| Research objective:After years of clinical observation,we found that there are many patients with primary Sjogren's syndrome(PSS)who have anxiety disorder,depression orcombined with anxiety and depression,which are closely related to the discomfort symptoms caused by the disease and adverse drug reactions.In traditional Chinese medicine,depression is related to the liver.We think that the main pathogenesis of PSS is the deficiency of liver yin(blood)and the loss of liver qi.We suggest that nourishing yin and soothing liver should be the treatment of PSS.This study:1.Under the guidance of the theory of treating from liver and nourishing yin and soothing liver,Yiguanjian modified formula was used to treat PSS.Through clinical observation and animal experiment,the theory and clinical basis of treating PSS from liver and nourishing yin and soothing liver were systematically discussed from multiple angles.2.Through JAK/stat and NF-?B signaling pathway,to observe the effect of triptolide on salivary gland injury in NOD mice,and to explore the mechanism of triptolide in treating PSS.Research method:1.The clinical study of Yiguanjian Decoction on primary Sjogren's syndrome of yin deficiency and liver depression and the influence of B lymphocyteIn this study,60 patients with PSS Yin deficiency and liver depression syndrome and 10 healthy controls were selected as the research objects.Peripheral blood 5-10ml was collected from patients with PSS Yin deficiency and liver depression syndrome and healthy controls.B cell activation,differentiation surface markers,TFH cell ratio,B cell proliferation ability,B cell apoptosis level and IFN-?,IL-4,IL-6,IL-10 and IL-27 levels were detected.The patients with PSS Yin deficiency and liver depression syndrome were divided into two groups,30 cases in the experimental group and 30 cases in the control group.The clinical efficacy was observed before and after treatment,and the B cell activation,differentiation surface markers.TFH cell ratio,B cell proliferation ability,B cell apoptosis level and serum IFN-?,IL-4,IL-6,IL-10 and IL-27 levels were measured before and after treatment.2.The effect of Yiguanjian Decoction on B lymphocyte of NOD miceSixty NOD mice and 10 normal ICR mice were selected.NOD mice were randomly divided into 6 groups with 10 mice in each group,namely model group,high-dose group,medium dose group,small dose group,Tripterygium wilfordii group and hydroxychloroquine group.ICR mice were the normal group.The mice were killed 8 weeks later.The spleen index and submandibular gland index were calculated.B cell activation,differentiation surface markers,TFH cell ratio,B cell proliferation ability,B cell apoptosis level and serum IgG,IFN-?,IL-4,IL-6,IL-10,BAFF and IL-27 were measured.3.Triptolide(TP)attenuates salivary gland injury in NOD mice through JAK/stat and NF-?B signaling pathways48 female NOD mice were randomly divided into 4 groups,12 in each group,namely control group(placebo),low-dose TP group,medium dose TP group and high-dose TP group.Salivary secretion was measured every 2 weeks.After 90 days,the mice were killed,the salivary gland and spleen were weighed,and the salivary gland index and spleen index were calculated;TNF-?,IL-2 and IL-6 were detected;the salivary glands were taken to evaluate the histopathology;the distribution of mouse salivary gland lymphocytes was analyzed;the proliferation test of spleen T cells and B cells was carried out;the salivary gland tissue protein was extracted and the protein expression was detected.Research results:1.The clinical study of Yiguanjian Decoction on primary Sjogren's syndrome of yin deficiency and liver depression and the influence of B lymphocyte1.1 Effect on ESR,CRP,globulin and immunoglobulin:after 12 weeks of treatment,ESR,C-reactive protein,globulin and IgG in the treatment group were significantly different from those before treatment(P<0.05),while IgA and IgM had no significant difference(P>0.05).There were significant differences in ESR,globulin and IgG in the control group compared with those before treatment(P<0.05),but there was no significant difference in C-reactive protein,IgA and IgM(P>0.05).There was no significant difference in ESR,C-reactive protein,globulin,IgG,IgA and IgM between the treatment group and the control group(P>0.05).1.2 Affect on tear flow rate and saliva flow rate:after 12 weeks of treatment,there were significant differences in both eyes' tear flow rate and saliva flow rate in the treatment group compared with before treatment(P<0.05),but there was no significant difference in the control group(P>0.05).There were significant differences between the treatment group and the control group after treatment(P<0.05).1.3 The influence on self rating anxiety and depression scale:after 12 weeks of treatment,the self rating anxiety and depression scale in the treatment group was significantly different from that before treatment(P<0.05),but there was no significant difference in the self rating anxiety and depression scale in the control