| Background and Objective: Gout is a series of clinical syndrome caused by the deposition of monosodium urate(MSU)crystals in articular joints and periarticular tissues or organs,including hyperuricemia,acute and chronic gouty arthritis,gouty nephropathy and nephrolithiasis.Recent years,the incidence and prevalence of gout is ascending in worldwide.Age of onset of gout was a younger trend.Epidemiological studies have suggested that the prevalence of gout was 1.4% in Europe,while that in China was 1% ~ 3%.Hyperuricemia was major biochemical basis and direct cause of gout attack.Studies have demonstrated that innate immunity is invovled in the MSU crystals triggering gouty inflammation.Innate immune cellular recognized MSU crystals by specific TLRs and activation NF-?B signaling pathway to induce multiple mediators of inflammation.A research found that in the absence of SOCS1,NF-?B phosphorylation was prolonged in response to LPS,which suggests SOCS1 could inhibit NF-?B signaling.A signicant characteristic of acute gouty arthritis is self-remission approximately 7-10 days after acute attack of gout,which is a distinguishing feature from other auto-inflammatory or autoimmune diseases,but the precise molecular mechnisms of self-remission and recurrence of acute attack are still unclear.Micro RNAs(mi RNAs)are a class of endogenous non-coding single-strand small RNAs of 18 to 25 nucleotides in length,which bind to the 3'-UTR of target m RNAs and form RNA-induced silencing complex(RISC)and invovle in the regulation of gene expressions by represses translation and/or degrade the target m RNAs.The regulation role of mi RNAs in the inflammatory response has been widely concerned.In recent years,several studies have found that mi RNAs play an important role in the pathogenesis.Previous studies of our team found that the level of mi R-150 was significantly increased in wild-type mouse bone marrow induced macrophages after MSU challenge,suggesting that mi R-150 could involve in MSU-induced inflammatory response.Gout belongs to arthromyodynia category in Traditional Chinese Medicine,which is caused by the abnormal function of spleen and stomach.The dampness-heat stasis and intermingled phlegm and blood stasis are the most common during the acute attack of gout.The patients with gout are treated using clearing heat and removing dampness detoxification prescription.The polygonum cuspidatum has the effect of clearing heat and reducing dampness.In recent years,the anti-inflammatory effects of resveratrol in regulating cellular signaling pathways and mi RNAs have become a research focus.Studies have shown that the anti-inflammatory effects of resveratrol by inhibit the activation of NF-?B and regulate regulate the TLR4 signaling pathway and inhibit the secretion of cytokines by macrophages,but the precise molecular mechnisms remains unclarified.In the study,we explore the role of mi R-150 and SOCS1 in gout inflammation,the potential regulatory mechanism of mi R-150 and the effect of resveratrol in gout inflammation.Materies and methods: 1.The peripheral venous blood samples of 60 cases of acute gout(AG)and 60 intercritical gout(IG)patients and 48 cases of healthy control(HC)for health examination were enrolled in this research and whose clinical and laboratory data were collected and blood samples as well.The level of mi R-150 was detected by RT-q PCR.The level of SOCS1,STAT3,NF-?B m RNA and protein were detected by RT-q PCR and western blotting.The correlation between mi R-150 and laboratory data was analyzed by Spearman.2.The target m RNA of mi R-150 in THP-1 cells were analyzed and identified by bioinformatics(1)Target genes associated with inflammation of mi R-150 were screened by bioinformatics online software,including RNAhybrid 2.2,Target Scan and mi Rbase and review related literature.(2)The targets dual luciferase reporter vector was constructed and co-transfected into 293 T cells with mi R-150 mimics(overexpression).Whether mi R-150 bind to the predicted site in the 3'-UTR region of the target gene were observe.(3)THP-1 cells were transfected with mi R-150 mimics(overexpression of mi R-150)and mi R-150 inhibitor(inhibition of mi R-150 expression).The level of mir-150 and SOCS1 m RNA were detected by RT-q PCR and the level of SOCS1 protein were detected by Western blotting.3.The role of mi R-150 in THP-1 macrophage inflammation after MSU challenge.