The Study On The Effect Of Zhuanggujianxi Formula On Proliferation Of Rat Chondrocytes And Its Collagen Type Ⅱ Expression Cultured With IL-1β

Objective:To observe the effect of ZhuangGuJianXi Formula(ZGJXF) on articular chondrocytes induced by IL-1β,and discuss the protection mechanism of ZGJXF on impaired articular chondrocytes through the aspects of cartilage cell proliferation,expression of typeⅡcollagen and its mRNA.Methods:1.To establish the vitro cultured system of chondrocytes.The Primary chondrocytes were got from four-week old SD articular cartilage with collagenase and trypsin digestion,then identified by toluidine blue staining and observe the biological characteristics of chondrocytes.2.MTT assay was performed to determine the effect of ZGJXF and "ZhuangGuGuanJie Pills" containing serum on proliferation of normal and IL-1β-induced chondrocytes.3.Immunohistochemistry was performed to observe the effect of ZGJXF and "ZhuangGuGuanJie Pills" containing serum on typeⅡcollagen of normal and IL-1β-induced chondrocytes.4.RT-PCR was performed to observe the effect of ZGJXF and "ZhuangGuGuanJie Pills" containing serum on expression level of typeⅡcollagen mRNA on normal and IL-1β-induced chondrocytes.Results:1.Culture four-week old SD rat's chondrocytes of articular surface with collagenase and trypsin digestion,and identified by toluidine blue staining and observe the biological characteristics of chondrocytes with invert microscope,And confirmed that it was conformed to the biological characteristics of chondrocytes.It's available to remain the phenotype of chondrocytes steadily within 3th generation from the performance of morphology, histochemical staining.2.The result of MTT assay is as below: The ZGJXF containing serum at final concentrations of 5%,10%and 20%could promote the normal and IL-1β-induced cartilage cells proliferation,without incremental effct.And at the 48h time,the10%concentration of ZGJXF containing serum could promote the cartilage cell proliferation best.The 10%concentration of ZGJXF was compared with the control group,there was no remarkable statistical difference(P＞0.05),and significant difference when compared with the blank serum group and the group of "10%ZGJXF +10ng/mlIL-1β"(P＜0.05).The 10ng/ml concentration of IL-1βcan perform the best inhibition effect on chondrocytes proliferation.3.The result of imunohistochemistry show as follows:The typeⅡcollagen expression was visible well in all six groups' specimens,and the expression site was only in the cytoplasm,with red-brown particles.The expression of typeⅡcollagen in "blank + inducement" group chondrocytes were lower than those in normal blank chondrocytes(P＜0.05).The expression of typeⅡcollagen in "ZGJXF + inducement" group was higher significantly in comparision with that of "blank+ inducement" group (P＜0.05),but no remarkable difference with "ZhuangGuGuanJie Pills + inducement" group(P＞0.05),the same result as the comparison between ZGJXF group and"ZhuangGuGuanJie Pills" group(P＞0.05),While there were a statistical significance between ZGJXF group and blank serum group(P＜0.05).4.The result of RT-PCR show as follows:The expected length of specific strip in typeⅡcollagen mRNA PCR products were visible in all six groups' pecimens.The expression of typeⅡcollagen mRNA in "blank+ inducement" group was lower significantly in comparision with that of blank group and"ZGJXF + inducement" group(P＜0.05),There were no remarkable difference below "ZGJXF + inducement" group and "ZhuangGuGuanJie Pills + inducement"group(P＞0.05),the same result as the comparison between ZGJXF group and "ZhuangGuGuanJie Pills" group(P＞0.05).There were significant statistical difference of typeⅡcollagen mRNA expression between ZGJXF group and blank serum group(P＜0.05).Conclusion:ZGJXF can significantly promote the proliferation of normal and IL-1β-induced chondrocytes,and effectively prevent the injury effect of chondrocytes induced by IL-1βcytokine at the level of transcription and translation of typeⅡcollagen.It can perform the protective effects well on articular cartilage,and it may be one of the mechanism to achieve the effect of prevention and treatment of osteoarthritis.