| Objective:Hyperuricemia is an independent risk factor for the progression of chronic kidney disease,and is involved in the process of renal tubular injury and renal interstitial fibrosis.Prevention and treatment of renal interstitial fibrosis characterized by massive fibroblast activation and excessive extracellular matrix(ECM)deposition is the key way to treat renal diseases caused by hyperuricemia.As we known,the renal tubular epithelial to mesenchymal transition(EMT),production and accumulation of extracellular matrix components in renal interstitium,inflammatory reaction,endoplasmic reticulum stress,apoptosis of renal tubular epithelial cells are important mechanisms of renal interstitial fibrosis.The activation of SMAD3 and STAT3 signaling played central roles in the pathogenesis of renal fibrosis,which have been recognized as potential targets for antifibrotic therapy.At present,there are two main kinds of uric acid lowering drugs,one is uric acid excretion enhancer represented by benzbromarone,which hinders uric acid reabsorption and reduces serum uric acid level by inhibiting uric acid transporter URAT1,but in some countries,it withdrew from the market because of its hepatotoxicity and side effects.The other is the uric acid production inhibitor represented by allopurinol,which reduces the production of uric acid by inhibiting the activity of xanthine oxidoreductase(XOR).However,allopurinol can cause fatal allergic reactions,especially in Asian populations.Therefore,natural products may provide a new strategy for the treatment of hyperuricemia induced renal injury.As we known,the potential of natural products as the candidates for drug discovery has been well recognized.As an inhibitor of STAT3,the effect and mechanism of pectolinarigenin(PEC)on renal fibrosis are unclear.In this study,we explored the renal protective effect and mechanism of PEC by establishing mouse models of HN and unilateral ureteral obstruction(UUO)and by employing NRK-49 F fibroblasts stimulated by TGF-β1 in vitro,thus searching a new therapeutic strategy for the prevention and treatment of renal fibrosis induced by hyperuricemia.Materials and Methods:Chapter 1: the effect of PEC on renal fibrosis in mice with HNMale C57BL/6J mice(8-10 weeks of age weighing 20-25g)were randomized into five groups with eight mice in each group.(1)control group: mice were orally administered by physiological saline at a dose of 200μL once every other day.(2)model group: the mice model of HN was induced by gavage of a mixture of adenine(160mg/kg/d)and potassium oxonate(2400mg/kg/d)dissolved in 200μL physiological saline once every other day for 28 days.(3)allopurinol group: after 3 hours treated with adenine and potassium oxonate,the mice were orally administered by allopurinol(10mg/kg)dissolved in 20% PEG-400 and diluted with physiological saline.(4)PEC low dose group(25mg/kg): after 3 hours treated with adenine and potassium oxonate,the mice were orally administered by PEC(25mg/kg)dissolved in 20% PEG-400 and diluted with physiological saline.(5)PEC high dose group(50mg/kg): the mice were orally administered by PEC(50mg/kg)on the basis of model group.Mice were sacrificed after treatment with the above intervention measures,kidney and blood samples were collected for detection.The level of blood urea nitrogen(BUN),serum creatinine(Scr)and serum uric acid(UA)were measured by automatic biochemical analyzer.HE,PAS,Masson and Sirius red staining were performed to evaluate the renal pathological changes.Immunohistochemistry(IHC)and Western blotting(WB)were used to evaluate the expression of epithelial mesenchymal transition(EMT)-related fibrosis proteins,such as α-SMA,COL-1,Ecadherin,FN.The expression of TGFβ/Smad3 and JAK2/STAT3 signaling pathway related proteins and apoptosis related proteins,as well as the m RNA transcription levels of KIM-1,NGAL and inflammatory factors IL-1β,IL-6,TNF-α,MCP-1 and FABP4 in kidney tissue were detected by RT-PCR and WB.Chapter 2: the effect of PEC on renal fibrosis in mice with UUOMale C57BL/6J mice(8-10 weeks of age weighing 20-25g)were randomized into five groups with six mice in each group.The UUO mice model was established in UUO model groups and PEC groups.Briefly,the abdominal cavity was exposed via a midline incision and the left ureter was isolated and ligated while sham operation group was treated with only left ureteral ligation.All mice were given the follow treatment after operation.(1)Sham operation group(sham): mice were orally administered by physiological saline at a dose of 200 μL once a day,and the mice were sacrificed,the kidneys were collected at day 7 or 14 for analysis.(2)model 7 day group(Model 7d):mice were given orally administered by physiological saline at a dose of 200 μL once a day,and the mice were sacrificed at day 7s.(3)model 14 day group(Model 14d): mice were given orally administered by physiological saline at a dose of 200 μL once a day,and the mice were sacrificed at day 14 s.(4)PEC treatment for 7 days group(PEC for 7d): after the establishment of UUO model,PEC was dissolved in 20%PEG-400,diluted with physiological saline.The mice were orally administered with PEC(25mg/kg)once a day,and the mice were sacrificed at day 7s.