| Chapter 1 Effect of method of Bushenjianpi and Jieduliyan on the distribution of IgA-secreting cells and expression of IgAObjective: Under the guidance of the theory of "larynx-kidney correlation" in Chinese medicine,to investigate the mechanism of action of the method of Bushenjianpi and Jieduliyan in the treatment of IgA nephropathy,taking IgA-secreting cells as the main line of research.Methods: 1.140 SD male rats were randomly divided into 20 in the blank group(Control group)and 120 in the modeling group,and an animal model of IgA nephropathy was prepared by mucosal immune modeling.The rats were randomly divided into 6 groups:Model group(Model group),Bushen Liyan recipe with high(BSLY-H group),medium(BSLY-M group),and low(BSLY-L group)dose groups,FTY720 group(FTY720group),lotensin group(Lotensin group),20 animals in each group,were given 4 ml/kg/d drug by gavage for 6 weeks,and kidney,small intestine and bone tissues were collected.Record the body weight and state changes of the rats,and the death situation.2.Flow cytometry for expression levels of CD19+ IgA-secreting cells(IgA+CD19+)and CD27+ IgA-secreting cells(IgA+CD27+)in Peyer’s tubercle,small intestinal lamina propria,and bone marrow.3.Western Blot for bone marrow and kidney IgA expression.Results: 1.Compared with the Control group,the rats in the remaining groups showed slow weight gain,yellowish and dull fur,hair loss and depressed mental status;after the drug intervention,the general condition improved and body weight increased.A total of 21 rats died.2.Flow cytometry of IgA+CD19+ lymphocytes and IgA+CD27+ lymphocytes in Peyer’s node,small intestine lamina propria and bone marrow showed a significant increase in expression in the Model group compared to the Control group(p<0.001).The expression was significantly lower in the Model group compared to the Model group(p<0.001).When comparing between the dosing groups,there was a statistical difference between the BSLY-H,BSLY-M and BSLY-L groups(P < 0.001,P < 0.01),no statistical difference between the BSLY-H and FTY720 groups(P ≥ 0.05),and a statistical difference between the Lotensin and BSLY-H and FTY720 groups(P < 0.001).3.The results of Western Blot showed that the expression of bone marrow and kidney IgA in the Model group increased significantly compared with the Control group(P<0.001,P<0.01).The expression of bone marrow and kidney IgA decreased in all dosing groups compared to the Model group(P<0.001,P<0.01,P<0.05).When comparing the bone marrow IgA administration groups,there was no statistical difference between the BSLY-M group and the BSLY-H and BSLY-L groups.There was no statistical difference between BSLY-M group and BSLY-H group and BSLY-L group(P≥0.05),no statistical difference between BSLY-H group,BSLY-M group and FTY720 group(P ≥ 0.05),and statistical difference between Lotensin group and BSLY-H group and FTY720 group(P<0.01,P<0.05).When comparing the renal IgA between the administration groups,there was no statistical difference between the BSLY-H,BSLY-M and BSLY-L groups(P≥0.05),and no statistical difference between the FTY720 and Lotensin groups and the BSLY-H,BSLY-M and BSLY-L groups(P≥0.05).Conclusion: 1.IgA-secreting cells increase and secrete large amounts of IgA,which eventually leads to the development of IgA nephropathy.2.Bushen Liyan recipe can effectively reduce the expression of IgA-secreting cells and IgA.It is speculated that the method of Bushenjianpi and Jieduliyan may achieve the effect of preventing and treating IgA nephropathy by inhibiting IgA-secreting cells.Chapter 2 The effect of chemokines SDF-1 and CXCR4 on the regulation of lymphocyte migration by the method of Bushenjianpi and JieduliyanObjective: To investigate the effect of the method of Bushenjianpi and Jieduliyan on the expression levels of chemokine SDF-1 and ligand CXCR4,and on the regulation of lymphocyte migration.Methods: 1.An animal model of IgA nephropathy was constructed,and small intestine and bone tissues were collected from rats.The expression levels of SDF-1 and CXCR4 in the small intestine and bone marrow were measured by immunohistochemistry.2.Real-time PCR was performed to detect the expression levels of SDF-1 m RNA in Peyer’s node,small intestine lamina propria and bone marrow.Results:1.The results of immunohistochemistry showed that the expression of SDF-1and CXCR4 in the small intestine and bone marrow of the Model group decreased significantly compared with that of the Control group(P<0.001,P<0.001,P<0.01).Compared with the Model group,the expression of SDF-1 in the small intestine was significantly increased in all dosing groups(P<0.001);compared among the dosing groups,there was a