21 a1 mRNA and the expression of CYP11 A1 protein were significantly decreased in the model group(P<0.01).Compared with the model group,the expression of Cyp21 a1 mRNA in the Epimedii Folium group and Curculiginis Rhizoma group was significantly up-regulated(P<0.05),and the expression of CYP1 A1 protein showed an upward trend in all Chinese medicine treatment groups and the compatibility group.(3)Compared with the control group,the mRNA expressions of Star and Npc1 and the protein expressions of StAR and NPC1 in adrenal gland of the model group were significantly decreased(P<0.05,P<0.01),and the protein expression of NPC2 was significantly increased(P<0.01).Compared with the model group,the expression of StAR protein was obviously increased in the Epimedii Folium group,Curculiginis Rhizoma group,Eucommiae Cortex group,Cuscutae Semen group and the compatibility group(P<0.05,P<0.01);the expression of NPC1 protein was increased in all Chinese medicine treatment groups,and the effect of Psoraleae Fructus was the most significant(P<0.05);except the Eucommiae Cortex group,the expression of NPC2 protein was significantly down-regulated in the other Chinese medicine treatment groups and the compatibility group(P<0.01).(4)Compared with the control group,the mRNA expressions of Ldlr and Scarb1 and the protein expressions of LDLR and SRBI in adrenal gland of the model group were significantly decreased(P<0.05,P<0.01).Compared with the model group,the expression of Ldlr mRNA was significantly down-regulated in the Curculiginis Rhizoma group,Psoraleae Fructus group,Cuscutae Semen group,Cistanches Herba group and the compatibility group(P<0.01),while the expression of SRBI protein was significantly increased in the Eucommiae Cortex group,Cuscutae Semen group and the compatibility group(P<0.05,P<0.01).(5)The expressions of Hmgcr mRNA and HSL protein in adrenal gland of the model group were significantly lower than those of the control group(P<0.01).Compared with the model group,the expression of Hmgcr mRNA was obviously decreased in the Curculiginis Rhizoma group,Morindae Officinalis Radix group,Eucommiae Cortex group,Psoraleae Fructus group and Cistanches Herba group(P<0.05,P<0.01),and the expression of HSL protein was significantly decreased in the Epimedii Folium group,Psoraleae Fructus group,Cuscutae Semen group,Cistanches Herba group and the compatibility group(P<0.05,P<0.01).(6)The expression of Abcg1 mRNA in adrenal gland of the model group was significantly higher than that of the control group(P<0.01).Except the Cistanches Herba group,the expression of Abcg1 mRNA in the other Chinese medicine treatment groups and the compatibility group was significantly lower than that in the model group(P<0.05,P<0.01).(7)The expression of Srebf2 mRNA in adrenal gland of the model group was significantly lower than that of the control group(P<0.01).Compared with the model group,the expression of Srebf2 mRNA was significantly decreased in the Psoraleae Fructus group,Cistanches Herba group and the compatibility group(P<0.05,P<0.01),and the expression of Nr1 h3 mRNA was significantly decreased in the Psoraleae Fructus group,Cuscutae Semen group,Cistanches Herba group and the compatibility group(P<0.05).Conclusion:Chinese medicines of warming and tonifying kidney yang can regulate the synthesis of adrenocortical hormone in deficiency syndrome mice induced by hydrocortisone.Among,Epimedii Folium,Curculiginis Rhizoma,Eucommiae Cortex and Cuscutae Semen show more significant effects.