group(P>0.05).There was a significant difference in the self rating anxiety and depression scale between the treatment group and the control group after treatment(P<0.05).1.4 The effect of B cell activation in peripheral blood:before treatment,compared with healthy control group,the activation rate of B cell in peripheral blood of treatment group and control group were significantly increased,the difference was statistically significant(P<0.05);compared with before treatment,the activation rate of B cell in peripheral blood of treatment group and control group was significantly decreased,the difference was statistically significant(P<0.05)There was no significant difference in the activation rate of peripheral blood B cells between the two groups(P>0.05).1.5 Effect of peripheral blood B cell differentiation:before treatment,compared with healthy controls,the differentiation rate of peripheral blood B cells(differentiated into memory B cells)in the treatment group and the control group was significantly lower(P<0.05);compared with before treatment,the differentiation rate of peripheral blood B cells in the treatment group and the control group was significantly increased after treatment(P<0.0)5)However,there was no significant difference in the differentiation rate of peripheral blood B cells between the treatment group and the control group(P>0.05).1.6 Effect of treatment on the ratio of peripheral blood TFH cells:before treatment,compared with healthy controls,the ratio of peripheral blood TFH cells(Q2 quadrant)in the control group and the control group was significantly increased,the differences were statistically significant(P<0.05);compared with before treatment,the ratio of peripheral blood TFH cells in the treatment group and the control group were significantly decreased after treatment,the differences were statistically significant(P<0.05);however,the treatment group and the control group had significant differences(P<0.05)There was no significant difference in the ratio of peripheral blood TFH cells between the treatment group and the control group(P>0.05).1.7 Effect of peripheral blood B cell proliferation:before treatment,compared with healthy control group,the proliferation rate of peripheral blood B cell in treatment group and control group was significantly increased,the difference was statistically significant(P<0.05);compared with before treatment,after treatment,the proliferation rate of peripheral blood B cell in treatment group and control group were significantly decreased,the difference was statistically significant(P<0.05);but the treatment group and the control group had significant difference(P<0.05)There was no significant difference in the proliferation rate of peripheral blood B cells between the two groups(P>0.05).1.8 Effect of peripheral blood B cell apoptosis:before treatment,compared with healthy control group,the apoptosis rate of peripheral blood B cell in treatment group and control group was significantly increased,the difference was statistically significant(P<0.05);compared with before treatment,after treatment,the apoptosis rate of peripheral blood B cell in treatment group and control group was significantly decreased,the difference was statistically significant(P<0.05);but the treatment group and the control group had significant difference(P<0.05)There was no significant difference in the apoptosis rate of peripheral blood B cells between the two groups(P>0.05).1.9 Effect on serum cytokines:before treatment,compared with healthy controls,the levels of serum IL-4,IL-6,IL-10,IL-27,IFN-? and other inflammatory cytokines in the treatment group and the control group were significantly increased,the differences were statistically significant(P<0.05);compared with before treatment,after treatment,serum IL-4,IL-6,IL-10,IL-27,IFN-? and other inflammatory cytokines in the treatment group and control group were significantly increased The difference was statistically significant(P<0.05),but there was no significant difference between the treatment group and the control group(P>0.05).2.The effect of Yiguanjian Decoction on B lymphocyte of NOD mice2.1 Effects on the water consumption,salivary flow,spleen index and submandibular gland index of NOD mice:compared with the blank group,the water consumption of NOD mice increased,the salivary flow decreased,and the spleen index and submandibular gland index were basically the same.2.2 Effect of B cell activation in peripheral blood:compared with the blank group,the activation rate of peripheral blood B cells in the model group was significantly increased,and the difference was statistically significant(P<0.05).Compared with the model group,the activation rate of peripheral blood B cells in the low concentration compound medicine group,the medium concentration compound medicine group,the high concentration compound medicine group,the Tripterygium wilfordii group and hydroxychloroquine group were decreased,and the difference was not statistically significant in the low concentration compound