Effectiveness And Safety Study For Kangzao No.1 Prescription And Anastrozole In Delaying The Progression Of Bone Age By Zebrafish Model

Objective:The aim of this study is to examine the effectiveness of Kangzao No.1 prescription(KZ)and aromatase inhibitor,anastrozole(ANZ),in delaying the progression of bone age and the safety of reproduction,bone metabolism and lipid metabolism using zebrafish animal model.Methods:Male zebrafish aged 45 dpf were randomly divided into the control group,72 mg/L Kangzao No.1 prescription extract(KZE)group(KZL group),180 mg/L KZE group(KZH group)and 0.7 μM ANZ group(ANZ group),with 70 males for each group.Drug exposure was started at 45 dpf to 75 dpf and continued to observe after stopping the drug exposure.Each group sacrificed 16 fish for testing at 45 dpf,60 dpf,75 dpf and 95 dpf.At 120 dpf,the mating behavior experiment was carried out with wild-type females.In addition,the osteoporosis group induced by 25 μM prednisolone(PNSL)was used as a negative control in bone metabolism safety test.The fatty liver group induced by 5%high cholesterol diet(HCD group)was used as the negative control in lipid metabolism safety test.Thirty male zebrafish in each group were treated from 35 dpf to 95 dpf,and sacrificed at various sampling time points.Experimental observation items include:(1)Reproductive safety(number of sperm,histological observation of testis by H&E staining,chasing number of mating,survival rate of fertilized eggs at 72 hpf,and expression levels of related genes);(2)Safety of bone metabolism(expression levels of genes related to osteogenesis,osteogenesis and bone remodeling);(3)Safety of liver lipid metabolism(H&E staining observation on liver fat distribution,expression levels of genes related to lipogenesis,lipolysis and lipid transport);(4)Observation of growth and development markers,including the effectiveness of delaying bone age and the safety of growth(double bone staining evaluation on growth potential,body length,weight,BMI,and expression levels of related-genes).Results:On reproductive safety,the sperm counts of the zebrafish in the ANZ group and the two KZE groups were significantly reduced during the treatment period,and they recovered after the drug was discontinued.In histological observation at 75 dpf,the germ cells of the two KZE groups were significantly immature than those of the other groups,and this phenomenon disappeared after drug withdrawal.The treatment of ANZ and KZE did not affect the tail-chasing number of fishes at sexual maturity and the survival rate of fertilized eggs at 72 h,indicating that ANZ and KZE did not adversely affect reproductive performance.Based on RT-PCR analysis,on the 30th day of treatment(75 dpf),the expression of vtgl and vtg2 genes,which are positively correlated to the amount of estrogen production,in the two KZE groups and ANZ group were significantly decreased.All groups had no difference in the expression of genes related to Hypothalamic-Pituitary-Gonadal Axis and steroid hormone conversion,including gnrh3,lhr,fshr,erα,erβ,cyp19a1a,cyp19a1b,star.At 75 dpf,the expression of sycp3 gene,which is positively correlated with the number of meiosis,in KZH group and ANZ group was lower than that in the control group.At 95 dpf,the expression of sycp3 gene in the two KZE groups and the ANZ group was decreased.The expression of dmc1 gene,which is positively correlated with the number of meiosis,in the ANZ group was also significantly decreased.These results showed that estrogen level in zebrafish would be reduced during treatment with ANZ and KZE,and the observed phenomenon further supports that ANZ and KZE can slow down the development of sexual characteristics without adversely affecting reproductive performance.On bone metabolism,the expression of col1a1a,alp,sp7 genes,which are related to osteogenesis,in the two KZE groups and ANZ group were reduced,but the degree of reduction was not as low as that in the PNSL group.The expression of runx2a and runx2b genes,which are related to osteogenesis,and opn and phex genes,which are related to bone remodeling,in the two KZE groups and the ANZ group were not significantly different from those in the control group.In terms of osteoclastogenesis,at 75 dpf,the expression of rank gene in KZH group and ANZ group was higher than that in the control group,while the PNSL group was lower than the two KZE groups and the ANZ group.There was no significant difference in the expression of ctsk and acp5b genes in each group during the treatment period.At 95 dpf,the expression of acp5b gene in the two KZE groups and the ANZ group was higher than that of the PNSL group.The above results showed that the bone metabolism performance of the two KZE groups and the ANZ group is different from that of the PNSL group.The KZE and ANZ did not cause osteoporosis.On liver lipid metabolism,with the progress of culture time,liver fat increased in both KZE groups and ANZ group,but the size of lipid droplets and the average area of each lipid droplet were smaller than those in the HCD group.The expression of fas,dgat2,mtp,apoba genes,which are related to fat synthesis,in the two two KZE groups and the ANZ group rised,but the increase was not as high as that in the HCD group.The expression of ppara and cpt1aa genes related to lipolysis also increased,the degree of increase was similar to that of the HCD group.The above results demonstrated that KZE and ANZ could increase the overall fat metabolism,and the fat synthesis would be less than that of the HCD group,while the lipolysis was similar,indicating that the manifestation of lipid metabolism is different from HCD group.On growth and development,KZE groups and ANZ group increased the ratio of cartilage area to the remaining caudal fin bone area after fin rays removal,and decreased body weight,but their BMI were not significantly different from the control group.In addition,the expression of igf-1 gene was increased in KZE groups and ANZ group.The above results showed that the two KZE groups and the ANZ group had more growth potential than the control group without causing changes in BMI.Conclusion:In terms of effectiveness,during the administration of ANZ and KZE,estrogen level in zebrafish would decrease,and the ratio of cartilage area to the remaining caudal fin bone area after fin rays removal would increase,indicating that KZE and ANZ are effective in inhibiting bone age progression.In terms of safety,this study supports the relative safety of ANZ and KZE in reproduction,bone metabolism,liver fat metabolism,and body development(includes weight and BMI).