Research Of Regulating Mechanism Based On IGF-1in Kidney Storing Essence And Governing Bones Theory And Intervention Effect Of Chinese Kidney-tonifying Drugs

Objective:To observe the level regulation of Zibu Shenjing Fang (滋补肾精方) on the kidney essence insufficient syndrome mice insulin-like growth factor-1(IGF-1), insulin-like growth factor binding protein-3(IGFBP-3) level according to the theory of the main bone of kidney essence and nourishing the kidney essence.And further research the effect of Zibu Shenjing Fang containing serum on mouse osteoblasts (MC3T3-E1) proliferation, cell cycle, expression of alkaline phosphatase (ALP) and IGF-lmRNA to explore kidney essence insufficient evidence based on IGF-I mechanism as well as effect of Chinese kidney-tonifying and replenishing essence drugs intervention to finally reveal the nature theory of storing essence and governing bones, which provide the basis to insufficient kidney essence card and the appropriate treatment of the disease.Methods:1. Animal experiments:50mice were randomly divided into five groups with10mice in each group:a normal group, a model group, a Jinkuishenqiwan(金匮肾气丸)group, a Zibu Shenjing Fang high dose group and a Zibu Shenjing Fang low dose group. In addition to the normal group, the other four groups established the kidney essence deficiency model by jointly exhaustive swimming and threat-injuring the kidney. In these four groups, the model group was given saline (0.02ml.g-1.d-1),Jinkuishenqiwan group given Jinkuishenqiwan suspension (2.7g.k g-1.d-1). Zibu Shenjing Fang high dose group and the low-dose group administered by the difference of Zibu Shenjing Fang (20g-kg-1d-1.10g.kg-1.d-1). all of which continued for21days.And then we observed the general condition of mice and weigh mice after the end of the experiment.In this experiment.the enzyme-linked immunosorbent assay (ELISA) was used to detect to levels of insulin-like growth factor-1(IGF-1) and insulin-like growth factor binding protein-3(IGFBP-3)in liver tissue. And at the same time, the the change of indicators were observed by using Chinese kidney-tonifying and replenishing essence drugs to treatment.2. Cell cxperiments:the optimal dose of Zibu Shenjing Fang containing serum was finally determined from former experiment, we next made preparation of containing serum containing and made recovery of mouse osteoblasts (in MC3T3-E1) with different concentrations of Zibu Shenjing Fang containing serum and saline control serum (2.5%,5%,10%,20%,40%) of the cultured cells48and72hours to observe the proliferation of cells and further determined the optimal concentration of serum containing. After osteoblasts were the intervention with the optimal concentration of serum containing and serum of normal saline about24,48and72hours, we used flow cytometry to determine the cell cycle, ALP activity detected by nitro phenol phosphate method and the expression of IGF-1mRNA by semi-quantitative RT-PCR analysis of cells. And further observed the change of indicators with cell culture extended.3Statistical Methods:All of data results were expressed as mean±standard deviation (x±s) using SPSS19.0statistical software to analyze, and were used by one-way ANOVA to conduct significant test and t-test for comparison between groups in which P<0.05was a significant difference.Results:1. General condition of the model mice conformed characteristics of the kidney essence deficiency syndrome in traditional Chinese medicine theory that IGF-1in the liver tissue and IGFBP-3in serum content were decreased. Zibu Shenjing Fang could improve the mouse kidney essence insufficience and enhance IGF-1and serum IGFBP-3levels in mouse liver tissue. And the research showed the best effect of the high dose group of using Zibu Shenjing Fang. Compared with the control group of the same concentration of saline serum, Zibu Shenjing Fang containing serum MC3T3-E1cell proliferation capability elevated in varying degree, which20%of Zibu ShenJing Fang containing serum was the most significant proliferation of promoting. Intervention with the optimal concentration of20%serum containing MC3T3-EI cells24h,48h.72h could significantly reduce the proportion of cells in G0/G1phase and improve the proportion of cells in S phase and proliferation index, ALP activity within the cell supernatants,and the IGF-1mRNA expression levels.These indicators changed more obviously with the time extension of the cell culture compared with saline serum.Conclusion:1. Nourishing the kidney essence parties may regulate the body of IGF-1, IGFBP-3, play a therapeutic effect on the kidney essence deficiency permit.2. Nourishing the kidney essence side containing serum can promote the proliferation of osteoblasts accelerated osteoblast cycle, regulate osteoblast ALP activity, IGF-1mRNA expression. Through animal experiments and cell experiments described from the perspective of endocrine main bone of kidney accuracy of the theory, solution to understand the nature of kidney essence insufficient evidence to provide a basis for the treatment of kidney essence insufficient card.