Study On The Mechanism Of "Sanshenyin" In The Treatment Of Primary Sjogren’s Syndrome Through Regulating P2X7/NLRP3 Inflammasome Pathway

Objectives:To observe the effect of Sanshen Yin(SSY)and Ruscogenin on P2X7/NLRP3 signaling pathway,and to preliminarily explore the immune mechanism of primary Sjogren’s syndrome(pSS).Methods:1.Effects of SSY on clinical symptoms and P2X7/NLRP3 signaling pathway in pSS patients 80 patients with pSS were randomly divided into SSY group(n=40)and control group(n=40).All patients in the SSY and control groups took hydroxychloroquine(HCQ),and the SSY group took Sanshen Yin decoction everyday.The clinical efficacy(ESSPRI,ESSDAI score),salivary gland(15min saliva flow)and lacrimal gland function(Schirmer test)were compared before and after treatment.At the same time,peripheral blood samples were collected to obtain serum,and the levels of cytokines and P2X7/NLRP3 signaling pathway proteins were detected by enzyme-linked immunosorbent assay.2.Effects of SSY in submandibular gland tissue and serum P2X7/NLRP3 signaling pathway on SS miceThe mice model of Sjogren’s syndrome was established by Freund’s incomplete adjuvant combined with DTP and treated with SSY.The body weight,salivary secretion,salivary gland index,submandibular reactive oxygen species(ROS),submandibular gland and serum cytokines,pathological changes of submandibular gland and P2X7/NLRP3 signaling pathway of submandibular gland were detected.3.Effects of Ruscogenin in P2X7/NLRP3 signaling pathway in submandibular gland tissues and acinar cells on SS miceNOD/ShiLtJ mice were treated with Ruscogenin,and acinar cells isolated from submandibular glands were treated with TNF-α and Ruscogenin.Salivary flow rate(SFR)was measured at the 11st,13rd,15th,17th,and 20th week respectively.Histological analysis of the submandibular glands was conducted by hematoxylin-eosin staining assay.IL-6,IL-17,TNF-α,and IL-1βmRNA expression was detected through qRT-PCR.AQP 5,AQP 4,P2X7R,NLRP3,caspase-1,IL-1β,Bax,and Bcl-2 protein levels were tested by western blot.Cell apoptosis was assessed through acridine orange and propidium iodide(AO/PI)staining assay and flow cytometry assay.Result:1.Effects of SSY on pSS patients in terms of clinical efficacy,salivary gland and lacrimal gland function,cytokines and P2X7/NLRP3 signaling pathway proteins in peripheral blood1.1 Effects of SSY in terms of disease activity on pSS patients:There was no significant difference in ESSPRI between the two groups before treatment,but the range of acceptable symptom status(PASS)in the SSY group was 52.5%after treatment,which was significantly higher than that in the control group(32.5%)(P<0.05).1.2 Effects of SSY in terms of salivary gland and lacrimal gland function on patients with pSS:Before treatment,there was no significant difference between the two groups,but after treatment,both groups showed some improvement compared with before.Despite of the mutual improvement,compared with the control group,the salivary flow rate in SSY group significantly increased within 2 minutes,with statistical difference(P<0.05).For evaluation of tear function,tear secretion in SSY group also significantly increased within 5 minutes compared with control group(P<0.05).1.3 Effects of SSY in cytokines on pSS patients:Before treatment,there was no significant difference between the two groups.However,after treatment,the levels of serum cytokines IL-1β,TNF-α and IL-6 decreased in both groups.Compared with the control group,the levels of serum cytokines IL-1β and TNF-α(P<0.05),however,were on a more dramatic decline in the SSY group,but there was no significant difference in the level of IL-6(P>0.05).1.4 Effects of SSY in P2X7/NLRP3 levels on pSS patients:Before treatment,there was no significant difference between the two groups.After treatment,both groups could inhibit the expression of P2X7/NLRP3.Compared with the control group,the SSY group could see a more significant reduction in the serum levels of P2X7,NLRP3,ASC and caspase-1 on pSS patients,and the differences between the two groups were statistically significant(P<0.05).2.Effects of SSY in terms of body weight,salivary secretion,salivary index,serum and submandibular gland cytokine levels,pathological changes of submandibular gland,and protein expression of submandibular gland P2X7/NLRP3 signaling pathway on SS model mice2.1 Effects of SSY in body weight on SS model mice:The body weight of normal mice grew steadily,while the body weight of model mice rose relatively mildly.Compared with the model group,SSY could significantly increase the body weight of SS mice(P<0.01).2.2 Effect of SSY in salivary secretion on SS model mice:Compared with the normal group,salivary secretion in SS mice significantly decreased.Compared with the model group,SSY significantly increased the salivary secretion of SS mice(P<0.01).2.3 Effect of SSY in salivary index on SS model mice:Compared with the normal group,salivary gland index of SS mice significantly decreased.Compared with model group,SSY significantly increased salivary gland index of SS mice(P<0.01).2.4 Effects of SSY in reactive oxygen species(ROS)in submandibular gland tissue on SS model mice:Compared with the normal control group,the ROS level in submandibular gland of SS mice significantly increased,and compared with the model group,the ROS level in SSY and HCQ groups both decreased to different degrees(P<0.01).2.5 Effects of SSY in cytokines on SS model mice:Compared with the normal group,the levels of IL-6,IL-1β and TNF-α in the submandibular gland and serum of SS mice significantly increased.Compared with the model group,the levels of IL-6,IL-1β and TNF-α in the SSY