| Objective: Primary Sj(?)gren’s syndrome(p SS)is a common chronic systemic autoimmune disease characterized by lymphocyte infiltration in the exocrine glands and the presence of multiple autoantibodies in the serum,mainly manifesting as dry mouth and dry eyes.The study found that in addition to a large number of lymphocyte infiltration,there is also adipose tissue infiltration in the salivary gland of p SS patients,and the destruction of salivary gland is often accompanied by the development of adipose tissue,which indicates that adipocytes may be involved in the pathogenesis of p SS.Although the pathogenesis of p SS is not well understood,there are numerous reports suggesting that inflammation and apoptosis play an important role in glandular injury.Therefore,exploring therapeutic approaches to alleviate glandular inflammation and apoptosis is important to improve glandular injury in p SS patients.Quercetin(QU)is a flavonol compound found in many fruits,vegetables,leaves and grains.QU can regulate inflammation,immunity and apoptosis.It is reported that QU can alleviate the damage of salivary gland caused by radiation,reduce the decrease of salivary flow rate and increase the expression of aquaporin 5(AQP5)and muscarinic receptor 3(M3R).However,whether QU plays a therapeutic role in p SS induced dry mouth has not been reported.Therefore,whether QU can alleviate dry mouth caused by p SS and regulate salivary gland inflammation and apoptosis is worthy of further study.Leptin(LP)is an adipokine secreted by white adipose tissue,which plays various physiological roles in metabolism and energy homeostasis.It has been reported that the LP signaling pathway is involved in the pathogenesis of p SS and that antagonizing the LP receptor(OB-R)alleviates the p SS induced decrease in salivary secretion and lymphocytic infiltration.LP signaling often functions by binding to OB-R and activating its major downstream pathway tyrosine kinase/signal transducer and activator of transcription(JAK/STAT).It has been shown that LP activated OB-R and its downstream JAK/STAT signaling pathway can mediate apoptosis and inflammation in salivary gland endothelial cells.QU can downregulate obesity induced elevated LP levels and LP induced OB-R expression in human umbilical vein endothelial cells.Moreover,QU has been shown to affect apoptosis and inflammation by downregulating the JAK/STAT signaling pathway.Taken together,we hypothesize that QU may exert an ameliorative effect on salivary gland apoptosis and inflammation through the inhibition of LP signaling pathway.This study focused on whether QU could alleviate pSS induced salivary gland injury,ameliorate salivary gland inflammation and apoptosis,and validate the involvement of LP signaling pathway in an IFN-γ induced in vitro model.Methods: Part I: Female BALB/c mice were selected as the control group and female NOD/Ltj mice as the SS animal model.The animals were divided into control,SS,SS+QUL and SS+QU-H groups,with 12 animals in each group.All mice(7 weeks old)were adaptively fed for 1 week before group administration.NOD/Ltj mice were gavaged with 50mg/kg QU(SS+QU-L)or 100mg/kg QU(SS+QU-H)once a day for 8 weeks to 16 weeks.In the administration group,QU was dissolved in dimethyl sulfoxide(DMSO).Control BALB/c mice and SS NOD/Ltj mice were given the same volume of DMSO as the administration group by daily gavage for 8 weeks.Mice were anesthetized by intraperitoneal injection of sodium pentobarbital(50mg/kg body weight),followed by stimulation of salivary secretion by intraperitoneal injection of sterile isoproterenol(20μg/100 g body weight).Dry swabs were weighed prior to saliva collection,and oral saliva was collected with the swabs for 10 min,and then weighed again.The volume of collected saliva was normalized to the corresponding mouse body weight(g)and expressed as: mg/g/10 min.Changes in salivary flow rate were monitored in each group of mice during administration(0,2,4,6,and 8 weeks).HE staining was performed to detect the submandibular gland(SMG)tissue and histological score in each group of mice.Western blot was used to detect the protein expression of salivary secretion markers AQP5 and M3 R in SMG tissues of mice in each group.Part II: TUNEL staining was used to detect the level of apoptosis in SMG tissue of mice in each group.The protein levels of Bcl-2,Bax,Caspase-3 and Cleaved Caspase-3 in SMG tissues of mice in each group were detected by Western blot.The transcript levels of TNF-α,IL-6 and IL-1β in SMG tissues of each group were measured by Real-time PCR,and the protein levels of TNF-α,IL-6 and IL-1β in SMG tissues of each group were measured by Western blot.Part III: ELISA was performed to detect LP levels in serum of each group of mice,Real-time PCR was performed to detect LP and OB-R transcript levels in SMG tissues of each group of mice,Western blot was performed to detect LP,OB-R,JAK2,p-JAK2,STAT3 and p-STAT3 protein levels in SMG tissues of each group of mice.After isolation of mouse salivary gland epithelial cells(SGECs),SGECs were identified by immunofluorescence.The maximum concentration of QU without significant effect on cell viability was determined by MTT method.After treatment with IFN-γ,LP,IFN-γ+LP,IFN-γ+QU and IFN-γ+QU+LP,cells were collected and TUNEL staining was performed to detect apoptosis.Western blot was performed to detect the protein levels of Bcl-2,Bax,Caspase-3,Cleaved Caspase-3,LP,OB-R,JAK2,p-JAK2,STAT3 and p-STAT3 in each group of cells.Realtime PCR was performed to detect the transcript levels of TNF-α,IL-6,LP and OB-R in each group of cells.Results: Part I: The salivary flow rate of SS mice decreased over time and was significantly lower than that of controls by week 8(P<0.01),and recovered significantly after high-dose QU administration(8 weeks)(P<0.01).The histopathological changes of SMG tissues in each