A Metabolic Insight Into The Neuroprotective Effect Of Jin-Mai-Tong On Diabetic Rats With Peripheral Neuropathy Based On Metabolomics

[Objective]1.The aim of this study is to explore the neuroprotective effect of traditional Chinese medicine(TCM)Jin-Mai-Tong(JMT)decoction on STZ-induced diabetic rats with peripheral neuropathy:2.Based on an untargeted metabolomics approach,the aim of the study is also to investigate the effect of JMT decoction on the metabolic phenotype of diabetic peripheral neuropathy(DPN)rats and elucidate the potential molecular mechanism of JMT.[Methods]1.Male Sprague-Dawley(SD)rats were induced to be diabetic rats by single intraperitoneal injection of STZ.Rats were randomly divided into 6 groups:the control group,model group,JMT low-dose group(JMT-L),JMT medium-dose group(JMT-M),JMT high-dose group(JMT-H),and neurotropin(NTP)group.The rats were intragastrically administrated once per day for consecutive 12 weeks.2.The body weight and blood glucose of rats in each group were measured before and at 4w,8w,12w after administration.After 12 weeks,withdrawal mechanical threshold testing was applied.At the end of the experiment,all rats sacrificed to collect blood from the abdominal aorta under deep anesthesia,and then harvest and fix the sciatic nerves(SN)and the skin of hind paw.H&E staining and transmission electron microscope(TEM)of SN were performed to observe the pathological changes.Protain gene products(PGP)9.5 immunofluorescence staining was used to observe the nerve profiles of skin and the loss of distal small nerve fibers were estimated by semi-quantitative calculation of the intra-epidermal nerve fibres density(IENFD)and subepidermal nerve fibres density(SNFD)of the skin of hind paw.3.According to the better efficacy of dosages of JMT(JMT-M and JMT-H)on improving peripheral neuropathy,untargeted metabolomics analysis based on UPLC/QTOF-MS was conducted on the serum samples of rats to further explore the effects of JMT on the metabolic phenotype of DPN rats.Data were collected under the positive and negative ion models in the mass spectrum,respectively.Then,the multivariate analysis method combined with one dimensional statistical analysis was used to compare the serum profiles among the control,model,JMT-M and JMT-H groups Furthermore,the potential biomarkers related to the efficacy of JMT were screened and identified,as well as the metabolic pathway was analyzed by using online database resources.At last,Spearman rank correlation analysis was conducted between the serum levels of these metabolic markers and the indicators of peripheral nervous injury,in order to explore the association between the regulatory effects of JMT on the serum metabolism and the neuroprotective effect on DPN rats.[Results]1.Body weght and blood glucose:At 4w,8w,and 12w after STZ injection,the body weight of diabetic rats was significantly lower than that of normal rats(p<0.01).Compared with the model group,the body weight of rats in JMT-L,JMT-M,JMT-H,and NTP groups didn,t change significantly(p>0.05).At 4w,8w,and 12w after STZ injection,compared with the normal group,the blood glucose levels of rats in the model,JMT-L,JMT-M,JMT-H,and NTP groups increased significantly(p<0.01).Compared with the model group,the blood glucose level in JMT-M group significantly reduced at 8w(p<0.01),and that in JMT-H group significantly reduced at 12w(p<0.01).There was no significant difference between other interventional groups and the model group(p>0.05).2.Withdrawal mechanical threshold(WMT):At 12w,compared with the normal rats,the WMT of diabetic rats significantly reduced(p<0.01).Compared with the model group,the WMT of rats in JMT-L,JMT-M,and JMT-H groups were significantly increased(p<0.01),as well as the WMT of rats in NTP group increased significantly(p<0.05),respectively.Compared with NTP group,the WMT of rats in JMT-L,JMT-M,and JMT-H groups was significantly increased(p<0.05,p<0.01,p<0.01,respectively).3.H&E staining of SN:Nerve fibers of rats in the normal group arranged closely and the axons were thick.Nerve fibers of rats in the model group loosely arranged,sparsely distributed,and were thinner and lighter in staining than that of the normal rats.diabetic rats in the model group exhibited obvious demyelination changes with axonal shrinkage.The fibers density was slightly improved in JMT-L,but there was no other difference in the pathologic changes between JMT-L group and the model group.Compared with the model group,demyelination and axonal shrinkage of nerve fibers were profoundly corrected in JMT-M and JMT-H group.The disordered and sparsed arrangement of nerve fibers in NTP group was mildly improved in NTP group,but the demyelination changes and axonal shrinkage didn't change significantly when compared with the model group.Compared with the NTP group,demyelination and axonal atrophy of nerve fibers significantly alleviated in JMT-M and JMT-H group.4.TEM of SN:In the normal rats,the ultrastucture of the nerve fibers of SN was clear,and the myelin sheath of myelinated fiber was concentric with a lamellar structure.The density of myelin sheath was uniform,and axons were thick,and the non-myelinated fibers were distributed in clusters in normal rats.In the model group,the ultrastucture of myelin sheath wsa fuzzy with lamellar separation,vacuolar-like defects,tumor-like changes and axonal deformation or shrinkages.Unmyelinated fibers bundles decreased and unmyelinated fibers shrinked significantly.Compared with the model group,the vacuolar-like defects decreased in JMT-L,but other pathologic