(1)THP-1 cells were transfected with mi R-150 mimic/inhibitor for 48 h,and then were induced with 100ng/ml PMA for 24 h before MSU stimulation 6h.The treated cells and supernatants were collected.(2)The apoptosis were detected by Flow cytometry after(1)treatment.The levels of mi R-150 and SOCS1 m RNA were detected by RT-q PCR.The levels of SOCS1,p-STAT3 and p-NF-?B p65 protein were detected by Western blot.The levels of IL-1?,TNF-? and IL-6 protein were measured by ELISA in supernatant.4.The role of resveratrol and SOCS1 si RNA in THP-1 macrophage inflammation after MSU challenge.(1)THP-1 cells were trasfected with SOCS1 si RNA for 48 h,and then were induced with 100ng/ml PMA for 24 h,100?M resveratrol conbination with 100?g/ml MSU stimulate THP-1 macrophage cells 6h,the treated cells and supernatum were collected.(2)The apoptosis was detect using flow cytometry after(1)treatment.The level of SOCS1 m RNA was detected by RT-q PCR.The levels of SOCS1,p-STAT3 and p-NF-?B p65 protein were detected by Western blot.In cell culture supernatant,the protein levels of interlukin-1?,tumor necrosis factor-? and interlukin-6 was detected using ELISA.5.The SPSS 13.0 software and Graph-Pad Prism5.0 software were used to analyze the data.The data were presented as the means ± SD.Numerical variables between the two groups were tested using an unpaired t-test.Multiple comparisons were performed using one-way analysis of variance(ANOVA)in combination with the Bonferroni posttest.Spearman was used for correlation analysis.Differences were considered significant at P< 0.05.Results: 1.Compared with HC group,with exception of increased inflammatory biomarkers,patients with gout were companied by metabolic disorders including high body mass index(BMI),hyperlipidemia,hyperglycemia and hypertension(P<0.01,P<0.05,respectively).2.The level of mi R-150 in AG group was significantly lower than that in IG group and HC group in PBMCs(P<0.05),there was no significant difference between IG group and HC group(P>0.05)3.SOCS1 was target gene of mi R-150 was predicted by Bioinformatics online software.The binding of mi R-150 to the predicted binding site onSOCS1 m RNA was confirmed by the construction of a dual-luciferase reporter vector containing the target of mi R-150.Compared with the Blank group,mi R-150 was significantly upregulated in mi R-150 mimic(P<0.01),and mi R-150 was significantly decreased in mi R-150 inhibitor(P<0.01).Compared with the Blank group,the levels of SOCS1 m RNA and protein significantly decreased in mi R-150 mimic(P<0.01,respectively),and the levels of SOCS1 m RNA and protein in mi R-150 inhibitor significantly increased(P<0.01,respectively).4.The role of mi R-150 in THP-1 macrophage inflammation after MSU challenge.(1)The level of mi R-150 was significantly increased in THP-1 cells after MSU challenge by RT-q PCR(P<0.01).The m RNA and protein levels of SOCS1 were significantly reduced in MSU-induced inflammation(P<0.05,respectively).The levels of IL-1?,TNF-? and IL-6 significantly increased in supernatant(P<0.01,respectively).(2)After mi R-150 mimic combination MSU treatment,we observed that the levels of SOCS1 m RNA and protein were significantly down-regulated by RT-q PCR and Western blot(P<0.01,respectively),the levels of p-NF-?B and p-STAT3 were significantly increased(P<0.05,respectively)and the levels of IL-1??TNF-??IL-6 were significantly increased by ELISA(all P<0.01,respectively).After mi R-150 inhibitor combination MSU treatment,we found that the m RNA and protein levels of SOCS1 were significantly increased(P<0.01,respectively),the levels of p-NF-?B and p-STAT3 were significantly reducd(P<0.05,respectively)and the levels of IL-1??TNF-? and IL-6 were significantly decreased by ELISA(P<0.01,respectively).