Discussion On The Mechanism Of Pheretima In Treatment Of Diabetic Kidney Disease Based On The Theory Of “Unsmooth Blood Circulation Results In Water Retention”

Objective:In this study,based on the “unsmooth blood circulation resulis in water retention”,the effect of effective components of Pheretima on TNF-α,IL-6and MCP-1 mRNA inflammatory factors and the expression levels of IKB,IKKβ and NF-κB in high-glucose HMC model cultured in vitro were observed from the point of entry of inflammatory response related factors and NF-κB signaling pathway.To study the specific transduction process and molecular mechanism of DKD treatment by effective components of Pheretima,and to provide reference for drug research and development of diabetic nephropathy.Methods:The cultured HMC cells were divided into Pheretima low-dose,medium-dose and high-dose groups and Fosinopri groups,and the normal group and the model group were set up for control.The specific groups were blank group: 89% DMEM(high glucose)+10% FBS+1% double antibody;Model group: on the basis of blank group,add 45 mmol/L D-glucose;Pheretima,medium-dose and high-dose groups: 80 μg,120 μg and160μg the effective ingredient of Dilong is dissolved solution were added to the model group,respectively;Fosinopril group: 10 g/ m L Fosinopril sodium was added on the basis of model group.MTT method was used to detect the growth and proliferation level of HMC cells,and the optimal dose group was obtained.At the same time,HE staining light microscopy was used to observe the HMC modeling in each group and the changes of morphology,structure and nucleus after drug intervention.The mRNA expression levels of TNF-α,IL-6 and MCP-1 in the remaining groups were detected by RT-qPCR.The protein expression levels of IKB,IKKβ and NF-κB were detected by Western Blot.Results:1.MTT assay showed that compared with blank group,the proliferation level of HMC cells in model group was increased(P<0.01).Compared with model group,the growth and proliferation level of HMC cells in DTH medium and high dose groups and Fosinopril groups was decreased(P<0.05).The average inhibition rate in the medium dose group was the highest.The results indicated that the medium dose group of Pheretima had the best intervention for HMC under high glucose.2.Under the light microscope of HE staining,it was found that the structure of the normal group was clear,the cytoplasm was multi-protruding,and the nucleus was in the center,and most of them were round or oval.The HMC cells with high glucose was fused and the nuclei were shrunk,indicating that the modeling was successful.Compared with the model group,after the intervention with Pheretima,the nuclei of HMC cells were significantly increased and the morphology tended to be normal.3.RT-qPCR technology showed that the contents of TNF-α,IL-6 and MCP-1 in cell culture medium of Pheretima group(Pheretima medium dose group)and Fosinopril showed a decreasing trend after intervention,compared with model group.The expression levels of TNF-α mRNA,MCP-1 mRNA and IL-6 mRNA in culture medium were decreased in ground Pheretima group(P<0.05).There was no statistical significance in the expression levels of TNF-α mRNA,MCP-1 mRNA and IL-6 mRNA between Pheretima group and Fosinopril group(P>0.05).Conclusion:1.The active components of Pheretima can inhibit the proliferation of HMC cultured with high sugar content.2.The effective components of Pheretima may be used to treat DKD by down-regulating the expression levels of TNF-α mRNA,MCP-1 mRNA and IL-6 mRNA factors.3.The effective components of Pheretima may be treated be used to treat DKD by down-regulating the expression levels of IKB,IKKβ and NF-κB.