Regulatory Effect Of P38MAPK/NF-κB Signaling Transduction Pathway On MCP-1-induced Cell Proliferation And The ECM Secretion In Cultured Rat Mesangial Cells.

Chemokines is a kind of cytokines secreated by different sorts of cells,a low molecular weight(8～10kD).They attract their own different sub-white blood cells i.e,neutrophil,monocyte and lymph cells.Monocyte chemoattrant protein-1(MCP-1) belongs to sub-families of chemokines,secreted by inflammatory cells and renal intrinsic cells such as endothelial cells,mesangial cells,potocyte cells,tubular epithelial cells and interstitial cells under the condition of immune complex, toxin, hypooxygen, ischemia, et al.More chemokines lead to the infiltration of mono-macrophage in the renal tissue;More infiltrating white blood cells can produce a large of chemokines,forming positive feed-back,aggrate the inflammatory reaction.Persistent inflammatory response lead to the accumulation of extracellular matrix,renal fibrosis ,glomeruler sclerosis and the progress of chronic renal failure.Recently founded that:MCP-1 not only attract the mono-macrophage in the circulation to renal tubullar-interstitial,but also directly stimulate the renal intrisic cells,lead to the accumulation of extracellular matrix and renal fibrosis.In the normal condition, Mesangial cells play roles of sustaince.swallow and maintenance of metabolism,but in the pathogenisis of mesangial proliferative glomerulo nephritis,Mesangial cells not only are victims,but also actively involve in the process, they secret many inflammatory factors and extracellular matrix causing the renal fibrosis.But wheather MCP-1 can directly stimulate the mesangial cells' proliferation,lead to the secretion of extracellular matrix ?the relavent reseach paper not founded yet. Our experiments are:observing whether MCP-1 can directly stimulate the mesangial cells' proliferation; whether MCP-1 can directly promote the mesangial cells to secret extracellular matrix such as Fibronectin (FN), Collagen Type I (COL I ).To illuminate MCP-1's role in the pathogenisis of mesangial proliferativeglomerulonephritis, To explore the mechanism of mesangial proliferative giomerulonephritis from another new view,To deepen the recognition to mesangial proliferative giomerulonephritis.Objective To explore the effect of MCP-1 on cell proliferation and the gene,protein expression of FN,COL I in cultured Rat MCs.Methods1.to explore the growth rules of the cultured Rat mesangial cells in vitro;draw the cells growth curve.2.the toxicity test of MCP-1.3.the concentration-gradient test of MCP-1.4.different concentration of MCP-1(12.5,25,50,100ng/ml)stimulate the cultured mesangial cells,observe the proliferative activity in differrent time (12h,24h,48h)by MTT.5.to examine the mRNA expression of FN, COL I in differrent time (12h,24h,48h) in the normally cultured Rat MCs by Semi-quantitive RT-PCR.6.to examine the protein expression of FN,COL I in differrent time (12h,24h,48h) in the normally cultured Rat MCs by Western Blot.7.to observe the mRNA expression of FN and COL I in MCs stimulated by different concentrations of MCP-l(25,50,100ng/ml) in differrent time (12h,24h,48h) by Semi-quantitive RT-PCR.8. to observe the protein expression of FN and COL I in MCs stimulated by different concentration of MCP-l(25,50,100ng/ml) in differrent time (12h,24h,48h) by Western Blot.Results1.The Rat mesangial cells can grow well in the concentration ranging from 2.5 to20% FCS RPMI 1640 solution,Cells begin to seed to grow in 12 hours after passing generation, during the log growth period , cell quantity increased rapidly and their volume bloomed, cells showing circular or shuttle shape,fibre connect the cells. after 72hours,cells go into the platform period.2.Trypan blue exclusion test shows that:different concentrations of MCP-1 (3.125,6.25,12.5,25,50,100,200ng/ml) have .no toxic effect on the cultured Rat mesangial cells,cell survival rate is above 97% after 72 hours.3.The concentration-gradient test shows that:different concentration of MCP-1 (3.125,6.25,12.5,25,50,100,200ng/ml) stimulate the mesangial cells,only when the concentration is up to 12.5ng/ml,the cell proliferativc activity is showing with the prolonged cultured time(24h,48h).the lower concentration groups have no the effect.The proliferative activity of MCP-1 (100ng/ml) group has no statistical difference compared to MCP-1 (200ng/ml)group' in 48hour and 72hour(p>0.05).4.Different concentration of MCP-1 (12.5,25,50,100ng/ml) stimulate the cultured mesangial cells,when the concentration of MCP-1 is 12.5ng/ml,the proliferative activity in 12 hours has no statistical difference compared to the normal group(p>0.05).But with the prolonged cultured time(24h,48h),they showed obvious statistic