Astragaloside ? Reduces Renal Tubulointerstitial Fibrosis By Inhibiting Toll Like Receptor Pathway In Vivo And In Vitro

Objective:1.To study the variation of Toll like receptor pathway and terminal inflammatory cytokines on mice model of renal fibrosis induced by unilateral ureteral obstruction?UUO?,and to investigate the effect of Astragaloside ??AS-??on Toll like receptor pathway and its terminal inflammatory cytokines and the degree of renal fibrosis induced by UUO in vivo.2.To observe the effect of AS-? on variation of Toll like receptor pathway and inflammation activated by lipopolysaccharide?LPS?in human kidney tubular epithelial?HK-2?cells in vitro.Methods:1.Establishment of UUO model and experiment groups in vivo:Male C57BL/650 mice were divided into five groups randomly,namely,sham-operated group,model group and groups of AS-??low-dose,medium-dose and high-dose?.All mice were anesthetized by 1%pentobarbital sodium intraperitoneal injection.In the model group and groups of AS-?,the mice were done the left ureter ligation though the left abdominal longitudinal incision.And then the muscle layer and skin layer of mice were sutured.Except for left ureter ligation,named shame ligation,the mice in sham-operated group were done all surgery procedures.After operation,the mice were kept in clean level lab for 14 days.From the day of surgery,the mice in groups of AS-??low-dose,medium-dose and high-dose?were treated by gavage of AS-? for 14 days in dosage of 10mg/?kg·d?,30 mg/?kg·d?and 50 mg/?kg·d?separately.The mice in sham-operated group and model group were treated with double distilled water containing 1‰ethanol instead of AS-?.2.Detection of renal function,renal fibrosis degree,Toll like receptor pathway related molecules and terminal inflammatory cytokines of renal tissure in each group mice:in order to estimate renal function changes,serum creatinine?Scr?and blood urea nitrogen?BUN?were detected by chemical methods.To investigate the degree of renal injury and fibrosis,histopathological changes and collagen deposition of affected kidney were observed under optical microscope with hematoxylin-eosin?HE?and MASSON staining,meanwhile,the protein and mRNA expressions of?-smooth muscle actin??-SMA?and fibronectin?FN?were tested by the methods of immunohistochemistry,western blot and reverse transcription-PCR.In order to observe the changes of Toll like receptor pathway in UUO renal fibrosis and to explore the roles of AS-? in this process,the protein and mRNA expressions of Toll-like receptor 4?TLR4?,Toll-like receptor 2?TLR2?,myeloid differentiation factor 88?MyD88?,TNF receptor–associated factor 6?TRAF6?,Toll-like receptors associated molecule?TRAM?,TIR-domain-conyaining adapter-inducing interferon-??TRIF?,nuclear factor-?B?NF-?B?,interleukin-6?IL-6?,transforming growth factor-??TNF-??and interferon-??IFN-??were tested by immunohistochemistry,western blot and reverse transcription-PCR methods.3.Establishment of cell inflammation model induced by LPS in vitro and experiment groups:HK-2 Cells were cultured with basic culture medium?10%FBS,1%mycillin and DMEM medium with high glucose?in 37?,5%CO₂ cell culture box.All cells in experiments were transferred to culture with 50%cell density and cultured with basic culture medium for 24 hours.At that moment the cell density will be around 80%.And it's the right time to start the experiment.In order to make sure the best dose of LPS for the establishment of an inflammatory model in vitro,cells were cultured in complete culture medium?0.1%DMSO,10%FBS,1%mycillin and DMEM medium with high glucose?containing gradient concentrations of LPS in pore plates.Activity status of these cells were analyzed by cell counting kit-8?CCK-8?;and protein expression of inflammatory cytokines IFN-?of these cells was also detected by western blot methods.Then 1?g/ml was identified as the best dose of LPS for the establishment of an inflammatory model in vitro.All cells in experiments were divided into five groups,namely,control group,LPS group,AS-? low-dose group,AS-? high-dose group and TAK-242group.These cells in each group were cultured for 48 hours in their corresponding culture medium.These cells in control group were cultured with complete culture medium;LPS group with complete culture medium+LPS 1?g/ml;AS-? low-dose group with complete culture medium+LPS 1?g/ml+AS-? 50?g/ml;AS-? high-dose group with complete culture medium+LPS 1?g/ml+AS-? 100?g/ml;and TAK-242 group with complete culture medium+LPS 1?g/ml+TLR4 receptor inhibitor TAK-242 5?M.4.Detection of Toll like receptor pathway related molecules?TLR4,MyD88,TRAM?and IFN-?of HK-2 cells in each group:the protein and mRNA expressions of Toll like receptor pathway related molecules and the protein expression of terminal inflammatorycytokineweretestedby Westernblotandreverse transcription-PCR methods.Results:1.The effect of AS-? on renal function,left?simulated ligation?renal tissue structure and degree of fibrosis in UUO mice:The results of renal function measurement in mice showed that there was no statistically significant difference between Scr and BUN in each group?P>0.05,same as below?.In sham-operation group mice,the result of HE staining showed that the structure of left kidney was normal;the result of MASSON staining showed that the collagen fibers were in blue and very few area was collagen positive staining,which was mainly located around the tubular basement membrane,the renal tubules and renal interstitial;the results of protein and mRNA expressions showed that?-SMA and FN in protein and mRNA levels of left kidney were both expressed,while the positive stained area was in brown and mainly located in renal interstitial.In model group mice,the renal glomeruli were atrophied or even disappeared and the renal interstitium was widened in affected kidney.Compared with sham-operation group,the results on affected renal tissue of model group mice showed that the stained area of collagen fibers was more and the protein and mRNA expressions level of?