| ObjectiveTo compare the subchronic nephrotoxicity induced by cinnabar and Wanshi Niuhuang Qingxin Pills in rats,and to provide reference for the clinical use of Wanshi Niuhuang Qingxin Pills.MethodsHealthy 96 SD rats were randomly divided into four groups,that was control group,cinnabar group,Wanshi Niuhuang Qingxin Pills(the whole recipe)group,and removal cinnabar of Wanshi Niuhuang Qingxin Pills group(the removal cinnabar recipe).Each group had 24 rats,including 12 male and 12 female.The rats were respectively given gavage solvent(0.5% sodium carboxymethyl cellulose),1.0 g/kg cinnabar,9.83 g/kg the whole recipe(containing 1.0 g/kg cinnabar)and 8.83 g/kg the removal recipe(without cinnabar)once a day for 12 weeks,intragastric administration volume was 2 m L/100 g.The rats were observed for 4 weeks after the end of the administration.During the experiment,the daily situation of rats such as feed and water intake were observed every day and the body weight were weighed twice a week.The samples of blood,kidney and urine of 6 rats(including 3 male and 3 female)in each group were individually collected after administered for 4,8,12 weeks and drug withdrawal for 4 weeks.The contents of mercury in blood,kidney and urine were determined by Zeeman atomic absorption mercury analyzer.Serum creatinine(SCr),blood uric acid(BUA)and 24 hours urinary albumin quantitation were determined by automatic biochemical analyzer.The content of retinol binding protein 4(RBP-4)in urine samples was tested by enzyme-linked immuno sorbent assay(ELISA).The content of hydroxyproline(Hyp)in renal tissue was determined by alkali hydrolysis method.The renal tissue sections were examinated by HE and Masson staining under microscope.The expression of TNF-? and IL-10 in renal tissue were detected by Real-time PCR.The data were analyzed by one-way ANOVA and compared any two groups difference,the interaction between drugs was analyzed by 2 × 2 factorial design variance with SPSS 17.0,significance level was ?=0.05.Results1.After administered for 4,8,12 weeks and drug withdrawal for 4 weeks,the contents of mercury in blood,kidney and urine of the cinnabar group were significant accumulated comparing with the whole recipe group,the removal cinnabar recipe group and the control group,the difference was statistically significant(P<0.05).After administered for 12 weeks,the contents of mercury in blood,kidney and urine of the whole recipe group were obvious higher than the control group and the removal cinnabar recipe group,the difference was statistically significant(P<0.05).A statistically significant interaction effect was found between cinnabar and the other 5 Chinese medicines(including bezoar,coptis chinensis,gardenia,turmeric and scutellaria baicalensis)in the Wanshi Niuhuang Qingxin Pills for the mercury content of blood,kidney and urine(P<0.05).2.After administered for 8,12 weeks and drug withdrawal for 4 weeks,the contents of RBP-4 and BUA of the cinnabar group were higher than the whole recipe group,the removal recipe group and the control group,the difference was statistically significant(P<0.05).Comparing with the whole recipe group,the contents of RBP-4 and BUA of the removal recipe group and the control group had no statistical differences(P>0.05).A statistically significant interaction effect was found between cinnabar and the other 5 Chinese medicines in the Wanshi Niuhuang Qingxin Pills for the content of RBP-4 and BUA(P<0.05).3.After administered for 12 weeks,under microscope,pathological changes in the cinnabar group including numerous infiltration of inflammatory cells,serious vacuolar degeneration,partial tubular epithelial cells swelling(several protein casts and necrosis)and renal interstitial mild fiber hyperplasia.The fiber hyperplasia degree score by Masson staining of the cinnabar group were higher than the whole recipe group,the removal recipe group and the control group,the difference was statistically significant(P<0.05).Little infiltration of inflammatory cells,slight vacuolar degeneration,separate tubular epithelial cells swelling were observed in the whole recipe group.Occasionally visible infiltration of inflammatory cells,vacuolar degeneration and fibroblasts were observed in the removal cinnabar recipe group.After drug withdrawal for 4 weeks,there was no significant change compared with 12 weeks except the infiltration of inflammatory cells decreased in each group.5.After administered for 12 weeks,the m RNA expression levels of TNF-? and IL-10 in renal tissue of the cinnabar group were obvious higher than that of the whole recipe group,the removal cinnabar recipe and the control group,the difference was statistically significant(P<0.05).The both expression levels of the female rats were higher than that of the male rats in the same group,the difference was statistically significant(P<0.05).The expression level of TNF-? m RNA of the whole recipe group was higher than that of the removal cinnabar recipe and the control group,and the expression level of IL-10 m RNA of the whole recipe group was higher than that of the control group in the female rats,the difference was statistically significant(P<0.05).A statistically significant interaction effect was found between cinnabar and the other 5 Chinese medicines in the Wanshi Niuhuang Qingxin Pills for the m RNA expression levels of TNF-? and IL-10 in renal tissue(P<0.05).ConclusionsLong-term overdose of cinnabar could cause mercury accumulation in kidney and result in kidney damage of rats.Comparing with cinnabar,the subchronic nephrotoxicity induced by Wanshi Niuhuang Qingxin Pills in rats was mild,might be related to its compatibility of bezoar,coptis chinensis,gardenia,turmeric and scutellaria baicalensis with cinnabar could antagonize the absorption of mercury in cinnabar and reduce the mercury accumulation of kidney. |
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