| Purpose:1.To observe the prevention and treatment effect of Yiqi Huoxue Tongluo Decoction on diabetic peripheral neuropathy through its effect on nerve conduction velocity,sciatic nerve tissue structure and pathological changes in diabetic rats.Besides,the potential targets of Yiqi Huoxue Tongluo Decoction for diabetic peripheral neuropathy were screened by network pharmacology and molecular docking.2.The AMPK/SIRT1/NF-κBp65 pathway was selected to observe the effect of Yiqi Huoxue Tongluo Decoction on the microcirculation disorder in diabetic rats.3.To observe the effect of Yiqi Huoxue Tongluo Decoction on energy metabolism of diabetic peripheral neuropathy rats and on the mitochondrial synthesis pathway AMPK/PGC1α/TFAM;To observe the effect of Yiqi Huoxue Tongluo Decoction on the autophagy of diabetic peripheral neuropathy rats based on AMPK/m TORc1/ULK1 pathway.4.Based on the theories of"qi-collaterals"and"blood-collaterals",the authors explored the correlation between the two and energy metabolism and microcirculation of diabetic peripheral neuropathy,and analyzed the possible mechanism of Yiqi Huoxue Tongluo Decoction in preventing and treating diabetic peripheral neuropathy.Material and method:1.Sixty male SPF Wistar rats were adaptively fed for one week.After one week,ten of them were randomly selected as the normal control group,and the remaining 50 rats were modeled based on diabetes mellitus induced by intraperitoneal injection of 53mg/kg streptozotocin(STZ).After 72 hours,blood was taken from the tail vein to determine the random blood glucose.A total of 45 rats(≥16.7mmol/L)for three consecutive times were considered to be diabetic model rats.The 45 rats were randomly divided into a model control group,a western medicine control group,and a Chinese medicine group,15 rats in each group according to blood glucose values.The Chinese medicine group was intragastrically administered with Yiqi Huoxue Tongluo Decoction(16.2ml/kg/d),the western medicine group was intragastrically administered with mecobalamin suspension(10.8ml/kg/d),and the model control group and the normal control group were intragastrically administered with equal volume distilled water for consecutive 8 weeks.During the drug intervention,the body weight of rats was measured every week and the intragastric dose was adjusted.Blood was taken from the caudal vein and the blood glucose was measured before and at 2,4,6,and 8 weeks after modeling.The mechanical pain threshold and sciatic nerve conduction velocity were measured at 4,6,and 8 weeks after modeling.After eight weeks of medication intervention,the rats were anesthetized with 20%urethane.Blood was taken from the abdominal aorta,centrifuged in a low-temperature ultracentrifuge for 10min,and the supernatant was subpackaged and frozen in a-80℃refrigerator for standby.The bilateral sciatic nerves of each rat were isolated,and a part of the sciatic nerves of each rat were stored in the fixative for pathological observation,and the rest of the tissues were frozen in the-80℃refrigerator for later use.Morphological changes of sciatic nerve in rats were observed by HE staining.Morphological changes of myelin sheath,unmyelinated nerve fibers and subcellular organelles in nerve cells were observed by a transmission electron microscope;Querying the chemical active substances and diabetic peripheral neuropathy related targets of the Yiqi Huoxue Tongluo decoction by using a traditional Chinese medicine system pharmacology technology platform(TCMSP)database and a genome annotation database platform(Genecards),screening common target genes of drugs and diseases,performing KEGG enrichment analysis,constructing a target-pathway network diagram,and verifying the docking condition of the screened core targets and chemical small molecules by using a molecular docking technology.2.The relative expression levels of AMPK,p-AMPK,SIRT1,and NF-κBp65 proteins in the sciatic nerve tissue of rats in each group were detected by Western-blot.The expressions of Sirt1 and NF-KB proteins in the sciatic nerve of rats in each group were detected by immunohistochemistry.The contents of serum IL-1β,IL-6,s VCAM-1 and TNF-αwere detected by ELISA.3.The relative expression levels of PGC-1α,TFAM,ULK1,and Drp-1 proteins in the sciatic nerve tissue were detected by Western-blot.The expressions of PGC-1α,TFAM,ULK1,and Drp-1 proteins in the sciatic nerve were detected by immunohistochemistry.The serum NA~+-K~+-ATPase level was detected by ELISA.Determination of ATP content in nerve tissue by luciferase bioluminescence assay.4.Expound and analyze the concepts and physiological functions of"qi-collaterals"and"blood-collaterals",trace the theoretical origin of the relationship between diabetic peripheral neuropathy and collaterals,then further explore the pathogenesis of diabetic peripheral neuropathy through the theory of"qi-collaterals and blood-collaterals".Results:1.After intraperitoneal injection with 2%STZ,a total of 45 rats were successfully modeled,with the modeling rate is 90%;After eight weeks of