Experimental Study On The Mechanism Of Action Of Mudan Granules In The Intervention Of Diabetic Peripheral Neuropathy And Network Pharmacolog

Purpose:To explore the optimal starting time and intervention mechanism of Mudan granule in the prevention and treatment of diabetic peripheral neuropathy.The changes of general state,body weight,blood glucose,latent period of heat contraction foot,sciatic nerve conduction velocity,sciatic nerve morphology,NF-κB protein content and serum inflammatory factors IL-6 and TNF-α in diabetic rats with different initial treatment time were observed.It provides experimental theoretical basis for mudan granule in clinical treatment of diabetic peripheral nerve and a new perspective for clinical treatment.Material and method:A total of 60 healthy SPF male SD rats weighing(230-270)g were selected.After 1 week of adaptive feeding,10 rats were randomly selected as the normal control group.The other 50 rats were fasting(overnight)for 12 hours and drinking water freely.Streptozotocin-1 %STZ solution(50 mg/kg)prepared by single injection of 0.1 mol/L Citric acid buffer PH4.2 in the left lower abdomen is developed.After 72 hours,the mice with fasting blood glucose ≥16.7mmol/L are considered as model rats.They were randomly divided into STZ model group,0W gavage group,2W gavage group,4W gavage group and6 W gavage group.The STZ model group was given normal saline at the same dose from 0W,and the group was given mudan granules at different starting times of 0W,2W,4W and 6W until 8W after model formation.At the end of 8 weeks,the changes of general state,blood glucose,body weight,latent period of heat contraction foot and conduction velocity of motor nerve were observed.The morphology of nerve fiber myelin sheath and axon were observed,and the content of NF-κB protein in sciatic nerve and the content of IL-6 and TNF-α in serum were detected.The effective ingredients and target information of Mudan granules were screened by TCMSP and chemical database,and the gene names corresponding to target were obtained by Uniprot database.Gene targets of DPN were obtained from Drug Bank,Gene Cards and OMIM disease gene databases.Venny2.1.0 software was used to draw venny diagram to obtain drug disease co-action targets.Using String database and Cytoscape3.7.1 software to draw "PPI" network diagram and "TCM-component-target" network diagram;The Metascape database was used to enrich the targets and further explore the mechanism of action of Mudan granule in treating DPN.Results:1.The rats in the normal control group were in good mental state with neat,smooth and shiny fur,significantly increased body weight,normal blood sugar and sensitive movement.Rats in STZ model group showed listless spirit,sluggish action,crouched back,dry fur,significant increase in food intake,water intake and urine volume,and relatively slow weight gain.All the other treatment groups showed the above symptoms,but compared with the STZ model,the symptoms were alleviated to varying degrees.The general state of the rats in the 0W and2 W intragastric groups was most significantly improved.2.Compared with normal control group,body weight of STZ model group and gavage groups was significantly decreased(P < 0.01);The body weight of each group was statistically different from that of the STZ model group(P<0.05),and the difference between the 0W gavage group and STZ model group was significant(P<0.01);3.Compared with normal control group,blood glucose levels in STZ model group and gavage groups were significantly increased(P < 0.01);There was no significant difference in blood glucose between all gavage groups and STZ model group(P > 0.05).4.Compared with normal control group,nerve conduction velocity in STZ model group and gavage group decreased significantly(P < 0.01),and nerve conduction velocity in EACH gavage group increased compared with THAT in STZ model group(P < 0.01).The nerve conduction velocity of the 0W group was significantly higher than that of the 2W and 4W groups(P < 0.01),and the nerve conduction velocity of the 2W group was higher than that of the 6W group(P< 0.05).The nerve conduction velocity of 4W group was higher than that of6 W group(P < 0.05).5.The incubation period of heat shrinkable foot in STZ model group,all gavage groups and normal control group decreased significantly(P < 0.01).Compared with STZ model group,the incubation period of heat shrinkable foot in all gavage groups was prolonged,with There were statistically significant differences(P < 0.05),and the incubation period of heat shrinkable feet was significantly prolonged in 0W,2W and 4W groups(P< 0.01).The latencies of heat shrinkable feet in 0W gavage group were longer than those in 2W gavage group(P < 0.05),and significantly longer than those in 4W and 6W gavage groups(P< 0.01).The incubation period of heat shrinkable feet in 2W group was significantly longer than that in 4W and 6W groups(P < 0.01).There was no statistical difference in heat shrinkable foot latency between the 4W group and the 6W group(P > 0.05).6.Normal resistance was observed under optical microscope,nerve fibers were normal,evenly distributed and dense,myelin sheath staining was uniform,and axons were clear;In STZ model group,nerve fibers were loosely arranged,and myelin density was uneven,thinning and even demyelination occurred.However,the nerve fiber injury was improved in the gavage group.The improvement was most significant in the 0w and 2w groups,and the arrangement of nerve fibers was orderly and the staining density was uniform.7.Compared with the normal control group,the protein content of NF-κB P65 in the sciatic nerve of the STZ model group and the gavage groups was significantly increased(P < 0.01),and the protein content of NF-κB P65 in the sciatic nerve of the STZ model group was significantly increased(P < 0.05).There were significant differences at 0W,2W and 4W(P <0.01).Compared with 0W group,NF-κB P65 protein was increased in 6W group,and the difference was statistically significant(P<0.05).8.Compared with normal control group,serum IL-6 content and TNF-A content in STZ model group and gavage groups were significantly increased,with statistically significant differences(P < 0.01).Compared with model group,il-6 content in 0W and 2W gavage groups was significantly decreased(P<0.01)and decreased in 4W group(P<0.05).Compared with 0W administration group,the content of 4W and 6W administration groups increased significantly(P<0.05).Tnf-a was detected as follows: compared with model group,the content of tnf-a in 0W and 2W groups decreased significantly(P< 0.05),compared with the0 W group,the contents of 2W,4W and 6W groups were relatively higher(P< 0.01).9.Mudan granule mainly regulates inflammation and repairs neurons to treat DPN by regulating TP53,TNF,RELA(NF-κB P65),JUN,MAPk1,Ak T1 and other key targets.10.Mudan granules mainly regulate age-rage signaling pathway,NF-Kappa B signaling pathway,Dopaminergic Synapse signaling pathway and other signaling pathways,regulate vascular endothelial factor,reduce blood glucose,reduce the accumulation of AGEs to treat DPN.Conclusion:1.The application of Muldan granule at different starting time can improve the motor nerve conduction velocity,the latent period of heat pain threshold response,the pathological morphology of sciatic nerve,the content of NF-κB P65 protein in sciatic nerve,the content of IL-6 and TNF-A in serum of diabetic rats.And the earlier the application effect is relatively obvious.Therefore,the application of Mudan granules in the early stage of diabetes can effectively delay the development of diabetic peripheral neuropathy.2.The mechanism of mutan granule delaying the occurrence of diabetic peripheral neuropathy may be related to its inhibition of NF-κB pathway,down-regulation of inflammatory factors IL-6 and TNF-A,and alleviation of inflammatory injury.