| ObjectiveThe objective of the study is to explore the therapeutic effects and molecular mechanism of Artesunate on Sjogren syndrome in model mice.MethodsNOD(non-obese diabetic)mice were selected as experimental subjects and randomly divided into 4 groups with 8 mice in each group,including negative control group,Artesunate low dose group,Artesunate high dose group and IL-2 positive control group.Among which,the negative control group was administered intragastrically with sterile water of 0.2 ml daily,the Artesunate low-dose group was given 25 mg/kg daily by intragastric administration,the high-dose group was 50mg/kg daily by intragastric administration,and the IL-2 positive control group was administered by subcutaneous injection of 1000IU/g every other day for 10 consecutive weeks since the NOD mice aged 8 weeks.The hair color,body weight and saliva flow rate were observed and measured;the activity endurance was evaluated by treadmill.The mice were sacrificed at the end of the 10 th week after drug administration,followed by evaluating the indexes of spleen and submandibular gland;HE staining was performed on the submandibular gland and lacrimal gland tissues,and then the histopathological score was assessed according to the degree of lymphocyte infiltration;indirect immunofluorescence assay was utilized to test the anti-nuclear and antibody level in mice;flow cytometry was used to detect changes in proportion of lymph nodes,spleen Th1,Th2,Th17,Treg,Tfh cells and germinal center-derived B-cell.SPSS20.0 software was used for statistic analysis with measurement data recorded as((?)±s)and tested by t.One-way ANOVA was used for the multi-group comparison and LSD test was for pairwise comparison,in which P<0.05 was considered of statistical significance.Results(1)General condition: the body weight of mice aged 18 weeks in Artesunate low-dose group,high-dose group and the IL-2 group were 25.25g±0.88 g,25.31g±0.88 g,25.71g± 1.72 g respectively,which were all significantly higher than those in sterile water group of 23.29 g ± 1.75g(P <0.05).The hair color was also brighter with less hair removal than sterile water group.(2)Saliva secretion volume: the saliva secretion volume of mice in Artesunate low-dose group,high-dose group and IL-2 group were 71.25mg±25.85 mg,84.50mg±21.85 mg,87.88mg±33.31 mg respectively,which were higher than those in sterile water group of 37.50 mg±18.00 mg.The difference was statistically significant P<0.05.(3)Running time: the activity endurance of mice in the low-dose and high-dose groups were significantly improved with longer running time(3.80min±1.66min;3.35min±0.56min)v.s.sterile water group(2.19min±0.68min).The difference was of statistical significance,P <0.05.(4)Spleen index: the spleen index(mg/10g)of mice in the low-dose group,high-dose group and the IL-2 group were 3.419±0.319,3.978±0.876,3.625±0.721,respectively.The spleen index of mice in low-dose group was lower than those in sterile water group of 4.013±0.612,the difference was statistically significant P<0.05.There was no statistical difference among the other groups,P>0.05.(5)Submandibular gland index: the submandibular gland index(mg/10g)of mice in the low-dose group,high-dose group and IL-2 group were 0.587±0.032,0.548±0.018,0.619±0.012 respectively,which were all higher than those in sterile water group of 0.419 ±0.041.The difference was statistically significant,P<0.05.(6)Quantity of lymphocytes infiltration foci in submandibular gland: the lymphocytes infiltration foci existed in the submandibular glands of mice in sterile water group;the quantity of lymphocytes infiltration foci of mice in Artesunate low-dose group,high-dose group and IL-2 group were 3.13±2.64,3.75±2.49 and3.13±1.64,respectively,which were all significantly lower than those in sterile water group of 9.63±4.47.The difference was statistically significant,P<0.001.(7)Histopathological score of submandibular gland: the histopathological scores of submandibular gland in Artesunate low-dose group,high dose group and IL-2group were 2.13±1.46,2.63±1.19 and1.63±1.19,respectively,which were all lower than those in sterile water group of 3.75± 0.46.The difference was statistically significant,P<0.05.(8)Lacrimal gland lymphocytes infiltration foci: disordered lacrimal gland arrangement,gland atrophy and gland necrosis all existed in the lacrimal glands of mice in sterile water group,while lymphocyte infiltration foci existed in most of them.The lacrimal glands of NOD mice in low-dose group,high-dose group and IL-2group were less damaged,and the quantity of lymphocyte infiltration foci were2.13±1.13,1.75±0.71 and1.50±0.93,which were all significantly lower than those in sterile water group of 5.25±1.39.The difference was statistically significant,P <0.05.