| Objective:To explore the mechanism of Jiawei Guizhi Poria Pills in the intervention of painful diabetic peripheral neuropathy(PDPN).The network pharmacology was used to explore the signal pathway of Jiawei Guizhi Poria Wan in the intervention of PDPN.By observing the behavioral and mental status,body weight,blood glucose,motor nerve conduction velocity,heat contraction latency,mechanical pain threshold,sciatic nerve morphology,expression of VEGF and IGF-1 in sciatic nerve and the changes of serum NGF content of PDPN rats during different treatment periods,It provides the experimental theoretical basis for the new idea of Jiawei Guizhi Poria pills in the clinical treatment of PDPN.Materials and METHODS:The active constituents and targets of Jiawei Guizhi Poria pills were screened by TCMSP and Herb database,and supplemented by Swiss target Protect database for target prediction.Converting targets into gene abbreviations in the Uni Prot database;Gene targets of PDPN were screened from Gene Cards,OMIM and Drug Bank databases.venny 2.1.0 was used to construct Venny diagram and derive an excel table of intersecting gene targets of drugs and diseases.Constructing "PPI" and Drug-Active Ingredient-Core Target" network diagrams via String database and CytoscaPe 3.9.1;The intersection targets of drugs and diseases were imported into DAVID database for GO enrichment analysis and KEGG pathway enrichment analysis,and the possible mechanism of Jiawei Guizhi Poria pills in the treatment of PDPN was discussed.Among 104 SPF healthy male SD rats(weight 200-220g),12 rats were selected as the normal control group by random number table method,and the remaining 92 rats were selected as the modeling group.After 1w of normal feeding,modeling began with a 12-hour fast.The chain urea with cephalosporins producing 2%(STZ StrePtozotocin)configured to STZ solution,abdominal sterilization,according to the single 50 mg/kg dose,intraperitoneal injection to induce diabetes,normal control group of rats injected to its with the same amount of excluding STZ solution.After 72 hours of modeling,caudal vein blood was collected to detect the blood glucose of the rats.The rats lower than 16.7mmol/L were excluded from the experiment,and the remaining rats were included in the diabetes model and fed with ordinary diet for 4w,and then the pain threshold(heat pain or mechanical pain)was determined.If the pain threshold was reduced by more than 50%,the rats could be included in the PDPN model.PDPN rats were randomly divided into model control group,Yikai(pancreatic kininogenase)group,low-dose Chinese medicine group,medium-dose Chinese medicine group and high-dose Chinese medicine group.During the experiment,all the rats were fed ordinary diet and were fed freely.The behavioral state and mental state of the rats were observed at 0w,2w,4w and 8w,respectively,and the weight and blood glucose were dynamically monitored.The effects of Jiawei Guizhu Poria pills on the pain threshold of the rats were detected by Von Frey fiber pain meter and thermal pain threshold meter.The morphological changes of sciatic nerve in PDPN rats were observed by HE staining,the content of serum NGF was detected by Elisa,the gene expressions of VEGF and IGF-1 in sciatic nerve were detected by RT-PCR,and the protein expressions of IGF-1 and VEGF in sciatic nerve were detected by Western blot.SPSS 26.0 was used for statistical analysis,and P<0.05 was statistically significant.Results:1.Jiawei Guizhu Poria Pills mainly regulate inflammation and repair neurons by regulating AKT1,STAT3,TP53,RELA(NF-kB P65),MAPK1,TNF,IL6 and other key targets to treat PDPN.2.Jiawei Guizhi Poria Wan mainly regulates HIF-1 signaling pathway and VEGF signaling pathway,down-regulates VEGF expression,up-regulates IGF-1 expression,improves hypoxia,reduces inflammation and treats PDPN.3.The rats in the model control group had poor mental state,strong odor,slow movement,dry and dull hair,and were accompanied by symptoms of polydipsia,polyphagia,polyuria and emaciation.After 8w of intragastric administration,the above symptoms still existed in all treatment groups,but the symptoms were reduced compared with the model control group.4.Compared with the normal control group,the body weight of the modeling group decreased significantly(P<0.01);After 8w of intragastric administration,compared with model control group,the body weight of rats in all treatment groups was increased(P<0.05),but there was no significant difference among all treatment