changes were not obvious.In JMT-M and JMT-H groups,the structure of myelin sheath lamellar layer damages was profoundly improved,including allevation in blurred,separated,tumor-like changes,and the axon disformation.The degree of pathological damages was similar between the JMT-M and JMT-H groups.In NTP group,the injuried in myelin sheath was slightly imporved,the vacuolar-like defects were reduced but the improvement on axonal atrophy was not obvious.The degree of improvement of demylination and axonal damages brought by JMT-M and JMT-H was more obvious than NTP.5.Distal small fibers injury:Compared with the normal group,the IENFD was significantly reduced in model group,JMT-L group and NTP group(p<0.01),and the SNFD was significantly reduced in the model group(p<0.01),as well as significantly reduced in the JMT-L,JMT-M,JMT-H and NTP groups(p<0.05).There was no significant difference in the IENFD between the normal group and JMT-M or JMT-H groups(p>0.05).There was no significant difference in the IENFD or SNFD between the JMT-L and model group(p>0.05).Compared with the model group,both of the IENFD and SNFD in JMT-M group significantly increased(p<0.05,p<0.01,respectively),as well as both of the IENFD and SNFD were significantly increased in JMT-H group(p<0.05).There was no significant difference in both of the IENFD and SNFD between the NTP and model groups(p>0.05).Compared with the NTP group,the IENFD was significantly higher in both of JMT-M and JMT-H group(p<0.05).But there was no significant difference in the SNFD between NTP and JMT-L,JMT-M,or JMT-H group(p>0.05).The IENFD in JMT-M group and JMT-H group were all significantly higher than that in JMT-L group(p<0.05),respectively.There was no statistically significant difference in the IENFD between JMT-M and JMT-H groups(p>0.05).There was no significant difference in the SNFD between JMT-L,JMT-M,or JMT-H groups(p>0.05).6.Differences of serum in metabolic phenotypes in each group:In this study,serum metabolomics analysis based on liquid chromatography coupled with mass spectrometry was established to investigate the serum metabolic changes of normal rats,DPN model rats,and DPN rats treated with medium and high dose of JMT.Principal component analysis(PCA)showed that the serum metabolic phenotypes in the normal and model groups were significantly different.The serum metabolic phenotypes of rats in JMT-M or JMT-H groups were also significantly different from the model group,respectively.Both medium-dose and high-dose JMT could corrected the abnormal metabolic phenotypes of DPN rats.7.Screening and identification of metabolic markers:Orthogonal partial least squares discriminant analysis(OPLS-DA)further characterized the metabolic profiles of separation between the normal rats and DPN rats,DPN rats and JMT administrated rats.And then 21 metabolites that were regulated to normal by high-dose of JMT were screened and identified,including 16 metabolites that were also callback markers by medium-dose of JMT(VIP?1,p<0.05).Based on KEGG database,the pathway enrichment analysis showed that the involved metabolic pathways included glycerolipid metabolism,tricarboxylic acid(TCA)cycle and amino acid metabolic pathway,and so on.8.Correlation analyses between the metabolic markers and nerve injury indicators:Correlation analysis was conducted between the serum levels of 11 metabolic markers which involved in lipids metabolism and energy metabolism pathways and the pharmacological indicators of nerve injury measured in first part of this study.The results showed that ten,nine and seven metabolic marker levels were negatively significantly correlated with the WMT,IENFD and SNFD(p<0.05).[Conclusion]1.12-weeks STZ-induced diabetic rats exhibited decreased mechanical pain threshod,diatal small nerve fibers density,and neuropathological structural changes of SN.Thus,the DPN rat model was successful established.2.JMT administration significantly improved the mechanical pain threshod,the loss of distal nerve fibers in skin,and the pathological changes of SN.The efficacy of medium-dose and high-dose of JMT on restoring sensory injury,small fiber neuropathy and neurodegenerative pathogical changes was superior to NTP.3.Metabolomics analysis showed that the serum metabolic profiles of DPN rats were significantly different from that of normal rats.JMT administration for 12w effectively regulated the serum metabolic disorders of DPN rats.The metabolic pathways involved included lipids metabolism,TCA cycle,and amino acid metabolism et al.4.The serum levels of multiple metabolic markers were significantly correlated with the indicators of peripheral neuropathy of DPN rats,indicating that the regulatory effects of JMT on metabolics were associated with its neuroprotective effects.[Innovation]This study confirmed the neuroprotective effects of JMT on diabetic rats with peripheral neuropathy.The present study also used an untarged metabolomics approach based on UPLC/QTOF-MS to explore the regulatory effects of JMT on the metabolic profiles in serum of DPN rats.The potential biological markers related to the therapeutic effects of JMT were analyzed and identified,and then the correlation between the nerve injury indicators of DPN and the serum levels of the mitabolites in important metabolic pathways markers were further analyzed.This study provided a new insight into the molecular biological mechanism of JMT in the treatment of DPN at the metabolic level.At present,there is no report about the influence of TCM compounds at the metabolic level of the animal model with DPN.