difference(p<0.01). when the concentration of MCP-1 is or above 25ng/ml,the proliferative activity showed distinct statistical difference(p<0.01) in dose- and time-dependent manners.5.The effect of MCP-1 on the mRNA expression of FN, COL I in cultured mesangial cells. In normally cultured condition,there are a little mRNA expression of FN,COL I in mesangial cells.But with the higher concentration of MCP-1 and the longer incubated time, the mRNA expression of FN,COL I in cultured mesangial cells increased remarkedly compared to the normal group(p<0.01),and the difference between the different concentration groups is obvious in dose- and time-dependent manners (p<0.05).6. The effect of MCP-1 on the protein expression of FN,COL I in cultured mesangial cells. In normal cultured condition,there are tiny protein expression of FN,COL I in cultured mesangial cells. when putting in different concentration of MCP-1(25,50,100ng/ml),Only the protein contents of FN.COL1 of MCP-1(100ng/ml) group increased remarkedly compared to the normal group after incubated 24hours(p<0.01) .But with the prolonged incubated time,the protein secretion of FN,COL1 in different concentration increased obviously compared to the normal group(p<0.01).Moreover the differences between different concentration of MCP-1 groups are remarkable(p<0.05).Conclusion MCP-1 can promote the mesangial cells proliferation and up-regulate the mRNA,protein expression of FN,COL I in cultured Rat mesangial cells in vitro.The experiment result indicated that:MCP-1 play certain role in the pathogenisis of mesangial proliferative glomerulonephritis.We probed some new pathological mechanism of mesangial proliferative glomerulonephritis from another new view, deepened the recognition to the pathological mechanism of mesangial proliferative glomerulonephritis.P38MAPK is a family member of mitogen-activated protein kinases.They participate in the cell regulatory reaction stimulated by the outer space through the transmission of cellular signaling transduction.They are common early signaling pathways to regulate cell proliferation,differentiation and apoptosis mediated by many cytokines and inflammatory factors. P38MAPK make the substrate phosphoralated to realize the rapid signaling transduction by turning themselves non-phosphoralatory state into phosphoralatory state.The lower stream phosphoralated factors are ATF₂,c-jun,c-fos, AP-1 and NF- κ B et al.SB203580 is a kind of pyrimizolum inhibitor, It can specifically inhibit the P38MAPK activity through competing with ATP to combine the ATP-combining site. SB203580 is widely used because of its specifity,safety and efficiency.NF - κ B is a kind of very important nuclear transcriptional factors mediated the intracellular signaling transtuction.In normal condition, NF -κ B, a triploymers combining with P50 - P65- I κ B, exsists in the plasma in a non-active state.At present,we have founded that NF -κ B regulates over 100 target genes expression,such as cytokines,chemoattractant factors,growth factors,adhesive factors,fibronectin factors and collegen factors ,et al. they all participate in the host immune and inflammatory response.The activity of NF - κ B increased in glomerular and renal interstial in many experimental glomerulonephritis models i.e Anti-GBM nephritis, mesangialproliferative glomerulonephritis, immune-compound glomerulonephritis, et al.From Part I experimental results,We obviously concluded that:Different concentration of MCP-1(25, 50, 100ng/ml) stimulated the mesangial cells proliferation and secretion of FN,COL1 in dose- and time-dependent manners in the cultured RatMesangial Cells in vitro.But whether the p38MAPK/NF- k B signaling transduction pathway is their intracellular signaling transduction pathway in the above-mentioned process?This section we mainly probe the. regulatory effect of p38MAPK/NF- k B signaling transduction pathway on MCP-1-induced cell proliferation and the expression of FN.COL I in cultured Rat mesangial cells.Objective To investigate the regulatory effect of p38MAPK/NF- k B signaling transduction pathway on MCP-1-induced cell proliferation and the expression of FN, COL I in cultured Rat mesangial cells in vitro.Methods1.To measure the effect of different concentration of pure P38MAPK inhibitor SB203580(l, 5, 10μmol/L)on the cell proliferation activity of the normally cultured mesangial cells in different time(12h,24h,48h)by MTT.2. To measure the effect of MCP-1(100ng/ml), MCP-1(100ng/ml)+SB203580(1, 5, 10μmol/L) groups on the cell proliferation activity of cultured mesangial cells in different time(12h,24h,48h)by MTT.3. To measure the P38MAPK phosphoralatory level in the normally cultured Rat MCs in different time(15',30',60',120')by Western Blot;4. To measure the protein expression