Effect Of Bupishen Huoxie Xiezhuo Traditional Chinese Medicine On Cell Proliferation And Apoptosis Of Rat Mesangial Cell

Chronical renal failure(CRF) results from the progressing deterioted of chronical renal lesions caused by all sorts of pathogeny. Western nation adopt dialysis and kidney transplant to treat the CRF and especially the end-stage renal disease(ESRD), for its expensive price and it limits existently, not yet carrying out extensively at the our country. How to interfere in the occurrence of the CRF and defer the development of CRF are always the unsolved problems in prevention and therapy the CRF. The main pathology changes of the earlier CRF were the glomerular mesangial cell(MC) over-proliferationg and the expansion of extracellular matrix (ECM) in the mesangial area, then developed into glomerulosclerosis(GS) at the end-stage with serious renal function failuie. So to inhibit the over-proliferation of the glomerular MC and suppress the expansion of the ECM is the key to defer the GS, prevent and therapy the CRF as well.The MC secretes the ECM mainly as the most active glomerular mesangial cell. As the study proved that the over-proliferation of the glomerular MC could synthesize and secrete various compositions of ECM; the glomerular MC's over-proliferation accompanied with the expansion of the ECM. As the glomerular MC over-proliferied and the ECM over-expanded, the suppressed-blocked vessel, decreased glomerular filtration rate, and the GS resulted. So the inhibiton of the MC's proliferation could decrease the expansion of the ECM, as to defer the GS progression.The cell apoptosis and proliferationg adjusts the dynamic equilibrium of the cell together. Along with the research continuouse development, the important role of the cell apopotosis mechanism in the procession of renal disease was noticed. During the earlier stage in the CRF, the over- proliferation of glomerular MC also accompanied with the imperfection of the glomerular MC'sapoptosis, so as a result this induced the over-proliferated glomerular MC's apoptosis, as to reduce the expansion of the ECM and defer the GS progress.At the present time, there isn't a safe and effective GS inhibitor. Chinese material have extensive source, and many Chinese material medica can decrease the BUN and Scr, relieve the symptom, defer the GS progress. For this reason, the studies of Chinese herbal inhibit GS have important clinical meaning. This study aims at investigate the effect of Bupishen Huoxie Xiezhuo traditional Chinese medicine (TCM) on cell's proliferation, cell cycle and apoptosis of rat glomerular MC. Thereby it will offer some theorical basis for applying the TCM to prevent and therapy the CRF.Part one: The effect of the TCM on rat mesangial cell's proliferationObjective:To observe the effect of Bupishen huoxie xiezhuo Chinese herbs on rat mesangial cell's proliferation, cycle and approach the possible mechanism.Methods:The rat MC which grew well at 3era, were exposed to medecine serum RPMI-1640 which contained the low,high dos TCM and west medecine respectively. Then study the effects on cell from the following aspects which include:1,The detection of the levity of cell with typan blue dying: Calculate the proportion of living cells dyed by typan blue.2,The detection of cell proliferation with MTT method: The testing cells were exeposed by MTT for 6h before the culture ending, then add 200μl DMSO. The values of absorbance at 570 nm were detected with an automatic microtiter plate reader.3,The detection of cell cycle progress with flow cytometry: The testing cells were harvested by centrifug for 5 min, fixde by cold ether, washed by PBS, hatched with RNAse at 37℃for 30min, dyed with PI for 30 min, detect cell cycle progress with flow cytometry.Result:1. The typan blue dye exclusion showed: The proportion of living cell wae over 96% exposed to the RPMI-1640 which contained 10% low dos or high dos the TCM serum and west medecine serum that wae not poisionouse to cells.2. The detection of MTT showed: The OD of the cell exposed to the RPMI-1640 which contained either 10% low dos or high dos herb serum is different frome to that of the cell exposeed to the normal rat serum(P＜0.01), indicating that both the low dos and high dos the TCM could inhibit the cell's proliferation and the effect depended on the dos of the TCM. And the difference between the herb and the westen medicine was also significent(P＜0.01), indicating that the surpressing effect of the TCM was stronger than that of western medicine. The surpressing effect of the herb was no difference between 24h and 48h(P＞0.05), indicating that the effect didn't depended on the time.3. The detection of flow cytometry assay showed: Some changes took place in the cell cycle distribution that the proportion of MCs in G₁ phase increased while the one in S phase decreased after exposed to either 10% low dos or high dos the TCM serum for 24h comeparing significantly with the control group(P＜0.01). The effect depended on the dos of the TCM, indicating that one of the mechanisms of cell proliferation inhibition was cell cycle progress inhibition ie, cell was detented in G₁ phase and the transition from G₁ phase to S phase slowed down.Part two: The effect of the TCM on rat mesangial cell's apoptosisObjectiye:To observe the effect of the TCM on rat mesangial cell's apoptosis, the expression of Bcl-2 and approach the possible mechanism.Methods:The rat MC which grew well at 3era, were exposed to medecine serum RPMI-1640 which contained low,high dos the TCM and west medecine respectively. Then study the effects on cell from the following aspects which include:1,The detection of rat mesangial cell's apoptosis with TUNEL method: The apoptosis of rat mesangial cells was detected with the in situ cell apoptosis kit according to the manufacturer's instructions.2,The detection of rat mesangial cell's apoptosis ratio by flow cytometry ass—ay with PI simple staining: The testing cells were harvested by centrifug for 5 min, fixde by cold ether, washed by PBS, hatched with RNAse at 37℃for 30min, dyed with PI for 30 min, detect cell cycle apoptosis with flow cytometry.3,The detection of rat mesangial cell's apoptosis-inhibiting Bcl-2 protein with Western blot: The testing cells washed by PBS were added the loading buffer, and turned to the EP tube to boil, then took the same quaintity protein to nitric acid cellulose film after polyacrylamide gel electrophoresis, next added buffer to block. at last add the anti-body and rabbit anti-Bcl-2 to hatch, flowing with color reaction and taking photo.Result:1. The detection of TUNEL showed: The apotosis took place in the MC exposed to medecine serum for 24h while that didn't in control group.2. The detection of flow cytometry assay with PI simple staining showed: The apoptosis specific hypodiploid peeks occurred in the MC exposed to medecine serum for 24h while that didn't in control group. The apoptosis ratio increased with the herb dos increasing comparing to the control group (P＜0.01), indicating that the effect depended on the dos of the TCM. And the difference between the TCM and the westen medicine was also significent(P＜0.01).3. The detection of Western blot showed: the expression of Bcl-2 protein in the MC decreased significantly after exposed to medecine serum for 24h while that didn't in control group.Conclusion:1. The TCM could inhibit the MC's proliferation and the one of the possible mechanisms of cell proliferation inhibition was cell cycle progress inhibition.ie, cell was detented in G₁ phase and the transition from G₁ phase to S phase slowed down.2. The TCM could induced the MC's apoptosis and the one of the possible mechanisms of cell apoptosis was supressing the expression of anti- apoptosis Bcl-2 protein.3. The MC was the target cell of the action of TCM. The possible mechanisms of TCM that defer the progression of GS and therapy CRF were by inhibiting the cell's proliferation as well supressing the expression of anti-apoptosis Bcl-2 protein to induce the MCs' apoptosis.