Study On The Suppressive Of Ginsenoside Rh1 On The Inflammation And Apoptosis Of Human Salivary Gland Cells Induced By Interferon-γ

Background:Primary sjogren’s sydrom（pSS）is a common inflammatory autoimmune disease,mainly occurring in postmenopausal women.It is characterized by chronic inflammation and exocrine gland injury,resulting in reduced secretion of saliva and tears,greatly reducing the quality of life of patients.The pathogenesis of pSS is complex.Studies have shown that interferon-γ（IFN-γ）induced salivary gland cell inflammation and apoptosis damage is an important mechanism of salivary gland secretion function impairment,and the inflammation and apoptosis of salivary gland cells will further accelerate the pathological process of immune response.Therefore,finding drugs that can inhibit salivary gland cell apoptosis,reduce inflammatory response and promote fluid secretion is a research hotspot in the treatment of this disease.There is no effective treatment for pSS.Traditional Chinese medicine has attracted more and more attention due to its few side effects and remarkable therapeutic effect on pSS,and has become a research hotspot in the treatment of pSS at present.Ginsenoside Rh1（Rh1）has become a research hotspot in autoimmune diseases such as systemic lupus erythematosus,rheumatoid arthritis and scleroderma due to its anti-inflammatory,anti-allergic and immunomodulatory effects.However,the effect of Rh1 on pSS is rarely reported.Objective:The anti-pSS targets of Ginsenoside Rh1 were predicted by network pharmacology,and the possible mechanism of action was discussed.In order to investigate the effects of Ginsenoside Rh1 on IFN-γ-induced inflammation,apoptosis and aquaporin 5（AQP5）expression of Human Salivary Gland Cells（HSGs）,an in vitro disease model of pSS was established.To study the potential mechanism of Rhl in treating pSS.Methods:1.Potential therapeutic targets of Rhl against pSS were screened through network pharmacology,and its potential mechanism of action was analyzed through gene body（GO）enrichment analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis.2.Screening the concentration of Rh1 that can effectively promote the proliferation of HSGs.HSGs was divided into blank group and Rhl group（the concentration was 1 μM,10 μM,50 μM and 100 μM,respectively）.The cell proliferation rate of each group was detected by CCK-8 method 24 h,48 h and 72 h after administration.3.To study the effect of Ginsenoside Rh1 on the expression of HSGs proinflammatory cytokines.HSGs was treated with IFN-γ（10 ng/ml）for 12 h to induce inflammatory injury model of pSS cells in vitro.The cells were divided into four groups,blank group;IFN-γ group;IFN-γ+Rh1 group;Rh1 group.Real-time PCR（qRT-PCR）was used to detect Interleukin 6（IL-6）,Interleukin 1β（IL-1β）,Tumor Necrosis Factorα（TNF-α）gene expression.4.To study the effects of Rh1 on PI3K/Akt signaling pathway and apoptosis of IFN-γ-induced HSGs.IFN-γ was used to induce HSGs to form pSS inflammatory injury model in vitro.The experimental groups were as follows:blank group;IFN-γgroup;IFN-γ+Rh1 group;Inhibitor group.Western blot was used to detect the protein expression of PI3K/Akt signaling pathway related proteins PI3K,p-PI3K,Akt and pAkt.The apoptosis rate of each group was detected by flow cytometry.Western blot analysis of apoptosis-related protein Caspase-3 and Bcl-xL protein expression levels,to explore the effect of Rh1 on PI3K/Akt signaling pathway and apoptosis and the relationship between the two.5.Effects of Rh1 on IFN-γ-induced expression of HSGs AQP5:The cells were divided into blank group,IFN-γ group,IFN-γ+Rh1 group and Rh1 group.The expression levels of AQP5 gene and protein were detected by qRT-PCR and Western blot assay,and the effects of Rh1 on their expression were analyzed.Results:1.Network pharmacology studies have shown that Rh1 can exert anti-pSS effect through regulating inflammation and apoptosis,and its effect on apoptosis may be realized through regulating PI3K/Akt signaling pathway.2.CCK-8 test results showed that after administration for 24 h,48 h and 72 h,the administration group showed a trend of promoting the proliferation of HSGs compared with the control group.Among them,the 10 μM group had the most obvious promoting effect on HSGs proliferation compared with other groups.Therefore,the concentration selected for subsequent experiments was 10 uM.3.qRT-PCR results showed that the expression of IL-6,IL-1β and TNF-α related genes of HSGs inflammation were increased after IFN-γ induction.Compared with IFN-γ group,IFN-γ+Rhl group decreased the expression of IL-6,IL-1β and TNF-α4.Western blot results showed that p-PI3K and p-Akt protein levels were significantly down-regulated after IFN-γ induction,p-PI3K and p-Akt protein levels were significantly increased in IFN-γ+Rh1 group,which could be reversed by LY294002,suggesting that Rh1 is associated with PI3K/Akt signaling pathway.5.Flow cytometry results showed that the apoptosis rate of HSGs was significantly increased after IFN-γ induction,and decreased in IFN-γ+Rh1 group,indicating that Rh1 has inhibitory effect on IFN-γ induced apoptosis of HSGs.Meanwhile,Western blot was used to detect the expression levels of apoptosis-related proteins Caspase-3 and Bcl-xL,and it was found that the expression of Caspase-3 increased in IFN-γ group,while Rh1 intervention on this basis decreased the expression of Caspase-3 and increased the expression of Bcl-xL.This effect can be reversed by LY294002.6.qRT-PCR and Western blot showed that Rh1 promoted gene transcription and protein expression of AQP5 in HSGs cells.Conclusion:1.Network pharmacology studies proved that Rh1 may exert anti-PSS effect by affecting PI3K/Akt signaling pathway in apoptotic signaling pathway.2.Rh1 can inhibit IFN-γ-induced inflammation of HSGs by down-regulating the gene expression of IL-6,IL-1β and TNF-α.3.Rh1 inhibits IFN-γ-induced apoptosis of HSGs by activating PI3K/Akt signaling pathway.4.Rh1 can promote the expression of AQP5.