of NF - k BP65 in the normally cultured Rat MCs in different time(12h,24h,48h)by Western Blot;5. To measure the effect of different concentration of MCP-1(25, 50, 100ng/ml). MCP-1(100ng/ml)+SB203580(10μmol/L) groups on the P38MAPK phosphoralatory level in different time(15',30',60',120')by Western Blot;6. To measure the effect of different concentration of MCP-1(25, 50, 100ng/ml), MCP-1(100ng/ml)+SB203580(10μmol/L) groups on the protein expression of NF - k BP65 in different time(12h,24h,48h)by Western Blot;7. To measure the effect of different concentration of MCP-1(25, 50, 100ng/ml), MCP-1(100ng/ml)+SB203580(10μmol/L) groups on the mRNA expression of FN,COLI in different time(12h,24h,48h)by Semi-quantitive RT-PCR;8. To measure the effect of different concentration of MCP-1(25, 50, MCP-1(100ng/ml)+SB203580(10μmol/L) groups on the protein expression of FN,COL I in different time(12h,24h,48h) by Western Blot.Results1.Different concentrations of SB(1, 5, 10μmol/L)have no effect on cell proliferation in the normally cultured Rat MCs in different time (12h,24h,48h) (p>0.05); But the cell proliferation of MCP-l(100ng/ml)+SB203580(1, 5, 10μmol/L) groups are inhibited remarkedly than that of MCP-1(100ng/ml) group(p<0.01);There is no statistical difference between different concentration SB groups and different time(p>0.05);2. The normally cultured Rat MCs have basic P38MAPK phosphoralation in different time (15',30',60',120'), but there is no statistical difference between different time(p>0.05);3. The protein expression of NF - k BP65 is not examined in the normally cultured Rat MCs in different time;4. The effect of MCP-1 on the P38MAPK phosphoralatory level in the cultrued Rat MCs:the P38MAPK phosphoralatory level increased obviously compared to the normal group when the MCP-1 concentration is 25ng/ml in 15'(p<0.01), the P38MAPK phosphoralatory level is the toppest in 30',they went down a little in 60'and 120',but there are obvious statistical difference compared to the normal group(p<0.01),but there is no statistical difference between 60'group and 120'group. In other MCP-1 groups ,we also founded the same changing regulation.In total, the higher concentration of MCP-1, the higher P38MAPK phosphoralatory level . there are obvious statistical difference between different concentration in the same time(p<0.01);5.The effect of MCP-1 on the protein expression of NF- κ Bp65 in the Rat MCs: the protein expression of NF- κ Bp65 is no difference compared to the normal group when the MCP-1 concentration is 25ng/ml in 12 hours(p>0.05),but with the higher concentration and the longer incubated time, the protein expression of NF-κBp65 increased remarkedly and there are obvious statistical difference between different concentration and different time(p<0.01);6. The effect of SB on the P38MAPK phosphoralatory level in the cultrucd Rat MCs:the P38MAPK phosphoralatory level obviously inhibited in MCP-1(100ng/ml) plus SB(10μmol/L) group,there are remarkable statistical difference compared to that of MCP-l(100ng/ml) group(p<0.01);7.The effect of SB on the protein expression of NF- k Bp65 in the Rat MCs: the protein expression of NF- k Bp65 obviously inhibited in MCP-l(100ng/ml) plus SB(10μmol/L)group, there are obvious statistical difference compared to that of MCP-l(100ng/ml) group(p<0.01).8.The effect of SB on the mRNA expression of FN,COL I in MCs: There are a little mRNA expression of FN,COL I in the normally cultured MCs.MCP-1 can remarkedly upregulate the mRNA expression of FN,COL I ,but the mRNA expression of FN, COL I decreased obviously when putting in SB(10μmol/L) compared to that of MCP-l(100ng/ml)group(p<0.01).9. The effect of SB on the protein expression of FN,COL I in MCs: There are basic protein secretion of FN, COL I in the normally cultured MCs.MCP-1 can remarkedly promote the secreation of FN,COL I ,but the protein secreation of FN,COL I decreased obviously when putting in SB(10μmol/L) compared to that of MCP-l(100ng/ml)group(p<0.01).Conclusion Different concentration of MCP-1 can promote MCs proliferation, the P38MAPK phosphoralation and activate Nuclear Factor- κB. MCP-1 upregulate the mRNA and protein expression of FN,COL I in dose- and time-dependent manners in cultured Rat MCs in vitro.The above-mentioned processes can be blocked by specific P38MAPK blocker SB203580,the results indicated that P38MAPK signal transduction pathway played a regulatory effect on cell proliferation and the synthesis of FN,COL I mediated by MCP-1.In the experiment,we prelimilarily investigated the intracellularsignal transduction mechanism in the pathogenisis of mesangial proliferative glomerulonephritis induced by MCP-1.To provide a new possible therapeutic target in treating mesangial proliferative glomerulonephritis.