(9)Histopathological scores of lacrimal gland: the histopathological scores of lacrimal glands of mice in Artesunate low-dose,high-dose group and IL-2 group were1.63±0.92,1.63±0.52 and 1.13±0.64,respectively,which were all significantly lower than those in sterile water group of 3.75± 0.46.The difference was statistically significant,P<0.001.(10)Anti-nuclear antibody positive rate and score: the positive rates of anti-nuclear antibody in the Artesunate low-dose group,high-dose group,IL-2 group and sterile water group were 25%(2/8),37.5%(3/8),25%(2/8)and 50%(4/8),respectively.The anti-nuclear antibody scores of mice in Artesunate low-dose group,high-dose group and IL-2 group were 0.75±1.49,1.00±1.51 and 0.50±0.93,which were all lower than those in sterile water group of 1.75±1.98.However,the difference was not significant,P>0.05.(11)Proportion of Th1 and Th2 cells in lymph nodes: the proportion of lymph node Th1 cells(%)in Artesunate low-dose group,high-dose group and IL-2 group were 1.15±0.42,1.29±0.58 and 1.28±0.45,which were all lower than that in sterile water group of 3.39±0.59.The difference was statistically significant,P<0.05.The proportion of lymph node Th2 cells(%)in Artesunate low-dose group,high-dose group and IL-2 group were 3.74±0.83,3.94±0.52,4.21±0.58,respectively,which were all higher than that in sterile water group of 2.13±0.97.The difference was statistically significant,P<0.05.(12)Proportion of Th17 and Treg cells in lymph nodes: the proportion(%)of Th17 cells in Artesunate low-dose group,high-dose group and IL-2 group were0.62±0.40,1.17±0.61,0.53±0.36,respectively,which were all lower than that in sterile water group of 1.78±0.75,P<0.05.The proportion(%)of Treg cells in Artesunate low-dose group,high-dose group and the IL-2 group were 13.76±1.93,13.23±2.33,14.35±2.33,which were all higher than that in sterile water group of9.67±2.31.The difference was statistically significant,P<0.05.(13)Proportion of Tfh and GCB cells in lymph nodes: the proportion of Tfh cells(%)in lymph nodes of mice in Artesunate low-dose group,high-dose group and IL-2 group were 16.10±3.12,13.94±3.35,12.74±3.33,respectively,which were all lower than those in sterile water group of 23.76±3.81,P<0.05.The proportion of GCB cells(%)in lymph nodes of mice in Artesunate low-dose group,high-dose groups and IL-2 group were 1.58±1.01,1.38±0.43,1.23±0.79,which were all lower than those in sterile water group of 2.91±0.75.The difference was statistically significant,P<0.05.(14)Proportion of Th1 and Th2 cells in the spleen: the proportion(%)of Th1 cells in the spleen of mice in Artesunate low-dose group,high-dose group and IL-2group were 1.54±0.40,1.50±0.70,1.09±0.37,which were all lower than sterile water group of 2.93±0.59,P<0.05.The proportion(%)of Th2 cells in the spleen of mice in Artesunate low-dose group,high-dose group and IL-2 group were 2.96±0.52,3.11±0.93,2.63±0.83,respectively,which were all higher than those in sterile water group of.67±0.41.The difference was of statistical significance,P<0.05.(15)Proportion of Th17 and Treg cells in the spleen: the proportion(%)of Th17 cells in spleen nodes of mice in Artesunate low-dose group,high-dose group and IL-2group were 0.76±0.65,0.45±0.36,0.79±0.54,respectively,which were all lower than those in sterile water group of 1.92±1.20.The difference was statistically significant,P<0.05.The proportion(%)of Treg cells in spleen nodes of mice in Artesunate high-dose group and IL-2 group were 13.03±1.09 and 13.50±2.03,which were both higher than those in sterile water group of 10.31±1.11.The difference was statistically significant,P<0.05.(16)Proportion of Tfh and GCB cells in spleen: the proportion of Tfh cells(%)in spleen nodes of mice in Artesunate low-dose group,high-dose group and IL-2group were 14.90±1.27,13.71±1.47,12.06±3.20,which were all lower than sterile water group of 17.55±2.70,P<0.05.The proportion of GCB cells(%)in spleen nodes of mice in Artesunate low-dose group,high-dose group and IL-2 group were0.36±0.34,0.32±0.20,0.40±0.40,which were all lower than those in sterile water group of 0.90±0.50.The difference was of statistical significance,P<0.05.ConclusionsArtesunate can improve general conditions of Sjogren syndrome in model mice by increasing the secretion volume of saliva and decreasing their fatigue level.Through reducing the quantity of lymphocyte infiltration focus in submandibular gland and lacrimal gland,inhibiting the generation of inflammatory lymphocytes Th1,Th17,Tfh and GCB,increasing the cellular level of immunosuppressive cell Treg,the Artesunate is able to regulate the balance of Th1/Th2,Th17/Treg,which may also be an effective treatment and specific mechanism of Sjogren symptom in NOD mice. |
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