| Research objective:Under the guidance of"tonifying stomach,relieving dryness and invigorating the circulation of blood",to observe the effect of "Sanshen decoction" on the regulatory mechanism of costimulatory molecular programmed death protein(PD-1)and ligand(PD-L1),and to probe into the immune mechanism of pSS.Research method:1.Clinical test40 clinical cases were randomly divided into two groups:the treatment group and the control group;meanwhile,another 10 healthy people were selected as the healthy control group.The treatment group(the TCM group and the western medicine group)was given the concentrated Sanshen decoction combined with total glycoside capsule while the control group(the western medicine group)was given only the total glycoside of paeony capsule.Then the patients,peripheral blood mononuclear cells were isolated and the flow cytometry was adopted to detect the expression of PD-1 on the T cell surface of peripheral blood and the expression of PD-L1 on the surface of CD 14+ monocytes and CD19+B cells,compare the expression change of PD-1 and PD-L1 before and after treatment,detect plasma sPD-1,IL-10 and IL-17 levels by ELISA,and analyze the clinical signi ficance.2.Animal Experiment30 NOD mice were randomly divided into three groups:the TCM group,the western medicine group and the control group with 10 mice in each group.The mice in the TCM group were given the concentrated Sanshen decoction,the mice in the western medicine group were given hydroxychloroquine solution daily according to the body weight,and the mice in the control group were given normal saline daily.The mice in each group were gavaged for a total of two months.Afterwards the effect of Sanshen decoction on the expression of PD-1 in NOD mice's submandibular gland was analyzed by means of HE staining and immunohistochemistry;besides,the flow cytometry was adopted to analyze the efrect of Sanshen decoction on the expression of PD-1 on the surface of CD4+of spleen cells of NOD mice.Research result:1.Effect of PD-1,PD-L1,plasma sPD-1,IL-10 and IL-17 on PBMC cells in patients with pSS;1.1 Effect on PD-1:upon comparison of the western medicine group and the Chinese and western medicine group before and after treatment,the result indicates that the expression of PD-1 on the surface of CD4+cells decreased significantly after treatment,but the decrease of that in the western medicine group(15.32%±2.16%vs 37.18%±10.960%)was lower than that in the Chinese and western medicine group(13.1%±1.022%vs 39.8%±1.125%),which was not statistically significant(P>0.05).The expression of PD-1 on the surface of CD8+cells decreased significantly after treatment,but the decrease of that in the western medicine group(20.91%±1.257%vs 34.05%±1.521%)was lower than that in the Chinese and western medicine group(20.7%11.542%vs 42.9%10.91%),which was statistically significant(P<0.05).1.2 Effect on PD-L1:upon comparison of the western medicine group and the Chinese and western medicine group before and after treatment,the result indicates that the expression of PD-1 on the surface of CD 14+ cells of both groups increased significantly after treatment,but the increase of that in the western medicine group(9.776%10.65%vs11.52%±0.683%)was lower than that in the Chinese and western medicine group(5.2%±1.04%vs16.2±1.804%),which was statistically significant(P<0.05).The expression of PD-L1 on the surface of C119+B cells of both groups decreased significantly after treatment,but the decrease of that in the western medicine group(2.21%10.0607%vs3.44%10.213%)was lower than that in the Chinese and western medicine group(1%±0.221%vs4.11%±1.21%),which was statistically significant(P<0.05).1.3 Effect on the plasma sPD-1,IL-10 and IL-17:upon comparison of the western medicine group and the Chinese and western medicine group before and after treatment,the result indicates the expression of sPD-1 of both groups decreased significantly after treatment,but the decrease of that in the western medicine group(0.355 ± 0.520ng/ml vs 0.618±0.040 ng/ml)was lower than that in the Chinese and western medicine group(0.287±0.012 ng/ml vs 0.702±0.002 ng/ml),which was not statistically significant(P>0.05).IL-10 of both groups increased significant after treatment,but the increase of that in the western medicine group(31.326±1.207 pg/ml vs 22.060±1.626 pg/ml)was lower than that in the Chinese and western medicine group(36.877±0.451 pg/ml vs 25.331±2.436 pg/ml),which was statistically significant(P<0.05).IL-17 of both groups decreased significant after treatment,but the increase of that in the western medicine group(24.850±0.670 pg/ml vs 38.4822±3.481 pg/ml)was lower than that in the Chinese and western medicine group(19.030±1.030 pg/ml vs 35.225±1.655 pg/ml),which was statistically significant(P<0.05).2 Effect on the expression of PD-1 on the salivary gland and spleen cells of NOD mice2.1 Effect on the expression of PD-1 on the salivary gland of NOD miceThe immunohistochemical section of salivary gland specimens of mice with sjogren syndrome showed that the expression of PD-1 in the TCM group was lower than that in the control group,which is statistically significant(P<0.05);the expression of PD-1 in the western medicine group was lower than that in the control group,which is statistically significant(P<0.05).The decrease of PD-1 in the TCM group was more significant than that in the western medicine group,which is statistically significant(P<0.05).2.2 Efect on the expression of PD-1 on NOD mice's spleen cellsWith spleen obtained from mice with sjogren syndrome,broken into single cell suspension to crack red blood cells,the flow cytometry was then adopted to detect the expression of PD-1 on the surface of CD4+.Upon comparison of the TCM group with the control group(1.625±0.264 vs1.694±0.158),the expression of PD-1 on the surface of NOD mice's spleen cells CD4+ was statistically significant(P<0.05).Upon comparison of the western medicine group and the control group(1.8±0.217 vs 1.694±0.158),the expression of PD-1 on the surface of NOD mice's spleen cells was up-regulated,which was statistically significant(P<0.05).Upon comparison of the western medicine group and the TCM group(1.8±0.217vs1.625±0.264),the decrease of the former was lower than that in the latter,but was not statistically significant(P>0.05).Conclusion:1.Sanshen decoction can down-regulate the expression of PD-1 in peripheral blood of patients with Sjogren's syndrome,and it has advantages compared with western medicine group;2.Sanshen decoction can down-regulate the expression of PD-L1 in peripheral blood of patients with Sjogren's syndrome,and it has advantages compared with western medicine group;3.Sanshen decoction can reduce the expression of sPD-1 in peripheral blood of patients with Sjogren's syndrome,increase the expression of IL-10 and decrease the expression of IL-17,and it has advantages compared with western medicine group;4.Sanshen decoction can down-regulate the expression of PD-1 in NOD mice's salivary gland,and it had obvious advantages compared with the western medicine group;5.Sanshen decoction can down-regulate the expression of PD-1 in NOD mice's spleen cells,and it had advantages compared with the western medicine group;6.PD-1/PD-L1 molecules expressed on T cells,B cells and mononuclear surfaces respectively participate in the pathogenesis and development of SS through interaction.Sanshen decoction can regulate the immunity and correct the immune disorder by reducing the expression of PD-1 and its ligand PD-L1 in peripheral blood.In pSS patients,it is possible to regulate Treg/Th17 through the PD-1/PD-L1 pathway in the development of SS. |
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