Glucocorticoid-induced Drug-induced Syndrome Evaluation In Mice And Research On The Material Basis Of Adrenal Cortex Function

Objective: To explore the standardized modeling method and evaluation index of glucocorticoid-induced syndrome in mice,and to evaluate the preliminary efficacy of TCM herbs related to kidney-tonifying and spleen-strengthening therapy on glucocorticoid-induced syndrome.Methods: Experiment One: ICR,BALB/c,C57BL/6J,KM and nude mice were selected.Prednisolone was used as the drug intervention.There were 16 mice of each strain,eight-each in the normal control group and prednisolone group.Prednisolone(0.5 mg/kg/d)was administered for 14 consecutive days in the prednisolone group.Experiment Two: ICR mice were administered with different doses of hydrocortisone(0.33,0.66,3.3,12.5 mg/kg/d),prednisolone(0.25,0.5,2.5,12.5 mg/kg/d)and dexamethasone(0.0125,0.05,0.25,1.25mg/kg/d).There were 16 mice in each group.Half of the mice in each group were administrated a drug for 7 days and the half for 14 days.Experiment Three: ICR mice with 1.65 mg/kg hydrocortisone for 14 days to induce drug-induced spleen and kidney deficiency syndrome was treated synchronously with Sijunzi Decoction,Liuwei Dihuang Decoction,Guifu Dihuang Decoction,Aconm Lateralis Radix Praeparaia-Cinnamomi Cortex,Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex,Ginseng Radix et Rhizoma-Astragali Radix,Rehmanniae Radix Praeparata-Corni Fructus.The body weight of the mice was observed daily during the experiment,and the TCM four diagnostic indicators such as body mass,holding power,axillary temperature,activity degrees of open field and infrared temperature were detected by the experimental methodology of mice syndrome differentiation;The mice were sacrificed whose spleen,thymus was weighed and the organ index was calculated.Serum corticosterone and ACTH were measured by ELISA.The gene expressions of Star,Cyp11a1,Cyp21a1,Cyp11b1,Cyp11b2 in the adrenal were detected by real-time fluorescent quantitative PCR.The proteins expressions of LDLR,SRBI,and St AR were detected by Western blotting.Results: Experiment One: Compared with the control group of the respective strains,1)the body weight of ICR mice decreased significantly 7 days after prednisolone administration(P<0.01);BALB/c mice decreased significantly 13 days after prednisolone administration(P<0.01);C57BL/6J mice decreased significantly 5 days after prednisolone administration(P<0.01);nude mice decreased significantly 13 days after prednisolone administration(P<0.05).2)Prednisolone administration resulted in a significant decrease in the grip strength and a significant decrease in the mean temperature of the body trunk of ICR mice(P<0.05).3)Giving prednisolone resulted in a significant decrease in the spleen mass of ICR mice and nude mice and a decrease in the spleen index of nude mice(P<0.05),whereas prednisolone administration resulted in a significant decrease in the thymic mass and thymic index of ICR,C57BL/6J and KM mice(P<0.01).4)Prednisolone administration significantly decreased the serum corticosterone level in BALB/c and KM mice(P<0.05),and significantly decreased serum levels of ACTH in BALB/c mice(P<0.01).5)Giving prednisolone significantly downregulated: Cyp21a1 expression in ICR mice(P<0.05);Star expression in C57BL/6J mice(P<0.01);Cyp11a1 and Cyp21a1 expression in KM mice(P<0.01);Star and Cyp21a1 expression in nude mice(P<0.05).In addition,prednisolone inhibited expression of LDLR and St AR in ICR and KM mice.Experiment Two: After 7 days and 14 days,compared with the normal control group: 1)hydrocortisone above a dose of 3.3mg/kg/d,prednisolone above a dose of 2.5mg/kg/d,and dexamethasone above a dose of 0.25mg/kg/d significantly inhibited the growth of body mass in mice within 14 days(P<0.05 or P<0.01).2)Significantly atrophic spleens were caused by hydrocortisone above a dose of 3.3 mg/kg/d,prednisolone above a dose of 2.5mg/kg/d,and dexamethasone above a dose of 0.25mg/kg/d(P<0.05 or P<0.01);Hydrocortisone above 0.66mg/kg/d,prednisolone above 0.25mg/kg/d,and dexamethasone above 0.25mg/kg/d led to significant thymus atrophy in mice(P<0.05 or P<0.01).3)According to the four diagnostic indicators of mice,hydrocortisone and dexamethasone at all doses decreased the body surface temperature of mice(P<0.05 or P<0.01);Prednisolone had little effect on body surface temperature of mice.4)Hydrocortisone above 3.3mg/kg/d,prednisolone above 0.25mg/kg/d significantly inhibited the secretion of corticosterone and ACTH(P<0.05 or P<0.01);dexamethasone above 0.0125mg/kg/d significantly inhibited the secretion of corticosterone(P<0.05).5)Hydrocortisone above 0.66 mg/kg/d,prednisolone above 0.25mg/kg/d and dexamethasone above 0.0125mg/kg/d significantly inhibited the expression of adrenal steroid hormone synthase(Star,Cyp11a1,Cyp21a1,Cyp11b1),and inhibited the expression of related proteins(LDLR,SRBI,St AR)during the conversion of adrenal cholesterol into hormones(P<0.05 or P<0.01).Experiment Three: 1)The effect of traditional Chinese medicine compound and traditional Chinese medicine combinations on the body mass decline of mice caused by hydrocortisone was not significant.2)Compared with the hydrocortisone group,t the Aconm Lateralis Radix Praeparaia-Cinnamomi Cortex group increased significantly(P<0.05);The axillary temperature of mice in Guifu Dihuang Decoction group increased significantly(P<0.01).3)Compared with hydrocortisone group,Sijunzi Decoction,Liuwei Dihuang Decoction,Guifu Dihuang Decoction and Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex significantly increased serum corticosterone(P<0.05 or P<0.01)and alleviate the decreased expression of adrenal steroid hormone synthase(Star,Cyp11a1,Cyp21a1,Cyp11b1,Cyp11b2)and related proteins(LDLR,SRBI,St AR)during the conversion of adrenal cholesterol into hormones.Conclusions Experiment One: ICR mice is suggested to be used for study of glucocorticoid-induced asthenia.Experiment Two: 1)The glucocorticoid-induced deficiency syndrome includes Yin deficiency,Yang deficiency and Qi deficiency(no evidence of Yang deficiency appeared in the prednisolone model),and its mainly related kidney and spleen states.2)Hydrocortisone-induced deficiency syndrome model is recommended to be administered at a dose of 0.66 to 3.3mg/kg/d for 14 days.The prednisone-induced deficiency syndrome model is recommended to be administered at a dose of 2.5mg/kg/d for seven days.The dexamethasone-induced deficiency model is recommended to be administered at a dose of 0.25mg/kg/d for seven days.The substance basis of glucocorticoid-induced deficiency syndrome is mainly the inhibition of the expression of adrenal corticosteroid synthase and the pituitary-adrenal cortical axis function.The body weight,spleen,thymus and body surface infrared temperature of mice,as well as the important enzymes in steroid hormone synthesis(St AR,CYP11A1,CYP21A1,CYP11B1)and cholesterol receptor(SRBI and LDLR)can be used as the reference indicators for the evaluation of glucocorticoid-induced syndromes.Experiment Three: From the adrenal cortex function,Sijunzi Decoction,Liuwei Dihuang Decoction,Guifu Dihuang Decoction and Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex all alleviated the effect of hydrocortisone-induced adrenal insufficiency.