drug group Statistical significance(P>0.05),medium concentration compound medicine group,high concentration compound medicine group,Tripterygium wilfordii group and hydroxychloroquine group were statistically significant(P<0.05).2.3 Effect of B cell differentiation in peripheral blood:compared with the blank group,the differentiation rate of peripheral blood B cells(differentiated into memory B cells)in the model group was significantly lower than that in the blank group(P<0.05);compared with the model group,the differentiation rate of peripheral blood B cells in the low concentration compound medicine group,the medium concentration compound medicine group,the high concentration compound medicine group,the Tripterygium wilfordii group and hydroxychloroquine group were increased There was no significant difference in low concentration compound medicine group,medium concentration compound medicine group and Tripterygium wilfordii group(all P>0.05);there was significant difference between high concentration compound medicine group and hydroxychloroquine group(P<0.05).2.4 Effect of TFH cell ratio in peripheral blood:compared with the blank group,the TFH cell ratio(Q2 quadrant)in the model group was significantly increased,and the difference was statistically significant(P<0.05);compared with the model group,the ratio of TFH cells in the low concentration compound medicine group,the medium concentration compound medicine group,the high concentration compound medicine group,the Tripterygium wilfordii group,and hydroxychloroquine group were all decreased,among which the low concentration of prescription drugs There was no significant difference among the three groups(all P>0.05),but there was significant difference between high concentration prescription group and hydroxychloroquine group(P<0.05).2.5 Effect of B cell proliferation in peripheral blood:compared with the blank group,the proliferation rate of peripheral blood B cells in the model group was significantly increased(P<0.05);compared with the model group,the proliferation rate of B cells in the low concentration compound medicine group,the medium concentration compound medicine group,the high concentration compound medicine group,the Tripterygium wilfordii group and hydroxychloroquine group were all decreased,among which the low concentration compound medicine group,the medium concentration compound medicine group,the high concentration compound medicine group,the Tripterygium wilfordii group and hydroxychloroquine group were all decreased There was no significant difference in the concentration of compound medicine group(P>0.05),but there were significant differences in high concentration compound medicine group,Tripterygium wilfordii group and hydroxychloroquine group(P<0.05).2.6 The effect of B cell apoptosis in peripheral blood:compared with the blank group,the apoptosis rate of peripheral blood B cells in the model group was significantly increased,and the difference was statistically significant(P<0.05);compared with the model group,the apoptosis rate of peripheral blood B cells in the low concentration compound medicine group,the medium concentration compound medicine group,the high concentration compound medicine group,the Tripterygium wilfordii group and hydroxychloroquine group were decreased,among which the low concentration of compound medicine group and the medium concentration compound medicine group were all decreased There was no significant difference between the two groups(P>0.05),but there were significant differences in high concentration compound group,Tripterygium wilfordii group and hydroxychloroquine group(all P<0.05).2.7 Effect on serum cytokines:compared with the blank group,the levels of BAFF,IFN-?,IgG,IL-4,IL-6,IL-10 and IL-27 in the model group were significantly increased,and the differences were statistically significant(all P<0.05);compared with the model group,the above indexes of low concentration compound medicine group,medium concentration compound medicine group,high concentration compound medicine group,Tripterygium wilfordii group and hydroxychloroquine group were decreased,among which the low concentration of compound medicine group was lower There was no significant difference in the levels of BAFF,IFN-?,IgG,IL-4,IL-6,IL-10 and IL-27 in group A(all P>0.05);there was no significant difference in the level of IL-4 in the medium concentration compound group(P>0.05),but there were significant differences in the levels of BAFF,IFN-?,IgG,IL-6,IL-10 and IL-27 in the medium concentration compound group,Tripterygium wilfordii group and hydroxychloroquine group(P<0.05)There were statistical differences(P<0.05).3.Triptolide(TP)attenuates salivary gland injury in NOD mice through JAK/stat and NF-?B signaling pathways3.1 The protective effect of TP on salivary gland index,spleen index and salivary flow of NOD mice:the salivary gland index of NOD mice in TP group was significantly higher than that of control group,and spleen index was significantly lower than that of control group.The