(3)Compared with HC group,the levels of SOCS1 m RNA and protein in AG and IG group were significantly lower in PBMCs(P<0.01,P<0.05,respectively),and those in AG group were significantly lower than those in IG group(P<0.05).The levels of STAT3 and NF-?B p65 m RNA in AG group were significantly higher than those in IG group and HC group(P<0.05,P<0.01,respectively),and the levels of p-STAT3 and p-NF-?B p65 protein in AG group were significantly higher than those in IG group and HC group(P value <0.01,respectively).5.The cell activity was greater than 95% and apoptosis rate was less than 5% after MSU,MSU combination differents concetration resveratrol,MSU combination with SOCS1 si RNA,MSU combination with SOCS1 si RNA and resveratrol treatment by Flow cytometry,indicating that those treatment had no significant effect on cell activity(P value >0.05,respectively).6.After 100?g/ml MSU combination with different concentrations resveratrol treatment(0,25,50,100?mol/L),compared with MSU group,we found that there were no difference in mi R-150 in differents Res.treatment group(P>0.05),and the m RNA and protein levels of SOCS1 were significantly increased in a dose dependent manner in MSU combination Res(50?mol/L and 100?mol/L)group(P<0.05,respectively).we also found that the levels of NF-?B p65 m RNA and p-NF-?B p65 protein in MSU combination Res(50?mol/L and 100?mol/L)group were significantly lower than that in MSU group(P <0.05,P <0.01,respectively).7.After 100?g/ml MSU combination with different concentrations resveratrol treatment THP-1 cells(0,25,50,100?mol/L),compared with MSU group,we found that the levels of IL-1??TNF-??IL-6 proteins were significantly reduced in a dose dependent manner in MSU combination Res(50?mol/L and 100?mol/L)group(P <0.01,respectively).8.After MSU combination with SOCS1 si RNA treatment THP-1 cells,compared with MSU group,we foun that the m RNA and protein levels of SOCS1 decreased significantly(P <0.01,respectively),whereas the protein expression levels of p-NF-?B p65 and p-STAT3 increased significantly(P <0.01,respectively).9.Compared with MSU combination with SOCS1 si RNA group,the levels of SOCS1 m RNA and protein were significantly increased in SOCS1 si RNA+Res100+MSU group(P <0.01,respectively),the levels of p-NF-?B p65 and p-STAT3 in THP-1cells(P <0.01,respectively)and the levels of IL-1??TNF-? and IL-6 in supernatant were significantly decreased(P <0.01,respectively).Compared with Res100+MSU group, we found that the m RNA and protein levels of SOCS1 were significantly decreased in SOCS1 si RNA group(P<0.05,respectively),whereas the protein levels of p-NF-?B p65 and p-STAT3 in THP-1cells(P<0.05,respectively)and the levels of IL-1??TNF-? and IL-6 were remarkablely increased in supernatant(P<0.01,respectively).Those results suggested that deletion of SOCS-1 expression by si RNA reversed the inhibition of STAT3 and NF-?B activation and proinflammatory cytokines secretion by resveratrol.Conclusion: 1.With the exception of increased inflammatory biomarkers,patients with gout were companied by metabolic disorders such as high body mass index(BMI),hyperglycemia,hyperlipidemia and hypertension.2.Abnormal expressions of mi R-150 in MSU-induced macrophage inflammatory response and in PBMCs of gout patients,suggesting that they might be involved in the pathogenesis of gout.3.It was predicted and verified that the SOCS1 was target gene of mi R-150.The levels of SOCS1 m RNA and protein remarkly decreased after transfection mi R-150 mimics in THP-1,indicating that mi R-150 might play an inhibition role by degrading SOCS1 m RNA.4.In patients with gout and MSU-induced inflammatory response,the levels of SOCS1 were significantly reduced,and the levels of NF-?B P65 and STAT3 m RNA and p-NF-?B P65 and p-STAT3 protein were significantly increased,suggesting that SOCS1?NF-?B P65 and STAT3 were closely associated with pathological of gout.5.The levels of SOCS1 were down-regulated by mi R-150,thereby promoting the activation of NF-?B and STAT3 signaling pathway,increasing the secretion of inflammatory factors,and thus aggravating gout inflammatory response.6.The levels of SOCS1 were upregulated by resveratrol in THP-1 macrophage in MSU-induced inflammation,resveratrol reduced the levels of p-NF-?B p65 and p-STAT3,as well as the inflammatory factors,including IL-1?,TNF-? and IL-6.Deletion of SOCS-1 expression by si RNA reversed the inhibition of STAT3 and NF-?B activation and proinflammatory cytokines secretion by resveratrol.It is indicating that the levels of SOCS1 were upregulated by resveratrol,inhibits NF-?B and STAT3 activation and ameliorates MSU-induced inflammatory response. |
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