group of mice were examined by HE staining,and the results showed that inflammatory cell infiltration was significantly increased in the SS group,while administration of QU resulted in a significant reduction of inflammatory cells,especially in the high-dose administration group,which had a denser and more normal tissue morphology than the SS group.The histological scores of SMG tissues in different groups were consistent with these morphological changes.Western blot results showed that the expression levels of salivary secretion related proteins AQP5 and M3 R in SMG tissues of SS mice were significantly decreased,while the expression of the above proteins was significantly increased after QU treatment(P<0.01).Part II: TUNEL staining results showed that the percentage of apoptotic cells in SMG tissues of mice in the SS group was significantly higher compared to the control group(P<0.001),while the percentage of apoptotic cells decreased significantly after administration of QU(P<0.05).Western blot results showed that Bcl-2 was decreased and Bax and Cleaved Caspase-3/Caspase-3 were increased in SMG tissues of mice in the SS group,and administration of QU reversed these effects,resulting in increased Bcl-2 and decreased Bax and Cleaved Caspase-3/Caspase-3.Real-time PCR results showed that the m RNA levels of TNF-α,IL-6 and IL-1β were significantly increased in the SMG tissues of mice in the SS group(P<0.001),and the m RNA levels of the above-mentioned pro-inflammatory cytokines were significantly decreased after the administration of QU,especially in the SS+QU-H group(P<0.001).Western blot results showed that the protein levels of TNF-α,IL-6 and IL-1β were also significantly increased in the SMG tissues of mice in the SS group(P<0.001),and the protein levels of the above-mentioned pro-inflammatory cytokines decreased significantly after the administration of QU,especially in the SS+QU-H group(P<0.001).Part III: ELISA results showed that serum LP levels were significantly increased in mice in the SS group(P<0.001),while their levels were significantly decreased in the QU administration group(P<0.001).The results of real-time PCR showed that there was no significant difference in the m RNA levels of LP in SMG tissues of mice in each group,but the m RNA expression levels of OB-R(OB-Ra and OB-Rb)in SMG tissues of mice in SS group increased significantly(P < 0.001),and the m RNA expression levels of OB-R in QU group decreased significantly,especially in SS+QU-H group(P < 0.001).Western blot showed no significant difference in the protein levels of LP in the SMG tissues of mice in each group,while the m RNA expression levels of OB-R(OB-Ra and OB-Rb)in the SMG tissues of mice in the SS group were significantly higher(P<0.001)and lower in the QU group,especially in the SS+QU-H group(P<0.001).The protein levels of JAK2,p-JAK2,STAT3 and p-STAT3 were significantly higher in the SMG tissues of mice in the SS group compared to the control group(P<0.001),while the protein expression levels of the above indicators decreased significantly after administration of QU,especially in the SS+QU-H group(P<0.05).Immunofluorescence results showed that SGECs were identified as positive for epithelial cell markers such as keratin 14,keratin 18,and p63,while myoepithelial cell marker α-SMA was negative.MTT assay was performed to detect the effect of QU on cell viability,and the results showed that 100 μM QU was the maximum concentration that had no statistically significant effect on the cell viability of SGECs,and then cells were treated with 100 μM QU for subsequent experiments.TUNEL staining of SGECs showed a significant increase in IFN-γ-induced apoptosis compared to the control group(P<0.001),which increased again after the administration of LP(P<0.05),while the administration of QU significantly alleviated apoptosis(P<0.001).Western blot results showed that the protein level of Bcl-2 was significantly decreased in the IFN-γ group compared to the control group(P<0.001),and its expression level decreased again after the addition of LP(P<0.05),while its expression level increased significantly after the administration of QU(P<0.01).The results of Bax and Cleaved Caspase-3/Caspase-3 were the opposite.Real-time PCR results showed that the m RNA levels of TNF-α and IL-6 were significantly higher in the cells of the IFN-γ group than in the control group(P<0.001),and their expression levels increased again after the addition of LP(P<0.001),while their expression levels decreased significantly after the administration of QU(P<0.01).The m RNA levels of OB-R(OB-Ra and OB-Rb)were significantly higher in the cells of the IFN-γ group compared with the control group(P<0.001),and their expression levels increased again after the addition of LP(P<0.01),while their expression levels decreased significantly after the administration of QU(P<0.01).The protein levels of OB-R(OB-Ra and OB-Rb)in the IFN-γ group cells were significantly increased compared to the control group(P<0.001),and their expression levels increased again after the addition of LP,while their expression levels decreased significantly after the administration of QU(P<0.05).The protein levels of JAK2,p-JAK2,STAT3 and p-STAT3 were significantly higher in the cells of the IFN-γ group compared with the control group(P<0.01),and their expression levels increased again after the addition of LP(P<0.01),while their expression levels decreased significantly after the administration of QU(P<0.05).Conclusion: 1.QU has a protective effect in p SS induced salivary gland injury and can alleviate p SS induced salivary gland injury.2.QU can alleviate apoptosis and reduce inflammation in SMG tissues of p SS model mice.3.The mechanism of action may be that QU alleviates p SS induced salivary gland injury by regulating the LP/OB-R signaling pathway. |
PDF Full Text Request