Study On The Effect Of Dioscin On UNC93B1/TLR7/TLR9 Signaling Pathway During Silica-accelerating SLE

Objective:Silicosis is one of the most common and serious occupational diseases.Long-term inhalation of high concentrations of silica can cause silicosis.Studies have shown that the occurrence of silicosis could increase the incidence of autoimmune diseases such as systemic lupus erythematosus(SLE)and scleroderma(SSC).Among them,SLE is one of the most important autoimmune diseases caused by silica.Therefore,exploring the pathogenesis of systemic lupus erythematosus triggered by silica and looking for its effective treatment are urgent tasks at present.Studies have found that toll-like receptors(TLRs)are closely related to SLE,and many studies have also shown that UNC93B1 plays an important role in self-antigen activation of TLR7/TLR9 signaling pathway.Therefore,we used J774A.1 macrophages and MRL/lpr lupus-prone mice to investigate the effects of Dioscin on UNC93B1/TLR7/TLR9 signaling pathway to explore the pathogenesis of systemic lupus erythematosus caused by dust and the therapeutic effect of Dioscin on the disease with in vitro and in vivo studies.Methods:J774A.1 cells were used in vitro,and different concentrations of silica suspension were firstly administered.After stimulation for different time,the cell proliferation activity was determined by MTS method to determine the silica concentration and stimulation time for subsequent experiments;after determining the concentration and time,the cells were stimulated,then the protein and mRNA were extracted to detect the expression level of UNC93B1 by Western Blot and RT-PCR methods.To explore the effect of Dioscin on UNC93B1/TLR7/TLR9 signaling pathway activated by silica,we treated the cells with silica and Dioscin,then extracted protein and mRNA to detect the expression level of UNC93B1 by Western Blot and RT-PCR.We detected cytokines IL-6 and TNF-αby ELISA with the extracted cell supernatant.The effects of Dioscin on UNC93B1/TLR7/TLR9 signaling pathway activated by silica were detected by immunofluorescence method.In vivo experiments we treated MRL/lpr mice with silica suspension or saline by intratracheal injection to establish the silica exposure model or the control model.On the next day,Dioscin solution or sodium carboxymethylcellulose(CMC-Na)was gavaged as a administration group or a control group.They were totally divided into four groups:group A was the control group(Saline+CMC-Na),group B was the control administration group(Saline+Dioscin),group C was the silica exposure group(Silica+CMC-Na)and group D was the group was a silica administration group(Silica+Dioscin).The mice were sacrificed after 7,28 and 56 days of feeding,and the left kidney was paraffin-embedded for hexamine silver staining(PASM staining)and immunofluorescence,to detect the activation of UNC93B1/TLR7/TLR9 signaling pathway;protein and mRNA were extracted from the spleen and right kidney to detect the expression level of UNC93B1 by Western Blot and RT-PCR.Results:1.Silica exposure could increase the expression level of UNC93B1 in J774A.1 cellsIn vitro experiments we firstly stimulated macrophages with appropriate silica stimulation concentrations according to the MTS method.The silica stimulating concentrations of 0,10,25,50μg/cm~2 and the silica stimulating time point of 6,24,48hours were selected according to the test results.The cells were stimulated with 0,10,25,50μg/cm~2 suspension of silica for 6,24 and 48 hours,then the mRNA and protein expression of UNC93B1 was detected by RT-PCR and Western Blot.The results showed that at the mRNA and protein levels,the expression of UNC93B1 increased with the increasing silica concentration and extending time period,and there was statistical significance at 24 hours and 48 hours stimulation groups(p<0.05).2.Dioscin could reduce the expression of UNC93B1 in J774A.1 cells stimulated by silica.The results of Western Blot and RT-PCR showed that the expression of UNC93B1was significantly up-regulated after administration of 50μg/cm~2 silica suspension compared with the control group.Compared with the control group,the expression of UNC93B1 was significantly down-graduated with the treatment of increasing concentrations of the Dioscin and in some intervention groups the difference had statistically differences(p<0.05).3.Dioscin could reduce the increasing inflammation level of J774A.1 cellular inflammation caused by silica stimulation.The results showed that the secretion of IL-6 was significantly increased at all stimulating time points with administration of 50μg/cm~2 of silica suspension,and the secretion decreased at 24 and 48 hours after