(5)PEC treatment for 14 days group(PEC for 14d): after the establishment of UUO model,the mice were orally administered by PEC(25mg/kg)once a day,and the mice were sacrificed at day 14 s.PAS staining and Masson staining were used to evaluate the pathological changes of the kidneys.The expressions of α-SMA and pSTAT3(Tyr705)were observed by immunohistochemistry(IHC),and the expressions of α-SMA,COL-1 and FN were detected by Western blot(WB).The expression of TGFβ/Smad3 and JAK2/STAT3 signaling pathway related proteins were also detected by Western blot(WB).Chapter 3: the effect of PEC on renal fibrosis in vitroIn Vitro,HEK293 cells intergrated with SMAD-responsive elements were designed and cultured in MEM medium.The cells were divided into 6 groups: TGF-β1(20ng/ml)group and TGF-β1+PEC group.The cells were incubated with PEC at concentrations of 2 μM,5 μM,10 μM,20 μM and 50 μM,respectively.After 24 hours,HEK293 cells were collected for luciferase reporter gene detection to study the effect of PEC on Smad3 activation.Rat renal stromal fibroblasts(NRK-49F)were cultured in DMEM medium containing 10% FBS.After passage for 3-4 times,NRK-49 F cells were inoculated into 6-well plates.The logarithmic phase cells were randomly divided into three groups and treated accordingly:(1)control group: NRK-49 F cells were not treated with other treatments.(2)model group(TGF-β1): NRK-49 F cells were exposed to TGF-β1(5 ng / ml)for 24 hours.(3)Intervention group(TGF-β1+PEC): NRK-49 F cells were exposed to TGF-β1(5 ng/ml)and PEC 50 μM for 24 hours.The above groups were exposed to TGF-β1 and treated with PEC for 24 hours.PEC in cellular experiments was dissolved in DMSO.The protein expression and m RNA levels of α-SMA,COL-1 and FN,and the expression levels of Smad3 and STAT3 signaling pathway related proteins were determined by Western blot and PCR.Results:Chapter 1: the effect of PEC on renal fibrosis in mice with HNDifferential gene expression and bioinformatics analysis revealed the key genes and related pathways in the process of chronic renal injury induced by hyperuricemia.Compared with the control group,the HN model group showed significantly increase in BUN,Scr and UA(p<0.001),which suggested that the model was successfully established.However,the levels of BUN,Scr and UA in PEC high-dose group,lowdose group and allopurinol group were significantly decreased(p<0.01).The pathological examination by PAS staining of kidney showed that the HN mice developed glomerulosclerosis,focal infiltration of inflammatory cells and serious tubular injury with tubular dilatation,epithelial atrophy when compared to control mice.Tubular injury score showed that a mixture of adenine/potassium oxonate for 4 weeks induced obvious renal tubular injury in HN mice(p<0.0001).Treatment with PEC at the dose of 25mg/kg and 50mg/kg markedly improved renal pathomorphological damages(p<0.0001),and the therapeutic effect of PEC treatment group was even better than that of the positive control allopurinol group.Masson’s trichrome staining and Sirius red staining of kidney showed that the HN mice developed significant interstitial collagen deposition,whereas the fibrotic score was significantly attenuated in the mice treated with PEC(p< 0.0001).Interestingly,the low dose of PEC was superior to the high dose of that,which was consistent with the biochemical results.The m RNA expression levels of renal injury markers such as KIM-1 and NGAL were significantly up-regulated by RT-PCR in the model group,whereas significantly down-regulated in the PEC group.Immunohistochemistry and WB results showed that the protein expression levels of FN,COL-1 and α-SMA in the model group were higher than those in the control group(p<0.0001),and PEC could significantly suppressed the above protein expression.The m RNA expression of TNF-α,IL-6,IL-1 β and MCP-1 was detected by RT-PCR,which showed that was significantly increased in the model group than in the control group.The m RNA expression of these inflammatory factors was significantly decreased in the PEC treatment group.In the previous study of our group,it was found that fatty acid binding protein 4(FABP4)was associated with renal injury caused by hyperuricemia.To determine whether PEC exhibited reno-protective effect by targeting FABP4,we further evaluated FABP4 expression in the kidney tissues of HN by western blot,immunohistochemistry,and RT-PCR.The results showed that expressions of FABP4 in the kidneys of control group were very low,but the expressions of FABP4 m RNA and protein were obviously up-regulated in the HN model group(p<0.0001).After PEC intervention,the expressions of FABP4 in renal tubular were significantly downregulated(p<0.0001).These results suggest that hyperuricemia-induced FABP4 overexpression in tubular epithelial cells,and PEC can alleviate the renal pathological injury and protect the renal function of HN mice.In order to further explore the protective mechanisms of PEC on hyperuricemia induced renal fibrosis,we further detected the expressions of STAT3 and p-STAT3(Tyr705)in the renal tissue of mice by immunohistochemistry,and detected the expression levels of TGFβ/Smad3 and JAK2/STAT3 signal pathway corresponding proteins by Western