Study On The Intervention Mechanism Of Qigui Tangtongning Granule On Diabetic Peripheral Nerve Demyelinating Disease Via MicroRNA-155-5p-Mediated PI3K/Akt/mTOR Autophagy Pathway

ObjectiveIn vivo and in vitro experiments were carried out to study the intervention of Qigui Tangtongning Granule on the demyelination of sciatic nerve in diabetic rats.Based on the PI3 K / Akt / m TOR autophagy signal pathway regulated by micro RNA-155-5p,the effect and mechanism of Qigui tangtongning on autophagy and apoptosis of high glucose cultured Schwann cells were discussed.Methods1.In vivo animal experimentThe diabetic peripheral neuropathy(DPN)model was established by feeding 50 SPF grade adult GK rats with high fat diet.10 normal Wistar rats were used as the control group.After 8 weeks of feeding,GK rats were divided into model group(MC),low dose group(qgt-l,2.25 g / kg · d),medium dose group(qgt-m,4.5g / kg · d),high dose group(qgt-h,9.0g / kg · d),Mecobalamin group(MEC,0.225 mg / kg · d),10 rats in each group The intervention was given for 6 weeks.Normal group and model group were given the same amount of normal saline.During the experiment,the general situation of rats was recorded,and the random blood glucose changes were detected every week.Before the 6th week,the neurophysiological function was detected,including sensory and motor nerve conduction velocity(SCV,MCV)and thermal and pain sensitive reaction time.After anesthesia,the sciatic nerve tissue was taken for pathological HE staining;the expression of Neuritin and lipin-1,the secretory factors of myelin sheath,the morphological structure of subcellular cells,the relative expression of Beclin-1,LC3Ⅱ/Ⅰ,PI3 K,p-Akt / Akt and p-m TOR / m TOR were detected by immunohistochemistry;the m RNA expression of mi R-155-5p,PI3 K,Akt and m TOR were detected by PCR.2.In vitro cell experimentExperiment 1: the method of high glucose culture of Schwann cells(SCS)was used to simulate the myelin injury model of DPN in vitro.Rsc96 Schwann cell line of rats was passed on for 3-5 generations.Wistar rats were given intragastric gavage for 3 days to prepare Qigui tangtongning containing serum.The normal serum culture group,high glucose culture group,low(5%),medium(10%)and high(20%)content group of Qigui tangtongning containing serum were established.The cell increment rate of each group was detected by CCK-8 method,the expression of beclin-1 was detected by immunofluorescence test,and the expression of miRNA-155-5p was detected by PCR.The target gene of miRNA-155-5p acting on PI3 K / Akt / m TOR pathway was predicted by Shengxin website,then wild-type and mutant target gene vectors were established,and then co transfected with miRNA-155-5p into Schwann cells for Luciferase Report experiment.Experiment 2: Constructing miRNA-155-5p overexpression mimics,inhibiting the expression of inhibitor and corresponding mimics NC and inhibitor NC plasmids.Schwann cells were divided into three groups: normal serum culture group(NC),high glucose culture group(MC),miRNA-155-mimics group(mi R-mimics),miRNA-155-inhibitor group(mi R-inhibitor),Qigui tangtongning group(20%containing serum),miRNA-155-mics-nc group(mi R-mimics-NC),miRNA-155-inhibitor-nc group(mi R-inhibitor-NC).PCR was used to detect the expression of miRNA-155-5p,PI3 K,ATG5 and atg12.Western blot was used to detect the relative expression of PI3 K,p-Akt / Akt,p-m TOR / m TOR and LC3Ⅱ/Ⅰ.Immunofluorescence was used to detect the expression of beclin-1 and p62.Flow cytometry was used to detect the level of apoptosis.Experiment 3: 1.The rescue experiment of autophagy mechanism: The high glucose group,Qigui tangtongning group and miRNA-155-inhibitor group in Experiment 2 were isolated and transfected with miRNA-155-inhibitor in Qigui tangtongning group.They were divided into high glucose group,miRNA-155-inhibitor group,Qigui tangtongning group and Qigui tangtongning + mi R-155-inhibitor group.The expression of beclin-1,p-Akt / Akt and p-m TOR / m TOR were detected by Western blot.2.The rescue experiment of apoptosis mechanism: The high glucose group and Qigui tangtongning group in Experiment 2 were separated and redivided into high glucose group,high glucose + 3MA group,Qigui tangtongning group and Qigui tangtongning + 3m A group by adding autophagy cutter 3-MA.The apoptosis rate of each group was detected by flow cytometry.Results1.In vivo animal experiment General observation showed that rats in model group had symptoms of polydipsia,polydipsia and polyuria,accompanied by emaciation and mental malaise,while the symptoms in low,medium and high dose groups of Qigui tangtongning were improved.The results of blood glucose monitoring showed that the blood glucose in the low,middle and high dose group of Qigui tangtongning was lower than that in the model group(P < 0.01)and showed a downward trend.From the third week,the blood glucose in the high dose group was significantly lower than that in the low dose group(P < 0.05),and that in the middle and high dose group was lower