the intervention of Dioscin.The groups which were treated with 400 nM and 800 nM Dioscin were statistically significant compared to the silica-stimulated group.The results of TNF-αassay showed that the secretion of TNF-αincreased at 24 and 48 hours after stimulation with silica,and the secretion decreased after the addition of Dioscin.At 24 hours,the difference between the 400 nM,800 nM groups and the silica-stimulated group had statistical differences(p<0.05).4.Dioscin could inhibit the activation of UNC93B1/TLR9 pathway in J774A.1 cells caused by silica stimulation.We used immunofluorescence to double-stain the expression of UNC93B1/TLR7and UNC93B1/TLR9 in J774A.1 cells.The results showed that UNC93B1/TLR7 and UNC93B1/TLR9 were expressed in cells and the inhibition of Dioscin treatment was not obvious after the cells were treated with 50μg/cm~2 of silica suspension and different concentrations of Dioscin for 6 hours;after the cells were treated with 50μg/cm~2 silica suspension and different concentrations of Dioscin for 24 hours,the co-expression of UNC93B1/TLR7 and UNC93B1/TLR9 increased and its express position in cells were consistent.With the increase of Dioscin concentration,the co-expression of UNC93B1/TLR7 and UNC93B1/TLR9 decreased.After administration of 50μg/cm~2silica suspension and different concentrations of Dioscin for 48 hours,the co-expression of UNC93B1/TLR7 and UNC93B1/TLR9 showed significant increase.And with the increase of Dioscin concentration,the co-expression of UNC93B1/TLR7 and UNC93B1/TLR9 decreased.5.Dioscin could alleviate lupus nephritis caused by silica stimulation.The results of renal PASM staining showed that there was no obvious pathological change in the kidneys of the control groups and the control administration groups.Mice with bronchial perfusion of silica for 7 days showed no change in typical lupus nephritis,and only a few glomerulus showed slight basement membrane thickening;compared with the control group and the control administration group,the mice showed obvious thickening of the mesangium and basement membrane,enlargement of the capsular space of Bowman’s capsule,and partial glomerular rupture of the basement membrane after the mice were exposed to silica for 28 days.After treatment with Dioscin solution,the thickness of the basement membrane and mesangial thinned and the cystic cavity decreased.The mice of silica exposed group exposed to silica for 56 days showed obvious cellulose deposition and a large number of glomeruli showed SLE characteristic"crescent body".After treatment with Dioscin,the"crescent body"disappeared,the mesangium became thinner,and the lesions were alleviated.6.Dioscin could reduce the increasing expression of UNC93B1 in the spleen and kidney of lupus-prone mice induced by silica stimulation.We firstly used RT-PCR and Western Blot to detect the expression of UNC93B1in the kidney and spleen at each time point and each group.The results showed that the expression of UNC93B1 in the silica exposed groups increased at each time point compared with the control groups and the control administration groups,and this increase was statistically significance at 28 days and 56 days compared with the control groups(p<0.05).At the same time,after administration of Dioscin,the corresponding expression of UNC93B1 in the silica-administered group decreased,and it was partically has statistical significance compared with the silica-exposed group(p<0.05).The results of this experiment showed that silica could up-regulate the expression of UNC93B1 in kidney and spleen from mRNA and protein levels,and the treatment with Dioscin could reduce the activity of UNC93B1 signaling pathway.7.Dioscin could inhibit the activation of the UNC93B1/TLR7/TLR9 pathway in lupus-prone mice induced by silica stimulation.Double staining was performed on the paraffin sections UNC93B1/TLR7 and UNC93B1/TLR9 of renal tissue by fluorescence staining.The results showed that the bronchial exposure to silica in lupus-prone mice resulted in activation of the UNC93B1/TLR7 and UNC93B1/TLR9 signaling pathways,and the activity of this pathway could be reduced after treatment with Dioscin.Conclusion:1.Silica exposure could increase the expression level of UNC93B1 in J774A.1 cells.2.Dioscin could inhibit the activation of UNC93B1/TLR7/TLR9 signaling pathway and reduce the expression of pro-inflammatory factors in J774A.1 cells induced by silica.3.Dioscin could alleviate lupus nephritis caused by silica.4.Dioscin could reduce the expression of UNC93B1 in kidney and spleen of lupus-prone mice,and inhibit the activation of UNC93B1/TLR7/TLR9 signaling pathway in lupus-prone mice induced by silica.