The Effect Of Huoxue Jiedu Recipe On LincR-Ccr2-5'AS-CCR5/CCL5 In Sjogren's Syndrome Model Mice

Background:Sjogren's syndrome(SS)is an autoimmune disease mainly involving exocrine glands.It is mainly characterized by dryness of mouth and eyes,and lymphocyte infiltration.The exact pathogenesis is not clear.There are various factors including genetic inheritance,immune dysfunction,viral infection and external environmental factors,and the interaction between them lead to imbalance of immune balance,so varieties of inflammatory cytokines and autoantibodies are produced,which damage tissues and ultimately cause gland dysfunction.In recent years,the role of long-chain noncoding RNA(LncRNA)in disease regulation has been emphasized.The study found that LincR-Ccr2-5'AS is specifically expressed in Th2 cells and regulates the expression of chemokine receptor 5(CCR5).CCR5 and its ligand CCL5 are highly expressed in the lacrimal gland of SS patients.The chemokine signaling pathway composed of LincR-Ccr2-5'AS and CCR5 and CCL5 may play an important role in the immune inflammatory response in SS.At present,symptomatic treatment is the mainly treatment of SS,and traditional Chinese medical(TCM)has unique advantages in treating SS because of its theory of syndrome differentiation.Dryness,toxicity and phlegm are the key pathogenesis of SS,so activate blood circulation and detoxify,and nourishing yin and moistening dryness are commonly used in treatment of SS.Objective:To research the effect of Huoxue Jiedu Decoction on the expression of LincR-Ccr2-5'AS,CCR5,CCL5 in the submandibular gland of Sjogren's syndrome(SS)model mice.Methods:Thirty-four NOD/Ltj mice were randomly divided into three group according to weight,including model group(n=10),control group(n=12)and therapy group(n=12),and twelve C57BL/6J mice were used as normal group.The mice in normal group and model group were given distilled water everyday.The mice in control group was given total glucosides of paeony every day.The mice in therapy group was given Huoxue Jiedu Decoction every day.The amount of drinking water was measured daily,and weigh was detected weekly.The saliva flow rate was measured at 9 weeks,12 weeks,and 16 weeks of age.After eight weeks,the mice in each group were executed by picking the eyeballs for'blood,and measured submandibular gland index.HE stained the submandibular gland tissues,and determined pathology score.The level of serum CCL5 and IFN-? were measured by ELISA.The level of LincR-Ccr2-5'AS,CCR5mRNA and CCL5 mRNA of submandibular gland was measured by RT-qPCR.Results:(1)At9 and 10 weeks of age,there was no significant difference in weight of mice.At 11 weeks of age,the weight of NOD mice was higher than that in the normal group(P<0.01).At 16 weeks of age,the weight of mice in therapy group was higher than that in control group(P<0.01).(2)At 9 weeks of age,the drinking volume of the mice in control group was lower than that of model group and therapy group(P<0.05).At 12 weeks of age,the drinking volume of the mice in control group was lower than that in model group.At 14 weeks of age,the drinking volume of mice in model group and treatment group was higher than that in normal group(P<0.05),and the drinking volume of the mice in therapy group was higher than that in control group(P<0.05).At 16 weeks of age,the drinking volume of the mice in therapy group was higher than that in normal group(P<0.05),and there was no statistical difference among each group of NOD mice.the drinking volume of mice at difference weeks of age was no significant difference comparing within group.(3)The saliva flow rate of mice in normal group was significantly higher than all NOD mice(P<0.01).The saliva flow rate of mice in therapy group was lower than that in model group(P<0.05).(4)The histological scores and submandibular gland index of mice in normal group were significantly lower than all NOD mice(P<0.01).There was no significant difference in the histological scores and submandibular gland index among three groups of NOD mice.(5)The level of serum CCL5 and IFN-? of the mice in the model group was significantly higher than that in the normal group(P<0.01).The level of serum CCL5 and IFN-? of mice in control group and therapy group was lower than that in the model group(P<0.01).The level of serum CCL5 and IFN-? in therapy group was significantly lower than that in control group(P<0.01).(6)The level of LincR-Ccr2-5'AS in submandibular gland of therapy group was lower than that in normal group(P<0.01).The level of CCL5 mRNA of submandibular gland in control group was lower than that in model group(P<0.05).There was no significant difference in the level of CCR5 mRNA in the submandibular glands between the two groups.(7)Correlation analysis showed that serum IFN-? expression level was positively correlated with that serum CCL5,submandibular gland CCL5 mRNA and submandibular gland LincR-Ccr2-5'AS expression(R=0.917,P<0.01;R=0.435,P<0.05;R=0.364,P<0.05;respectively),serum CCL5 was positively correlated with CCR5 mRNA and CCL5 mRNA expression of submandibular gland(R=0.413,P<0.05;R=0.377,P<0.05,respectively).CCR5 mRNA of submandibular gland was positively correlated with CCL5 mRNA expression of submandibular gland(R=0.512,P<0.01),there was no significant correlation between the remaining indicators.Conclusion:Huoxue Jiedu Decoction can reduce the expression of serum CCL5 and IFN-y,and down-regulate the immune inflammatory response in NOD mice.Huoxue Jiedu Decoction can down-regulate the expression of LincR-Ccr2-5'AS in the submandibular gland of NOD mice.The LincR-Ccr2-5'AS-CCR5/CCL5 signaling pathway may be involved in the regulation of immune inflammatory responses through interaction with IFN-y.