| Objective: To explore the mechanism of abnormal migration of Ig A-secreting cells from mucosa to bone marrow mediated by abnormal chemokine CCL28/CCR10,and to reveal the mechanism of Bushen Jianpi、Jiedu Liyan,in the treatment of Ig AN.Methods: 1.Modeling method: 140 SPF male SD rats with a body weight of 180±20g were randomly divided into 20 blank rats and 120 model rats;the Ig AN rat animal model was prepared by Lu Huiyu’s improved method.2.Grouping and treat method: After the model was successfully verified,the rats were randomly divided into 7 groups,blank group,model group,high-dose,medium-and low-dose Chinese medicine group,FTY720 group,and Benazepril Hydrochloride Tablets(BHT)group;The blank group and the model group were given normal saline 4ml/kg by gavage once a day;FTY720 group was given FTY720 0.5mg/kg by gavage once a day;BHT group was given BHT 1.041mg/kg by gavage once a day;TCM low-dose group was given TCM 4ml/kg,containing drug 10.4g,once a day;Chinese medicine medium-dose group was given TCM 4ml/kg,containing drug20.81 g,once a day;TCM high dosage group was given TCM 4ml/kg,containing drug41.62 g,once a day;continuous administration for 6 weeks.3.Observation indicators:(1)Validation model: Ig A deposition in kidney tissue was observed by Immunofluorescence method;(2)TLR9 expression levels in small intestine propria,Peyer’s nodules and bone marrow were detected by Flow Cytometry;(3)Chemokines and ligands CCL28/CCR10,adhesion molecules VCAM-1,and integrins α4 and β1 that mainly mediate lymphocyte migration and homing were detected by RT-PCR,expressed in the lamina propria of small intestine,Peyer’s nodules and bone marrow;(4)Ig A protein expression levels in bone marrow and kidney tissue were detected by Immunohistochemistry;(5)Transferrin receptor(Tf R)and transforming growth factor-β(TGF-β)expression in kidney tissue.Results:1.The results of renal tissue immunofluorescence showed that there was no obvious Ig A immunofluorescence in the glomeruli of the rats in the blank group,and a large number of green fluorescence signals were seen in the glomeruli of the Ig AN model rats,with discontinuous granular deposition.2.The results of Flow Cytometry showed that compared with the blank group,the expression levels of TLR9 in the small intestinal lamina propria,Peyer’s nodules and bone marrow tissues of the rats in the model group increased(P<0.001).After medication intervention,compared with the model group,the expression levels of TLR9 in the above three tissues were significantly decreased in the high,medium and low dose groups of traditional Chinese medicine,the FTY720 group and the BHT group(P<0.001,P<0.01,P<0.05).There was no significant difference between the FTY720 group and the high-dose Chinese medicine group in each tissue(P>0.05).3.RT-PCR test results showed that compared with the blank group,the expressions of CCL28 and CCR10 in the small intestine lamina propria and Peyer’s nodules of the rats in the model group were significantly decreased(P<0.001),and the expression of α4,β1 and VCAM-1 were significantly increased(P<0.001,P<0.01,P<0.05).The expressions of CCL28 and CCR10 in the bone marrow of rats in the model group increased significantly(P<0.001),while the expressions of α4 and β1 showed a downward trend,but there was no statistical significance(P>0.05).The expression of VCAM-1 Significantly increased(P<0.05).3.1 In the small intestine lamina propria,after drug intervention in each group,compared with the model group,the expression levels of CCL28 and CCR10 in the small intestine lamina propria tissue of the rats in each group increased significantly(P<0.001,P<0.01,P <0.05),the expression of α4,β1 and VCAM-1 was significantly decreased(P <0.05),and there was no difference between the FTY720 group and the high-dose Chinese medicine group(P>0.05).The middle-dose group was better than the low-dose group(P<0.05).3.2 In Peyer’s nodules,after drug intervention in each group,compared with the model group,the expressions of CCL28 and CCR10 in Peyer’s nodules of rats in each group increased significantly(P<0.001),the expression level of α4,β1,VCAM-1 were significantly decreased(P<0.05),and there was no difference between the FTY720 group and the high-dose traditional Chinese medicine group(P>0.05).Compared with the traditional Chinese medicine group,the high and medium dose groups of traditional Chinese medicine were better than the low dose group(P<0.05).3.3 In the bone marrow,after the intervention in each group,compared with the model group,the expressions of CCL28 and CCR10 in the bone marrow of the rats in each group were significantly decreased(P<0.001).The expression of α4,β1 and VCAM-1 were significantly decreased(P<0.01,P<0.05),and there was no difference between the FTY720 group and the high-dose Chinese medicine group(P>0.05).4.The detection results of Immunohistochemistry showed that compared with the blank group,the Ig A staining area and average optical density of the kidney and bone marrow tissue of the rats in the model group were significantly increased(P<0.001).Compared with model group,the area and average optical density of Ig A staining in the kidney and bone marrow tissue of rats in each group were significantly decreased,among which the high,medium and low dose groups of traditional Chinese medicine,FTY720 group and BHT group significantly decreased(P<0.001,P<0.01,P< 0.05),and there were differences between the FTY720 group,the Luo Ting Xin group and the high,medium and low dose groups of traditional Chinese medicine(P > 0.05).5.The results of Immunohistochemical technology showed that compared with the blank group,the staining area and average optical density of Tf R and TGF-β1 in the kidneys of the rats in the model group were significantly increased(P<0.001);Compared with other groups,the staining area and average optical density of Tf R and TGF-β1 in the kidneys of rats in each group were significantly decreased(P<0.001,P<0.01,P<0.05),and there was no difference between the FTY720 group and the high-dose Chinese medicine group(P>0.05).Conclusion:1.The method of Bushen Jianpi、Jiedu Liyan can improve the symptoms of hematuria and proteinuria,relieve inflammatory stimulation,and alleviate the disease by downregulating the expression of small intestinal mucosal lamina propria,Peyer’s nodules and bone marrow TLR9 in Ig AN model rats.2.The method of Bushen Jianpi 、 Jiedu Liyan can regulate the distribution of chemokines and ligands CCL28/CCR10,adhesion molecules VCAM-1,integrin α4,β1 in the mucosa-bone marrow axis and play a therapeutic role.3.The method of Bushen Jianpi、Jiedu Liyan can indirectly reduce the deposition of Ig A or Ig A-based immune complexes and protect the kidneys.4.TfR and TGF-β1 may be involved in the process of Ig AN renal fibrosis;the methods of Bushen Jianpi、Jiedu Liyan can reduce kidney damage by down-regulating the expression of the two factors in the kidney,delay renal fibrosis,protect the kidneys. |
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