salivary flow of NOD mice in the control group decreased with time.The salivary flow rate of TP group was significantly lower than that of TP group from 6 to 12 weeks.With the increase of TP concentration,the salivary flow rate was significantly different from that of the control group.3.2 TP inhibited autoimmune injury in NOD mice:there was a small amount of lymphocyte infiltration and no lymph node formation in TP treatment group.The histopathological scores were 2.4 ± 0.18,1.8 ± 0.17 and 1.5± 0.19,respectively.Compared with the control group,the levels of IgG,IgM,IL-2,IL-6 and TNF-? antibodies in serum of NOD mice in TP treatment group were significantly decreased.CD4+,CD8+and B220+/CD45+cells decreased significantly.Compared with 0.05%DMSO,the proliferation of T cells and B cells in spleen of NOD mice treated with TP was significantly decreased.3.3 TP inhibits JAK/stat and NF-?B signaling pathways in salivary glands of NOD mice:Western blot results showed that TP significantly inhibited JAK/STAT signaling pathway by inhibiting phosphorylation of Jak1/2 and stat,and significantly inhibited NF-?B signaling pathway by inhibiting phosphorylation of p65 and I ?B ?.3.4 TP reduces IFN-?-induced inflammatory injury of salivary gland epithelial cells by inhibiting JAK/stat and NF-?B signaling pathways:there was no significant change in cell activity after treatment with 10 ng/ml,20 ng/ml and 40 ng/ml TP for 72 h.TP significantly inhibited the expression of inflammatory factors.TP had no effect on the expression of Jakl/2,STAT3,I ?B a and NF-?B,but p-jak1/2,p-STAT3,P-I ?B ? and p-nf-?B were significantly inhibited(P<0.001).Conclusion:1.The clinical study of Yiguanjian Decoction on primary Sjogren's syndrome of yin deficiency and liver depression and the influence of B lymphocyte1.1 Compared with healthy people,the activation,proliferation and apoptosis of B cells in PSS patients were significantly increased,memory B cells were decreased,and the ratio of TFH cells was significantly increased.After treatment,the number of activated B cells decreased,the proliferation level decreased,the apoptosis decreased,memory B cells increased,plasma cells decreased,and the ratio of blood TFH cells decreased.1.2 The levels of IL-4,IL-6,IL-10,IL-27,IFN-? cytokines in serum of patients with PSS were increased,while the levels of IL-4,IL-6,IL-10,IL-27 and IFN-? were decreased after treatment.1.3 Yiguanjian Decoction can reduce erythrocyte sedimentation rate,C-reactive protein,globulin and IgG index in patients with PSS,and has a regulatory effect on B lymphocytes.Compared with hydroxychloroquine,there is no significant statistical difference,but it shows a good effect in improving the dry symptoms and emotions of patients.It provides a good theoretical basis for Yiguanjian Decoction in the treatment of primary Sjogren's syndrome with Yin deficiency and liver depression.2.The effect of Yiguanjian Decoction on B lymphocyte of NOD mice2.1 Compared with the blank group,NOD mice had more drinking water,less salivary flow,and the spleen index and submandibular gland index were basically the same.2.2 The activation rate of peripheral blood B cells,the ratio of peripheral blood TFH cells,the proliferation rate of peripheral blood B cells,the apoptosis rate of peripheral blood B cells were significantly increased,the differentiation rate of peripheral blood B cells(differentiated into memory B cells)was significantly decreased,and the levels of BAFF,IFN-?,IgG,IL-4,IL-6,IL-10 and IL-27 were significantly increased in NOD mice.2.3 The activation rate of peripheral blood B cells,the ratio of peripheral blood TFH cells,the proliferation rate of peripheral blood B cells and the apoptosis rate of peripheral blood B cells in low,medium and high concentration compound groups,Tripterygium wilfordii group and hydroxychloroquine group were decreased,the differentiation rate of peripheral blood B cells was increased,and the levels of BAFF,IFN-?,IgG,IL-4,IL-6,IL-10 and IL-27 were also decreased.The high concentration compound group,Tripterygium wilfordii group and hydroxychloroquine group showed good curative effect3.Triptolide(TP)attenuates salivary gland injury in NOD mice through JAK/stat and NF-?B signaling pathways3.1 With the increase of age,the salivary flow of NOD mice in the control group gradually decreased,and more serious lymphocytic infiltration lesions appeared in the salivary glands.The concentration of IgG and IgM in serum increased gradually,and the expression of inflammatory factors increased.3.2 Compared with the control group,TP treatment group significantly improved salivary gland flow rate,decreased lymphocyte infiltration,IgG and IgM concentrations and the expression of inflammatory factors.TP inhibited the proliferation of T and B lymphocytes and improved the dry symptoms of NOD mice without obvious tissue damage.3.3 TP attenuates inflammation and tissue damage in NOD mice by inhibiting JAK/STAT pathway and NF-?B pathway. |
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