drug intervention,41 rats were finally left,including 10 rats in the normal control group,13 rats in the model control group,14 rats in the western medicine control group and 14 rats in the Chinese medicine group.During the experiment,the body weight of rats in the normal control group increased steadily,while that in the model group decreased gradually.Compared with the model control group,the body weights of rats in the Chinese medicine group and western medicine control group were increased significantly(P<0.01),and those in the Chinese medicine group were higher than those in the western medicine control group(P<0.05).The results of blood glucose showed that the blood glucose of the normal control group was stable before and after medication,while the blood glucose of other groups was significantly increased(P<0.01).Six weeks after medication,the blood glucose level of rats in the Chinese medicine group was significantly lower than that in the model control group(P<0.05).Mechanical pain threshold and nerve conduction velocity measured at the 4th,6th and 8th week showed that the values of the mechanical pain threshold and nerve conduction velocity in the model control group were significantly lower than those in the normal control group(P<0.01),while those in the western medicine control group and the Chinese medicine group were significantly higher than those in the model control group(P<0.05).Moreover,the improvement degree in the Chinese medicine group was higher than that in the western medicine control group(P<0.05).The results of HE staining showed that the nerve tissues in the model control group were sparsely distributed and lightly stained.The improvements in both the western medicine control group and the Chinese medicine group were significant,and the improvement in the Chinese medicine group was due to the improvement in the western medicine control group.The results of transmission electron microscopy showed that compared with the normal control group,the myelin sheaths of the sciatic nerve in rats of the model control group were seriously separated,and the nuclear and nuclear membranes in Schwann cells were shrunken,and the mitochondria were swollen and dissolved.The distribution of unmyelinated nerve fibers was sparse and damaged.Compared with the model control group,the improvements in the western medicine control group and the Chinese medicine group were significant,and the improvement in the Chinese medicine group was better than that in the western medicine control group;Network pharmacology results showed that the chemical substances such as quercetin,luteolin,kaempferol,wogonin,and licochalcone A contained in Yiqi Huoxue Tongluo Decoction had a high degree of correlation with diabetic peripheral neuropathy targets.KEGG enrichment results included AGE-RAGE signaling pathway,fluid shear stress and atherosclerosis,IL-17 signaling pathway,TNF signaling pathway,and others,which were complications of diabetes.The results of target-pathway network construction showed that the targets with the highest degree of core to the above pathways were RELA and AKT1.The molecular docking results showed that the binding energies at quercetin-AKT1(-9.6),quercetin-RELA(-7.4),luteolin-AKT1(-9.6),and glycyrrhetinic chalcone A-RELA(-7.5)were the lowest.2.Western-blot results showed that,compared with the normal control group,the expression levels of p-AMPK,AMPK,p-AMPK/AMPK and SIRT1 in nervous tissues of rats in the model control group were significantly reduced,and the expression level of NF-κBp65 was significantly increased(P<0.01).Compared with the model control group,the expression levels of p-AMPK,AMPK,p-AMPK/AMPK and SIRT1 in the Chinese medicine group were significantly increased,while the expression level of NF-κBp65 was significantly decreased(P<0.01).The expression of each index in the western medicine control group was slightly improved,but there was no significant difference(P>0.05).Immunohistochemical results showed that,compared with the normal control group,the brown staining of AMPK and SIRT1 proteins in paraffin sections of nerve tissue in the model control group was lighter,and the positive expressions were not significant.The average optical density value was significantly reduced(P<0.01).The staining of NF-κBp65 protein was significantly deepened,and the positive expression was increased,with the average optical density value significantly increased(P<0.01).Compared with the model control group,the yellowish-brown staining of AMPK and SIRT1 proteins in the Chinese medicine group was relatively deepened,and the positive expressions were increased,with the average optical density value significantly increased(P<0.01).The staining of NF-κBp65 protein was significantly lighter,and the positive expressions were decreased,with the average optical density value significantly decreased(P<0.01).ELISA results showed that compared with the normal control group,serum IL-1β,s VCAM-1,IL-6,and TNF-αlevels of rats in the model control group were significantly increased(P<0.01).Compared