-SMA and FN were higher,these differences were all statistically significant?P<0.05,same as below?.In groups of AS-?,histopathological changes with HE staining showed that there were different degrees of renal structure change in affected renal of mice;but compared with model group,the renal interstitial edema,glomerular atrophy and inflammatory cell infiltration were significantly reduced.In renal tissues of AS-? groups mice,the degree of collagen deposition in renal interstitial and the protein expression level of?-SMA and FN was lower than those in the model group.In the renal tissues of the medium-dose and high-dose AS-? groups mice,the mRNA expression of?-SMA and FN was less than those in model group.However there was no statistically significant difference in the mRNA expression of?-SMA and FN in renal tissue between low-dose AS-? group and model group mice.Compared with sham-operation group,the protein and mRNA expressions of?-SMA and FN in renal tissue of mice in low-dose AS-? group were higher,however,there were no statistical difference in those of medium-dose and high-dose AS-? groups.2.The effects of AS-? on Toll like receptor pathway related molecules and terminal inflammatory cytokines in the renal tissue of UUO mice:In sham-operation group,the protein and mRNA of Toll like receptor pathway related molecules were both expressed in the renal tissue of the mice.In renal tissue of model group mice,the protein and mRNA expressions of MyD88-dependent receptor signaling pathway molecules?TLR4,TLR2,MyD88,TRAF6,NF-?B?and MyD88-independent receptor signaling pathway molecules?TLR4,TRAM,TRIF,NF-?B?and the protein expression of terminal inflammatory cytokines?IL-6,TNF-?,IFN-??were significantly higher than those in sham-operation group.In renal tissue of high-dose AS-? group mice,the protein expression of Toll like receptor pathway related molecules was significantly less than those in model group,and the protein expressions of TLR4,TLR2,MyD88,TRAF6,TRAM and NF-?B had no significant difference with those in sham-operation group;meanwhile the mRNA expression of Toll like receptor pathway related molecules was significantly less than those in model group and had no significant difference with those in sham-operation group.In renal tissue of medium-dose AS-? group mice,the protein expression of Toll like receptor pathway related molecules was significantly less than those in model group,and the protein expression of TLR4,TLR2,TRAF6,TRAM and NF-?B had no statistical difference with those in sham-operation group;meanwhile the mRNA expression of Toll like receptor pathway related molecules including TLR4,TRAF6,TRAM,TRIF and NF-?B was significantly lower than those in model group,and the mRNA expression of Toll like receptor pathway related molecules had no statistical difference with sham-operation group.In renal tissue of low-dose AS-? group mice,the protein expression of Toll like receptor pathway related molecules also showed a decreasing trend in comparison with model group,the protein expression of TLR4,TLR2,MyD88and TRIF was significantly less than those in model group,but the protein expression of Toll like receptor pathway related molecules was higher than those in sham-operation group;meanwhile the mRNA expression of Toll like receptor pathway related molecules had no statistical difference with model group,and the mRNA expression of MyD88,TRAF6,TRAM,TRIF and NF-?B had no statistical difference with sham-operation group.In renal tissue of groups of AS-?,the protein expression of terminal inflammatory cytokines?IL-6,TNF-?,IFN-??was significantly less than those in model group;meanwhile compared with sham-operation group,the protein expression of terminal inflammatory cytokines in renal tissue of high-dose AS-? group had no statistical difference but those in medium-dose and low-dose AS-? groups were significantly higher.3.The variation of Toll like receptor pathway related molecules and terminal inflammatory cytokine in each group of HK-2 cells:In the cells of control group,the protein and mRNA of TLR4,MyD88,TRAM and the protein of IFN-?were both expressed.In the cells of LPS group,the protein and mRNA expressions of Toll like receptor pathway related molecules and the protein expression of IFN-?were significantly higher than those in control group.TAK-242 group was a positive control group.In the cells of TAK-242group,the protein and mRNA expressions of Toll like receptor pathway related molecules and the protein expression of IFN-?were significantly less in comparison with LPS group,and compared with control group,the protein and mRNA expressions of TLR4,MyD88 and the protein expression of IFN-?had no significant difference.Compared with LPS group,the protein and mRNA expressions of Toll like receptor pathway related molecules and the protein expression of IFN-?in the cells of AS-? high-dose group reduced significantly;meanwhile,those in the cells of AS-? low-dose group also showed a decreasing trend,including the protein expression of TRAM,IFN-?and the mRNA expression of all tested molecules were decreased significantly.Compared with control group,in the cells of AS-? high-dose group,the protein expression of TLR4,MyD88,IFN-?and the mRNA expression of Toll like receptor pathway related molecules had no significant difference;meanwhile the protein and mRNA expressions of all tested molecules in the cells of AS-? low-dose group had no significant difference either.Compared with TAK-242 group,the protein and mRNA expressions of Toll like receptor pathway related molecules and IFN-?in the cells of AS-? high-dose and low-dose groups had no significant difference.Conclusion:1.Toll like receptor pathway and inflammatory response induced though Toll like receptor pathway participated in the renal fibrosis process of UUO mice.2.AS-? can inhibit the expression of Toll like receptor pathway and the release of inflammatory cytokines and reduce renal interstitial fibrosis in the affected kidney of UUO mice.3.AS-? can inhibit the up-regulated of Toll like receptor pathway and the inflammatory response induced by LPS in HK-2 cells.4.In UUO mice,the mechanism of AS-? in reducing renal interstitial fibrosis may be that AS-? can inhibit the expression of Toll like receptor pathway and reduce inflammatory response.