blotting.The results showed that PEC significantly reduced the phosphorylated proteins level and inhibited the activation of the above signal pathways.In order to further explore whether PEC can alleviate renal tubular damage by regulating apoptosis,we detected the expression levels of Bax,Bcl-2 and cleaved caspase 3 by Western blotting.The results showed that the expressions of proapoptotic proteins Bax and caspase3 were up-regulated,and the expression of antiapoptotic protein Bcl-2 was down regulated in the model group;PEC could regulate the expression of these apoptosis related proteins.These results suggest that hyperuricemia can induce apoptosis of renal tubule cells in mice.The protective effect of PEC on renal fibrosis induced by hyperuricemia may be related to inhibition of related signaling pathways and regulation of apoptosis.Chapter 2: the effect of PEC on renal fibrosis in mice with UUOThe PAS staining showed that the kidneys of UUO mice exhibited the massive tubular degeneration,atrophy,and interstitial inflammatory cell infiltration,whereas they could not be detected in the contralateral kidneys of sham operation group.After treatment with PEC for 7 or 14 days after operation,the renal tubular injury score was significantly improved(p<0.0001).It can be observed that there was significant collagen deposition in the model group,but fibrogenesis could not be detected in the contralateral kidneys of sham group by staining with Masson.After treatment with PEC,either 7 days or 14 days after operation,the fibrotic score was also significantly attenuated in the obstructed kidneys of mice(p<0.0001).PEC treatment could effectively suppressed the expression of α-SMA in the renal interstitium of UUO mice by immunohistochemistry staining.Furthermore,the intervention with PEC significantly inhibited the upregulation of α-SMA,COL-1,and FN protein in obstructed kidneys of UUO mice at day 7 and day 14.These findings suggested that PEC treatment alleviated tubulointerstitial fibrosis in UUO mice.In order to further explore the mechanism of PEC in UUO mice,we first studied whether PEC affected TGFβ/SMADs signaling pathway in the kidney of UUO mice.Compared with sham operation group,the UUO surgery of vehicle remarkably induced the protein expression of TGF-β1,and SMAD3 phosphorylation.while treatment with PEC significantly inhibited the TGF-β1 and phosphorylated activation of Smad3,as well as Src at day 7 or day 14.In addition,we examined the effect of PEC on JAK2/STAT3 signaling pathway in UUO mice.The results showed that p-JAK2 and p-STAT3(Tyr705)were highly expressed in the obstructed kidneys at day 7 or 14 after operation.Treatment with PEC significantly inhibited the phosphorylated activation of JAK2/STAT3 signaling in the kidneys of UUO mice.In addition,significant nuclear staining of phosphorylated STAT3 was observed in tubule and interstitial cells of UUO mice,while PEC significantly inhibited the number of p-STAT3(Tyr705)positive cells in the obstructed kidneys.These above results indicate that renal protecting effect in UUO mice was involved in suppression of TGFβ/Smad3 and JAK2/STAT3 signaling pathways.Chapter 3: the effect of PEC on renal fibrosis in vitroTo examine the effect of PEC on myofibroblast activation and ECM production,NRK-49 F fibroblasts were treated with TGF-β1 and PEC(50μМ)for 24 h.It is as expected that TGF-β1 treatment notably induced the high expression of α-SMA,COL-1 and FN,conversely,PEC significantly suppressed the corresponding gene and protein levels of α-SMA,COL-1 and FN.These results indicated that PEC treatment could inhibit myofibroblast activation and ECM production by TGF-β1 stimulation in NRK-49 F fibroblasts.In order to investigate antifibrotic effects of PEC on the activation of SMAD3 in vitro,we firstly designed and used the SMAD binding element(SBE)reporterHEK293 cell line for detecting the activity of TGFβ/SMADs signaling.It was shown that TGF-β1(20 ng/ml)induced the higher TGFβ/SMADs luminescence compared to control cells and upregulated the phosphorylation of SMAD3 in NRK-49 F cells(p< 0.0001).The treatment with PEC at a concentration of 50 μM effectively suppressed the TGF-β1 protein expression(p<0.0001)and the SMAD3 phosphorylation(p< 0.0001),whereas there were no detectable changes of total SMAD3 protein.In addition,we found that the expression of p-JAK2(p<0.001)and p-STAT3(Tyr705)(p< 0.0001)were significantly increased in NRK-49 F cells stimulated by TGF-β1 and PEC suppressed the activation of JAK2/STAT3 signaling.These results indicate that PEC could significantly suppress myofibroblast activation and ECM production by inhibiting the activation of SMAD3 and STAT3 signaling pathways in TGF-β1-induced NRK-49 F fibroblast.Conclusion: In summary,our results demonstrated that PEC effectively inhibited myofibroblast activation and ECM production in TGF-β1-induced NRK-49 F cells and in mouse models of HN and UUO for the first time.Our findings also indicated that PEC alleviated renal interstitial fibrosis by inhibiting the activation of SMAD3 and STAT3 signaling pathways.It is suggested that PEC is a potential therapeutic strategy for renal fibrosis. |
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