with the model control group,each index of rats in the Chinese medicine group was significantly reduced(P<0.01).3.Western-blot results showed that,compared with the normal control group,the expression levels of PGC-1α,TFAM and ULK1 proteins in the nervous tissues of rats in the model control group were significantly reduced,and the expression level of DRP-1 was significantly increased(P<0.01).Compared with the model control group,the expression levels of PGC-1α,TFAM and ULK1 proteins in the traditional Chinese medicine group were significantly increased(P<0.01).There was no significant change in the expression of DRP-1(P>0.05).In the western medicine control group,the expression of PGC-1αprotein was significantly increased(P<0.05),and no significant change was observed in the other indicators(P>0.05).Immunohistochemical results showed that,compared with the normal control group,the brown staining of PGC-1α,TFAM and ULK1 proteins in paraffin sections of nerve tissue in the model control group was lighter,and the positive expressions were not significant.The average optical density value was significantly reduced(P<0.01).Compared with the model control group,the staining of PGC-1α,TFAM and ULK1 proteins in paraffin sections of nerve tissue in the Chinese medicine group was deepened,the positive expressions were increased,and the average optical density value was significantly increased(P<0.01).Compared with the normal control group,the results of ELISA showed that the Na~+–K~+-ATPase levels of rats in the model control group were significantly reduced(P<0.01).Compared with the model control group,NA~+-K~+-ATPase levels in the western medicine control group and the Chinese medicine group were significantly increased(P<0.01).Compared with the normal control group,the results of fluorescence detection showed that the ATP level in nervous tissue of rats in the model control group was significantly reduced(P<0.01).Compared with the model control group,the ATP level in the western medicine control group was significantly increased(P<0.05),and the ATP level in the Chinese medicine group was significantly increased(P<0.01).4.Qi collateral is the place where the essence,qi,blood,body fluid and liquid transformed in the whole body,promoting material exchange and energy metabolism of the body.Blood collateral,also known as venation,has the function of containing and running blood;Because it has the pathogenesis characteristics of collateral disease,diabetic peripheral neuropathy commonly uses collateral disease theory to study and explain its pathogenesis.The normal operation of qi collaterals can be maintained only by the stimulation and promotion of qi,so the function of qi collaterals is closely related to energy metabolism.The lesion of qi collaterals can be regarded as energy metabolism disorder.The pathological changes of blood collaterals are closely related to the microcirculation disorders caused by hemodynamic abnormalities and vascular endothelial damage.Conclusion:1.Yiqi Huoxue Tongluo Decoction can significantly improve the mechanical pain threshold and sciatic nerve conduction velocity in rats with diabetic peripheral neuropathy and delay the pathological damage of sciatic nerve tissue in rats with diabetic peripheral neuropathy,and has a good prevention and treatment effect on diabetic peripheral neuropathy.At the level of biological information,it was found that the main components of Yiqi Huoxue Tongluo Decoction could affect the gene expression of RELA(p65),and treat DPN by anti-oxidation,anti-inflammation and anti-apoptosis pathways.2.Animal experiments showed that Yiqi Huoxue Tongluo Decoction might down-regulate the expressions of serum s VCAM-1,IL-1β,IL-6,TNF-αand other factors by regulating the AMPK/SIRT1/NF-κBp65 signaling pathway to exert the anti-inflammatory effect,reduce vascular endothelial damage,improve hemodynamics,improve microcirculation disorders,and play a role in the prevention and treatment of diabetic peripheral neuropathy.3.Yiqi Huoxue Tongluo Decoction may activate AMPK/PGC-1α/TFAM signaling pathway,increase mitochondrial biogenesis and improve mitochondrial function,provide enough ATP for nerve tissue cells and improve NA~+-K~+-ATPase activity;At the same time,the AMPK/m TORc1/ULK1 signaling pathway was activated to promote autophagy and cell energy metabolism,thereby playing a role in improving nerve conduction velocity and inhibiting nerve injury.4.The symptoms and disease location of diabetic peripheral neuropathy are very similar to collateral diseases,and the medical mechanism is closely related to the lesions of qi and blood collaterals.Modern medicine pathogenesis shows that diabetic peripheral neuropathy is closely related to abnormal cell energy metabolism and microcirculation disorder.The function of qi meridian can reflect whether the energy metabolism of the body is normal or not,and the function of blood meridian can reflect the circulation of the body,especially microcirculation. |
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