statistical difference between the BSLY-H,BSLY-M and BSLY-L groups(P<0.001),between the BSLY-H and FTY720 groups(P<0.001),and between the Lotensin and FTY720 groups(P<0.001).The BSLY-H and FTY720 groups were statistically different(P < 0.001).Compared with the Model group,the expression of CXCR4 in the small intestine increased in the BSLY-H,FTY720,Lotensin and BSLY-M groups(P<0.001,P<0.01)and in the BSLY-L group,but there was no statistical difference(P≥0.05);compared with the Model group,the expression of CXCR4 in the BSLY-H,FTY720,Lotensin and BSLY-L groups was statistically different from that in the BSLY-M and BSLY-L groups(P<0.01).There was a statistical difference between the BSLY-H group and BSLY-M and BSLY-L groups(P<0.001),no statistical difference between the BSLY-M and BSLY-L groups(P≥0.05),a statistical difference between the BSLY-H and FTY720 groups(P<0.01),and a statistical difference between the Lotensin and BSLY-H and FTY720 groups(P<0.001).Compared with the Model group,the expression of SDF-1 in bone marrow increased in the BSLY-H,BSLY-M,FTY720 and Lotensin groups(P<0.001,P<0.01),and in the BSLY-L group,but there was no statistical difference(P≥0.05);compared between the dosing groups,there was a statistical difference between the BSLY-H and BSLY-M and BSLY-L groups(P<0.001),and between the BSLY-M and FTY720 groups(P≥0.05).There was a statistical difference between the BSLY-H group and BSLY-M and BSLY-L groups(P<0.001),no statistical difference between the BSLY-M and BSLY-L groups(P≥0.05),a statistical difference between the BSLY-H and FTY720 groups(P<0.01),and a statistical difference between the Lotensin and BSLY-H and FTY720 groups(P<0.001).Compared with the Model group,the expression of bone marrow CXCR4 increased in each administration group(P<0.001,P<0.01);compared with each administration group,the BSLY-H group was statistically different from the BSLY-M and BSLY-L groups(P<0.001),the BSLY-M group was not statistically different from the BSLY-L group(P≥0.05),and the BSLY-H group was statistically different from the FTY720 group.There was a statistical difference between the BSLY-M group and the BSLY-L group(P ≥ 0.05),between the BSLY-H group and the FTY720 group(P<0.05),and between the Lotensin group and the BSLY-H and FTY720 groups(P<0.001).2.Real-time PCR results showed that SDF-1 expression in Peyer’s node,small intestine lamina propria and bone marrow was significantly lower in the Model group compared to the Control group(p<0.001).The expression of SDF-1 in Peyer’s node was significantly increased in all dosing groups compared with that in Model group(P<0.001);the expression of SDF-1 in BSLY-H group was statistically different from that in BSLY-M and BSLY-L groups(P<0.001),while there was no statistical difference between BSLY-M and BSLY-L groups(P≥0.05)and BSLY-H and FTY720 groups(P≥0.05).The Lotensin group was statistically different from the BSLY-H and FTY720 groups(P < 0.001).Compared with the Model group,the expression of SDF-1 in the small intestinal lamina propria was increased in the BSLY-H,FTY720,Lotensin and BSLY-M groups(P<0.001,P<0.05),and increased in the BSLY-L group,but there was no statistical difference(P ≥ 0.05);compared between groups,the expression of SDF-1 in the BSLY-H group was statistically different from that in the BSLY-M and BSLY-L groups.The BSLY-L group was statistically different from the BSLY-M and BSLY-L groups(P<0.001),the BSLY-M group was not statistically different from the BSLY-L group(P≥0.05),the BSLY-H group was statistically different from the FTY720 group(P<0.001),and the Lotensin group was statistically different from the BSLY-H and FTY720 groups(P<0.001).Compared with the Model group,the expression of SDF-1 in bone marrow increased in the BSLY-H,FTY720,Lotensin and BSLY-M groups(P<0.001,P<0.01)and in the BSLY-L group,but there was no statistically significant difference(P ≥ 0.05).There was a statistical difference between the BSLY-H group and BSLY-M and BSLY-L groups(P<0.001),no statistical difference between the BSLY-M and BSLY-L groups(P≥0.05),a statistical difference between the BSLY-H and FTY720 groups(P<0.01),and a statistical difference between the Lotensin and BSLY-H and FTY720 groups(P<0.001).Conclusion: 1.The chemokine SDF-1 and the ligand CXCR4 may not be the key molecules causing the mis-migration of IgA-secreting cells and their homing to the bone marrow.2.Bushen Liyan recipe can effectively increase the expression of SDF-1 and CXCR4 in small intestine tissues.It is speculated that the method of Bushenjianpi and Jieduliyan may regulate the expression of chemokine SDF-1 and ligand CXCR4 in small intestine tissues and the disorder of chemotaxis