Effect of compound preparation of huangqi and dahuang on proliferation and secretion of extracellular matrix in mesangial cells of cultured rats

Wei XIAO; Yun MA; Lianbo WEI; Haibo LONG

ABSTRACT

BACKGROUND:

Diabetic nephropathy is one of the most serious vascular complications of diabetes mellitus. Compound preparation of huangqi and dahuang, a traditional Chinese medicine, has been used to preventing or treating diabetic nephropathy for several years, and has a certain protective effect on the kidney of diabetes mellitus patients. But its exact mechanism remains unknown and needs to be studied more.

OBJECTIVE:

To investigate the effect of compound preparation shenkang wan on the proliferation and secretion of extracellular matrix in cultured rat mesangial cells induced by high glucose.

DESIGN:

Randomized and controlled study.

SETTING:

Center of Integrated Traditional and Western Nephrology of Zhujiang Hospital and Medicine Department of Nanfang Hospital, Southern Medical University.MATERIALS The serum pharmacological experiment was performed in Animal Experimental Center of Southern Medical University in A pril 2005.The cell culture experiment was conducted in Cell culture room of Southern Medical University from April 2005 to July 2005. Totally 16 normal Wistar male rats, weighted varied from 190 g to 220 g, were used in the study.

METHODS:

Sixteen normal Wistar male rats were randomly divided into 4 groups normal serum group, capoten group, shenkang wan group (high dose and low dose); shenkang wan was mainly constituted of huangqi,dahuang, leech, gordon guryale seed and corn stigma and made in Pharmacy Department of Zhujiang Hospital of Nanfang Medical University, agent number 20031214). ① The rats in capoten group and high and low dose shenkang wan group were given the corresponding drugs respectively according to 5 mg/kg, 2.4 g/kg, 1.2 g/kg weight. The rats in normal serum group were given the same volume water. After treated 7 days, all rats were hocused and separated medication serum. ② Mesangial cell was cultured in vitro with different concentrations of glucose (10, 20, 30 and 40 mmol/L).The proliferation of mesangial cell was observed with the methyl-thiazoltelrazolium colorimetric assay at 24, 48, 72 hours and 96 hours. ③ Then the cultured mesangial cells were divided into six subgroupsLow glucose control group (10 mmol/L glucose), high glucose group (30 mmol/L glucose);normal serum group (30 mmol/L glucose); capoten group (30 mmol/L glucose); shenkang wan group (high dose and low dose, 30 mmol/L glucose).After cultured 72 hours, the proliferation of mesangial cell was detected with the methyl-thiazol-telrazolium colorimetric assay, the secretion and mRNA gene expression of fibronetin levels in mesangial cell were respectively detected by enzyme linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) method.MAIN OUTCOME

MEASURES:

①Proliferation of mesangial cell induced by different concentrations glucose. ② Proliferation and secretion and mRNA gene expression of fibronectin in every group.RUSULTS ① Effect of different concentrations glucose on the prolifera-tion of mesangial cell Compared with low concentrations glucose(10 mmol/L), 20 mmol/L glucose could accelerate the proliferation ofmesangial cell during 96 hours experiment period, but only had a statisti-cally significant difference at 72 and 96 hours (P ＜ 0.05). 30 mmol/L glu-cose could significantly accelerate the proliferation of mesangial cell thanthat of 10 mmol/L glucose from 24 hours to 96 hours (P ＜ 0.05 or P ＜ 0.01),and this effect was increasing with time in 72 hours and reduced after 72hours. 40 mmol/L glucose could significantly increase the proliferation ofmesangial cell than of low concentrations glucose in 48 hours (P ＜ 0.05),and this effect was reduced after 48 hours and even conversed to restraineffect. ② Effect of different medication serum on the proliferation ofmesangial cell The optical density value in high glucose group is obviouslyhigher than that of low glucose control group (P ＜ 0.01). Compared withhigh glucose group, the optical density value in capoten, shenkang wangroup (high dose and low dose) was decreased markedly (P ＜ 0.01 or P＜ 0.05). While the optical density value in normal serum group was showedno difference with the high glucose group (P ＞ 0.05). ③ Effect of differentmedication serum on secretion of fibronectin in mesangial cell Content offibronectin in high glucose group was increased more markedly than that oflow glucose group (P ＜ 0.01). Compared with high glucose group, contentof fibronectin in capoten and shenkang wan group (high dose and low dose)was showed a significantly decrease (P ＜ 0.01 or P ＜ 0.05), while contentof fibronectin in normal serum group was showed no difference with thehigh glucose group (P ＞ 0.05). ④ Effect of different medication serum onexpression of fibronectin mRNA in mesangial cell The optical density val-ue of fibronectin strip in high glucose group was brighter than that in lowglucose group and the ratio of it and β-actin were increased markedly too(P ＜ 0.01). Compared with high glucose group, the optical density value offibronectin strip in capoten and shenkang wan group (high dose and lowdose) was showed a significantly decrease and the ratio of it and β-actinwas reduced distinctly too (P ＜ 0.01), while the ratio of it and β-actin innormal serum group was showed no difference (P ＞ 0.05).

CONCLUSION:

High glucose could accelerate proliferation, increase thesecretion and mRNA gene expression of fibronectin in mesangial cell,while shenkang wan could inhibit proliferation and secretion of the extra-cellular matrix in mesangial cell induced by high glucose.