| Backgroud and ObjectiveGout is a series of clincal syndrome casued by deposition of uric acid or monosodium urate (MSU) crystal into tissue or organ, including hyperuricemia, gouty arthritis, urate nephropathy and nephrolithiasis. The prevalence of gout is ascending trend all around the world. Epidemiological studies show that prevalence of gout is 1.4% in European, and 1.14% in the Shandong coastal cities of Eastern China. A signicant self-remission characteristic of acute gouty arthritis is approximately one week after acute attack of gout, which is a distinguishing feature from other arthropathy or auto-inflammatory diseases, but the precise molecular mechnisms of self remission and recurrence of acute attack are not illuminated yet. Studies have shown that innate immunity is involved in the MSU crystals triggering gouty inflammation. NLRP3, which is one member of NOD-like receptors (NLRs) family, could recognize MSU and activate NLRP3 inflammasome to release abundant pro-inflammatory cytokine IL-1?. In vitro, MSU irritates the NLRP3 inflammasome activity and increases autophagy activity as well. Autophagy can directly or indrectly regulate NLRP3 inflammasome negatively to dampen IL-1? production. It suggested that autophagy could participate in regulation of gouty inflammation. The traditional Chinese medicine (TCM) considered that the pathogenesis of gout is caused by dysfunction of spleen and stomach. The most common syndrome of TCM in acute attack of gout is accumulation of dampness and heat, and the treatment is primarily cleaning heat,eliminating dampness and detoxification. resveratrol has the effect of cleaning heat, eliminating dampness and strengthens the functions of spleen and stomach. Studies have shown that resveratrol has a good anti-inflammatory effect on collagen induced arthritis and osteoarthritis in rats. resveratrol serves as natural agonist of Sirtuin 1 (SIRT1) can irratiate adenosine monophosphate activated protein kinase (AMPK)/SIRT1 signalling pathway to induce autophagy, but the mechnism of that is not clarifed yet. In this research, changes of autophagy in patients with gout, autophagy regulating NLRP3 inflammasome activity and resveratrol regulating the level of autophagy are investigated to explore the regulatory role of autophagy in gouty inflammation.Materials and Methods1.48 sample of acute gout (AG) and 40 intercritical gout (IG) patients and 80 sample of healthy control (HC) for health examination were enrolled in this research and whose clnical and laboratory data were collected and blood samples as well.6 samples of IG patients were used to stimulate with MSU, co-stimulate with MSU and autophagy regulator, co-stimulate with MSU and resveratrol for functional study.2. The ratio of live cells in peripheral blood of IG patients with MSU stimulation, MSU and autophagy regulator co-stimulation, MSU and resveratrol co-stimulation were detected by flow cytometer. The real-time quantitative PCR (RT-qPCR) and western blot were used to detect the gene expression and protein levels of beclin-1? LC3?NLRP3?ASC?SIRT1?IL-1? in peripheral blood mononuclear cell (PBMC). IL-1? concentrations in plasma of IG patients with different stimulatory factors were tested by enzyme-linked immuno sorbent assay (ELISA).3. Statistical analysis was performed using SPSS 13.0 and GraphPad Prism 6 statistic analysis software. Data were displayed as median (quartile range) or meanąstandard deviation. analysis of variance and unpair student's t-test were used for statistical analysis. and P<0.05 is defined as statistical significance.Results1. In comparison with HC, with the exception of increased inflammatory cells, gout patients were companied with metabolic disorders such as high body mass index(BMI), hypertension, hyperglycemia, hyperlipidemia (P<0.05 or 0.01, respectively).2. In comparison with HC, beclin-1 mRNA expression and protein levels in PBMC of AG and IG patients are lower(P<00.01, respectively), but the mRNA expression of LC3 in AG and IG patients are higher (P<0.01, respectively), LC3-II protein levels in AG patients increase compared to IG patients and HC (P<0.01, respectively), between IG patients and HC have no significant difference(P>0.05).3. The protein levels of Beclin-1 increased in PBMC of IG patients with MSU stimulation in 1,3 hours (P<0.01, respectively) compared to non-stimulated MSU, but beclin-1 decreased in 6 hours (P<0.01) compared to non-stimulated MSU.4. In comparison of placebo (DMSO, PBS), the levels of LC3-? protein in MSU stimulation, co-stimulation of MSU and autophagy agonist (rapamycin), co-stimulation of MSU and autophagy antagonist [3-methyladenine (3-MA), Bafilomycin A1(Baf A1), E64d+pepsin A(pep A)] of IG patients increased (P<0.01, respectively), the levels of beclin-1 protein in MSU stimulation, co-stimulation of MSU and rapamycin decreased (P<0.01, respectively), but the levels of beclin-1 proteins in co-stimulation of MSU and autophagy antagonist (3-MA?Baf A1?E64d+pep A) increased compared to MSU stimulation, co-stimulation of MSU and rapamycin (P<0.01, respectively). However, in the HC, beclin-1 protein levels in MSU stimulation, co-stimulation of MSU and rapamycin were no significant difference compared to placebo (DMSO, PBS) (P<0.01, respectively), the levels of beclin-1 protein in co-stimulation of MSU and autophagy antagonist (3-MA?Baf A1?E64d+pep A) decreased compared to placebo (DMSO, PBS), MSU stimulation, co-stimulation of MSU and rapamycin (P<0.01, respectively).5. The mRNA expression of NLRP3 in AG patients is lower than HC (P<0.05), but there is no significant different between AG and IG, IG and HC. However, expression of ASC mRNA increased more than IG?HC(P<0.01,0.05; respectively), between IG patients and HC have no significant difference(P>0.05). In comparison with HC, the NLRP3 and ASC protein levels decreased significantly in AG?IG patients (P<0.01, respectively). Morever, ASC protein levels in IG patients were lower than AG(P<0.05). Additionally, the levels of ASC isoform (ASC-b) protein in AG patients increased significantly compared to IG, HC (P<0.01, respectively), and ASC-b in IG patients is lower than HC(P<0.05).6. ASC protein levels decreased significantly in IG patients with MSU stimulation, co-stimulation of MSU and rapamycin compared to placebo (DMSO) (P<0.01, respectively), and ASC protein levels in co-stimulation of MSU and rapamycin were lower than MSU stimulation (P<0.05), however, ASC protein levels in co-stimulation of MSU and autophagy antagonist (3-MA?Baf A1?E64d+pep A) increased compared to placebo (DMSO), MSU stimulation, co-stimulation of MSU and rapamycin (P<0.01, respectively), there were no significant difference among 3-MA?Baf A1 and E64d+pep A.7. The protein levels of caspase-1 p20 were not detected in placebo (DMSO), the levels of caspase-1 p20 protein in PBMC of IG patients with MSU stimulation increased significantly compared to placebo (P<0.01), however, caspase-1 p20 levels in co-stimulation of MSU with rapamycin were lower than MSU stimulation (P<0.01). In comparison of placebo (DMSO) in the plasma, IL-1? levels of IG patients with MSU stimulation and co-stimulation of MSU with rapamycin were higher, however, co-stimulation of MSU with rapamycin were lower compared with MSU stimulaltion (P<0.01). morever, IL-1? levels increased in co-stimulaiton of MSU with and autophagy antagonist (3-MA?Baf A1?E64d+pep A) compared to placebo and co-stimulation of MSU with rapamycin (P<0.01, respectively).8. The mRNA expression of SIRT1 in AG and IG patients is lower than HC (P<0.05), and in comparison of IG patients, the expression of SIRT1 mRNA in AG patients decline, but had no significant difference (P>0.05). The protein levels of SIRT1 in AG and IG patients decreased significantly compared to HC (P<0.01,0.05; respectively), and SIRT1 protien levels in AG patients decreased than IG patients (P<0.01). In IG patients with co-stimulation of MSU with different concentration of resveratrol, the SIRT1 protein levels in MSU with 200 ?g/ml and 400 ?g/ml resveratrol increased markedly than MSU stimulation (P<0.01, respectively), althrough SIRT1 in co-stimulation of MSU with 100?g/ml resveratrol increased, but did not different (P>0.05).9. The beclin-1 and LC3 mRNA expression in IG patients with MSU stimulation increased dramatically compared to placebo. In comparison of MSU stimulation, beclin-1 mRNA expression decreased significantly in co-stimulation of MSU with 200?g/ml and 400?g/ml resveratrol(P<0.01, respectively), however, the high levels of LC3 mRNA expression had no change. Beclin-1 protein levels in co-stimulation of MSU with100?g/ml and 200?g/ml resveratrol increased markedly compared to placebo and MSU stimulation (P<0.01, respectively), but not MSU with 400?g/ml resveratrol (P>0.05). The LC3-II protein levels in MSU and co-stimulation of MSU with different concentration of resveratrol increased significantly than placebo (P<0.01, respectively) and the LC3-II protein levels were negtively correlated with increasing concentration of resveratrol.10. In comparison of MSU stimulation, the protein levels of IL-1 P p31 in co-stimulation of MSU with 100?g/ml and 200?g/ml resveratrol increased (P<0.01, respectively), but 400?g/ml resveratrol decreased (P<0.05). However, levels of IL-1? p17 protein in co-stimulation of MSU with 200?g/ml and 400?g/ml resveratrol decreased (P<0.01, respectively). Morever, IL-1? levels in plasma were detected, IL-1? levels in MSU stimulation increased markedly compared to placebo(P<0.01). IL-1? levels in co-stimulation of MSU with 200 ?g/ml and 400? g/ml resveratrol increased compared to MSU stimulation (P<0.01, respectively).Conclusions1. with the exception of increased inflammatory cells, gout patients were companied by metabolic disorders such as high body mass index (BMI), hypertension, hyperglycemia, hyperlipidemia.2. Autophagy increased in acute gout patient. Autophagy was involved in pathogenesis of gouty inflammation.3. In gout patients, autophagy influx increased, and autophagy signalling pathway was not blockaded, both beclin-1 and LC3-? were degraded through autophagosome pathway.4. Autophagy regulated NLRP3 inflammasome function negtively to dampen gouty inflammatory response. It may be involved in self-remission of gouty inflammation.5. Resveratrol can enhance SIRT1 levels in gout patients. It suggested that SIRT1 serves as a target of resveratrol dampened gouty inflammatory response.6. Resveratrol enhanced autophagy and dampened IL-1? production. It suggested that resveratrol increased SIRT1 levels to regulate autophagy positively and then dampen gouty inflammatory response. |
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