regulation,and selectively induce the related lymphocytes to nest to secondary lymphoid organs,thus achieving the effect of preventing and treating IgA nephropathy.Chapter 3 Effects of the method of Bushenjianpi and Jieduliyan on the TLR4/My D88/NF-κB signalling pathway in the kidney tissue of rats with IgA nephropathy and the intervention mechanism of kidney injuryObjective: To investigate the effect of the method of Bushenjianpi and Jieduliyan on the molecular regulation of TLR4/My D88/NF-κB signaling pathway in the kidney tissue of rats with IgA nephropathy and the mechanism of anti-kidney injury intervention.Methods: 1.An animal model of IgA nephropathy was constructed and blood samples and kidney tissues were collected from rats.The expression levels of IL-6 in rat serum were measured by enzyme-linked immunoassay.2.The expression levels of TLR4,My D88,NF-κB and IL-6 in kidney tissues were measured by Western Blot.3.Immunohistochemical assay to detect TLR4,My D88,NF-κB and IL-6 expression levels in the kidney.Results: 1.Serum IL-6 was measured by enzyme-linked immunoassay and the results showed a significant increase in the Model group compared to the Control group(P<0.001).The results of enzyme immunoassay showed that: compared with the Control group,the Model group showed a significant increase(P<0.001),compared with the Model group,the administration groups showed a significant decrease(P<0.001);compared with the Model group,the administration groups showed a statistically significant difference(P<0.001),compared with the BSLY-M and BSLY-L groups,the BSLY-M and BSLY-L groups showed a statistically significant difference(P<0.01),the FTY720,Lotensin and BSLY-H groups showed no statistically significant difference(P≥0.05).-H group were not statistically different(P≥0.05).2.The results of Western Blot showed that the expression of renal TLR4,My D88,NF-κB and IL-6 in the Model group increased significantly compared with that in the Control group(P<0.01,P<0.001).Compared with the Model group,the expression of renal TLR4,My D88,NF-κB and IL-6 decreased in the BSLY-H,FTY720,BSLY-M groups and Lotensin groups(P<0.01,P<0.05),and decreased in the BSLY-L groups,but there was no statistical difference(P≥0.05);When comparing the renal TLR4 and My D88 groups in each dosing group,there was no statistical difference between the BSLY-H,BSLY-M and BSLY-L groups(P≥0.05),and there was no statistical difference between the FTY720 and Lotensin groups and the BSLY-H and BSLY-M groups(P≥0.05);When comparing the renal NF-κB and IL-6 between the dosing groups,there was no statistical difference between the BSLY-M,BSLY-H and BSLY-L groups(P≥0.05),there was a statistical difference between the BSLY-H and BSLY-L groups(P<0.05,P<0.01),and there was no statistical difference between the FTY720 and Lotensin groups and the BSLY-H and BSLY-M groups(P≥0.05).There was no statistical difference between the FTY720 and Lotensin groups and the BSLY-H and BSLY-M groups(P≥0.05).3.The results of immunohistochemistry showed that the expression of renal TLR4,My D88,NF-κB and IL-6 was significantly increased in the Model group compared with the Control group(P<0.001).Compared with the Model group,the expression of renal TLR4,My D88,NF-κ B and IL-6 decreased in the BSLY-H,FTY720,BSLY-M and Lotensin groups(P<0.01,P<0.05)and the BSLY-M group(P<0.05).There was no statistical difference between the groups of BSLY-H,BSLY-M and BSLY-L(P≥0.05)and between the groups of FTY720 and Lotensin and the groups of BSLY-H,BSLY-M and BSLY-L(P≥0.05)in the comparison of renal TLR4 and My D88 administration.There was no statistical difference(P≥0.05)between the groups of renal NF-κB and IL-6 administration,and there was no statistical difference(P≥0.05)between the groups of BSLY-M and BSLY-H and BSLY-L,and a statistical difference(P<0.05)between the groups of BSLY-H and BSLY-L,and between the groups of FTY720 and Lotensin and BSLY-H and BSLY-L.There was no statistical difference between the FTY720 and Lotensin groups and the BSLY-H,BSLY-M and BSLY-L groups(P≥0.05).Conclusion: 1.Antigen activates the TLR4/My D88/NF-κB signaling pathway and drives up the expression of the downstream cytokine IL-6,thus causing the development of renal injury in IgA nephropathy.2.Bushen Liyan recipe can effectively reduce the expression of serum IL-6 and effectively reduce the expression of TLR4,My D88,NF-κB and IL-6 in kidney tissues.It is hypothesized that the method of Bushenjianpi and Jieduliyan may achieve the effect of preventing and treating IgA nephropathy and anti-kidney injury by blocking the expression of TLR4/My D88/NF-κB signaling pathway in kidney tissues. |
PDF Full Text Request