and HCQ groups decreased to varying degrees(P<0.01).2.6 Effects of SSY in pathological changes of submandibular gland on SS model mice:Compared with the normal control group,the lymphocyte infiltration in the tympanic gland of the model group increased,the cells were necrotic and shed,and the lesions were obvious.However,improvement can be shown in the SSY and HCQ groups.2.7 Effects of SSY in transmission electron microscopy of submandibular gland on SS model mice:Compared with the normal group,the submandibular gland mitochondria in the model group showed atrophy and fusion,etc.However,the deterioration could be significantly improved in the SSY and HCQ groups.2.8 Effects of SSY in P2X7/NLRP3 signaling pathway in the submandibular gland on SS model mice:Compared with the normal control group,the protein expressions of P2X7,NLRP3,ASC,caspase-1 and IL-1β in the submandibular gland of the model group significantly increased.However,compared with the model group,the protein expressions of P2X7,NLRP3,ASC,caspase-1 and IL-1β(P<0.01)significantly decreased in the SSY and HCQ groups.3.Effects of Ruscogenin in salivary secretion,inflammation of submandibular gland,acinar cells of submandibular gland tissue,AQPs and inflammatory related factors on SS model mice3.1 Effects of Ruscogenin in salivary secretion and submandibular gland inflammation on SS model mice:The SFR of NOD/ShiLtJ and Vehicle groups gradually decreased,while the SFR of Ruscogenin treated mice did not decrease compared with Vehicle treated mice(P<0.05),suggesting that Ruscogenin could restore SS-like symptoms.Histological analysis of submandibular gland showed that both NOD/ShiLtJ and Vehicle groups showed a large number of lymphocytic infiltrates and more inflammatory lesions with larger area.However,0.3mg/kg Ruscogenin treatment could reduce the infiltration of lymphocytes and reduce the area and number of inflammatory centers,and lmg/kg Ruscogenin could further strengthen the reduction effect.Similarly,in NOD/ShiLtJ and Vehicle group,IL-6,IL-17,TNF alpha and beta IL-1 mRNA expression,there were no significant differences compared with Vehicle group,Ruscogenin 0.3 groups and Ruscogenin 1 set of IL-6,IL-17,The mRNA expressions of TNFa and IL-1β significantly decreased,and the effect of 1 mg/kg Ruscogenin was better than that of 0.3 mg/kg Ruscogenin(P<0.01).3.2 Effects of Ruscogenin in acinar cells,AQPs and inflammatory factors in submandibular gland tissue on SS model mice:Western blot analysis showed that there were no differences in AQP5,AQP4,P2X7R,NLRP3,Caspase 1 and IL-1β protein levels between NOD/ShiLtJ and Vehicle groups.However,compared with Vehicle treated mice,the protein expressions of AQP5 and AQP4 increased in Ruscogenin treated mice,and expression levels of P2X7R,NLRP3,Caspase 1 and IL-1βdecreased.It is also noticeable that Ruscogenin 1 group showed a better effect(P<0.05).3.3 Effects of Ruscogenin in apoptosis of acinar cells on SS model mice:Compared with normal mice(BALB/c),apoptosis of acinar cells in NOD/ShiLtJ mice significantly increased,and 1 mg/kg Ruscogenin significantly inhibited apoptosis of acinar cells in NOD/ShiLtJ mice.Acinar cells were cultured with different doses of Ruscogenin before being exposed to TNF-α.The results of flow cytometry showed that the apoptosis rate of TNF-αtreated cells significantly increased(P<0.001),and with the increase of the concentration of Ruscogenin,Ruscogenin treatment could inhibit the apoptosis induced by TNF-α(P<0.01).Furthermore,compared with the control group,the TNF-α group showed lower AQP5 and AQP4 protein levels,and higher NLRP3,Caspase 1 and IL-1β expression levels(P<0.001).With the increase of Ruscogenin dose,the levels of AQP5 and AQP4 in the cells treated with TNF-α and Ruscogenin increased,and the expressions of NLRP3,Caspase 1 and IL-1β decreased(P<0.05).Conclusion:1.Effects of SSY on clinical manifestations and P2X7/NLRP3 signaling pathway in pSS patientsCompared with the control group,SSY can significantly improve the clinical efficacy,improve the function of salivary gland and lacrimal gland,and reduce the levels of serum cytokines and P2X7/NLRP3 signaling pathway protein in patients with Sjogren’s syndrome.In conclusion,SSY can significantly improve the clinical symptoms of patients with Sjogren’s syndrome,and its mechanism is related to the level of cytokines and the regulation of P2X7/NLRP3 signal.2.Effects of SSY in submandibular gland tissue and serum P2X7/NLRP3 signaling pathway on SS miceSSY can significantly increase the body weight of Sjogren’s syndrome model mice,increase the salivary secretion and salivary index of Sjogren’s syndrome mice,reduce the serum and submandibular gland cytokine levels of Sjogren’s syndrome mice,improve the pathological changes of submandibular gland of Sjogren’s syndrome mice,and inhibit the expression of P2X7/NLRP3 signaling pathway protein in submandibular gland of Sjogren’s syndrome mice.In conclusion,SSY can significantly improve the symptoms of Sjogren’s syndrome in mice,and its mechanism is related to the regulation of P2X7/NLRP3 signal.3.Effects of Ruscogenin in P2X7/NLRP3 signaling pathway in submandibular gland tissues and acinar cells on SS miceRuscogenin ameliorated the SFR and submandibular gland in flammation of NOD/ShiLtJ mice.Ruscogenin promoted the preservation of acinar cells and suppressed in flammationrelated factors(P2X7R,NLRP3,caspase 1,and IL-1β)in submandibular gland tissues of NOD/ShiLtJ mice.Ruscogenin inhibited acinar cell apoptosis in NOD/ShiLtJ mice and reversed TNF-α-induced apoptosis and in flammation of acinar cells.Ruscogenin ameliorated SS by inhibiting clinical signs and symptoms.