groups(P>0.05).5.Compared with the normal control group,blood glucose in the modeling group was significantly increased(P<0.01);After 8w of intragastric administration,compared with model control group,the blood glucose level of rats in all treatment groups decreased(P<0.05),and there were significant differences between high-dose group and Yikai group(P<0.01).6.Compared with the normal control group,the nerve conduction velocity in the modeling group decreased significantly(P<0.01);After 8w of intragastric administration,compared with model control group,nerve conduction velocity of rats in treatment groups was increased(P<0.05),and there were significant differences between high-dose group and Yikai group(P<0.01).7.Compared with the normal control group,the incubation period of heat contraction in the modeling group was significantly shortened(P<0.01);After 8w of intragastric administration,the incubation period of heat contraction was significantly prolonged in all treatment groups compared with model control group(P<0.01),and there were significant differences between high-dose group and Yikai group(P<0.01).Compared with the normal control group,the mechanical pain threshold in the modeling group was significantly decreased(P<0.01).After 8w of intragastric administration,compared with model control group,mechanical pain threshold of all treatment groups was significantly increased(P<0.01),and there were significant differences between high-dose group and Yikai group(P<0.01).8.Compared with the normal control group,the nerve fibers in the model control group were disordered,with different sizes and incomplete shapes.Obvious pathological changes were observed,a large number of nerve fibers were edema,and the myelin sheath structure was shed and lost into vacuoles.Compared with the model control group,the arrangement of nerve fibers in each treatment group was more regular,the shape was more uniform,the degree of myelination was higher,and the degree of edema was less.9.Compared with the normal control group,the content of NGF in serum of the model group was significantly decreased(P<0.01);After 8w intragastric administration,compared with model control group,serum NGF content in all treatment groups was significantly increased(P<0.01),and there were significant differences between high-dose group and Yikai group(P<0.01).10.Compared with the normal control group,the content of VEGF mRNA in sciatic nerve of the modeling group was significantly increased(P<0.01).After 8w of intragastric administration,the content of VEGF mRNA in sciatic nerve of the treatment groups was significantly decreased compared with the model control group(P<0.01),and the high-dose group was significantly different from the Yikai group(P<0.01).The IGF-1 mRNA content in sciatic nerve of the modeling group was significantly decreased(P<0.01).Compared with model control group,the IGF-1 mRNA content in sciatic nerve of all treatment groups was significantly increased after 8w intragastric administration(P<0.01),and there were significant differences between high-dose group and Yikai group(P<0.01).11.Compared with the normal control group,the content of VEGF protein in sciatic nerve of the modeling group was significantly increased(P<0.01).After 8w of intragastric administration,the content of VEGF protein in sciatic nerve of the treatment groups was significantly decreased compared with the model control group(P<0.01),and there were significant differences between the high-dose group and Yikai group(P<0.05).The content of IGF-1 protein in sciatic nerve of modeling group was significantly decreased(P<0.01);After 8w of intragastric administration,the content of IGF-1 protein in sciatic nerve of all treatment groups was significantly increased compared with model control group(P<0.01),and there were significant differences between high-dose group and Yikai group(P<0.05).Conclusion:1.Jiawei Guizhi Poria Pills can treat painful diabetic peripheral neuropathy,and its mechanism may be related to inhibiting HIF-1 pathway,down-regulating VEGF,up-regulating IGF-1 and NGF content,alleviating hypoxia injury and reducing inflammation.2.Jiawei Guizi Poria Wan can improve blood glucose and body weight,motor nerve conduction velocity,heat contraction latency,mechanical pain threshold,pathological morphology of sciatic nerve,contents of sciatic VEGF and IGF-1 and serum NGF content in PDPN rats,so Jiawei Guizi Poria Wan can treat PDPN. |
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