than that in the low dose group(P< 0.05).The results showed that the SCV and MCV of the model group were significantly slower than those of the normal group,and the response time of heat sensitivity and pain sensitivity was longer(P The response time of heat and pain in Mecobalamin group was lower than that in low dose group(P < 0.05).Morphological observation showed that the myelin sheath of he stained model group was broken and dissolved,the nerve fibers were arranged disorderly,the density was decreased,and atrophy and swelling were also seen,forming irregular type.Under electron microscope,mitochondria were swollen,endoplasmic reticulum was broken,and apoptotic bodies were observed in some cells.Compared with the model group,the demyelinating lesion and organelle damage were significantly reduced in the low,middle and high dose group,and a certain number of autophagy bodies and vesicles were observed in the high dose group.Immunohistochemistry showed that the expression of neiritin and lipin-1 in the model group was lower than that in the normal group(P < 0.01),while the expression of lipin-1 in the low,middle and high dose groups of Qigui tangtongning was higher than that in the model group(P < 0.05,P < 0.01),which was dose-dependent.Western blot showed that the expression of LC3 Ⅱ / I in model group was significantly lower than that in normal group(P < 0.01),and the expression of PI3 K,p-Akt / Akt and p-m TOR / m TOR was increased(P < 0.01);the expression of LC3Ⅱ/Ⅰ in model group was significantly higher than that in model group(P < 0.01);the relative expression of PI3 K,p-Akt / Akt and p-m TOR / m TOR was decreased(P The relative expression of Beclin1,LC3Ⅱ/Ⅰin high dose group was higher than that in low dose group(P < 0.05),while the relative expression of PI3 K and p-m TOR / m TOR decreased(P < 0.05).PCR detection showed that the expression of mi R-155-5p in the model group was lower than that in the normal group,and the m RNA expression of PI3 K,Akt and m TOR was higher than that in the model group;the expression of mi R-155-5p in the low,middle and high dose Qigui tangtongning group was higher than that in the model group(P < 0.01),and the m RNA expression of PI3 K,Akt and m TOR was lower,and the expression of mi R-155-5p in the high dose group was higher than that in the low dose group(P <0.05),and the m RNA expression of PI3 K was higher than that in the low dose group(P< 0.05)The dosage group decreased(P < 0.05).2.In vitro cell experimentExperiment 1: CCK-8 experiment showed that the value-added rate of Schwann cells in high glucose group was lower than that in normal group,while the value-added rate in low,medium and high dose groups was higher than that in model group(P < 0.01),which was dose-dependent.The results of immunofluorescence showed that the expression of Beclin1 in Schwann cells in high glucose group was significantly lower than that in normal group.The results of PCR showed that the expression of miRNA-155-5p in the model group was lower than that in the normal group,and the expression of miRNA-155-5p in the low,middle and high dose groups of Qigui tangtongning was higher than the normal group(P < 0.05,P < 0.01).Luciferase reporter gene experiment showed that in Schwann cells,miRNA-155-5p had a direct binding effect with the target gene PI3 KCA and inhibited the expression of PI3 KCA.Experiment 2: PCR results showed that the expression of miRNA-155-5p,ATG5,atg12 and PI3K in high glucose group was lower than that in normal group(P < 0.01);the expression of miRNA-155-5p,ATG5,atg12 in Qigui tangtongning group was higher than that in high glucose group(P < 0.01),and the m RNA expression of PI3 K was lower(P < 0.05);the expression of miRNA-155-5p,ATG5,atg12 in mi R MICs group was higher than that in mi R MICs NC group,and the expression of PI3 K was lower The expression of miRNA-155-5p,ATG5,atg12 in mi R inhibitor group was lower than that in mi R inhibitor NC group,and the expression of PI3 K was increased(P < 0.05,P <0.01).Western The results showed that the relative expression of PI3 K,p-Akt / Akt,p-m TOR / m TOR in high glucose group was higher than that in normal group,and the relative expression of LC3Ⅱ/Ⅰwas lower(P < 0.01);compared with high glucose group,the relative expression of PI3 K,p-Akt / Akt,p-m TOR / m TOR in Qigui tangtongning group was lower,and the relative expression of LC3Ⅱ/Ⅰwas higher(P <0.05,P < 0.01);the relative expression of PI3 K,p-Akt / Akt,p-m TOR / m TOR in mi R MICs group was higher(P < 0.05,P < 0.01)The relative expression of PI3 K,p-Akt /Akt,p-m TOR / m TOR in mi R inhibitor group was higher than that in mi R inhibitor NC group,and the relative expression of LC3Ⅱ/Ⅰwas lower than that in mi R inhibitors NC group(P < 0.05,P < 0.01).Immunofluorescence showed that the expression of p62 in high glucose group increased,the expression of beclin-1 decreased(P < 0.01);compared with high glucose group,the expression of p62 in Qigui tangtongning group decreased,the expression of beclin-1 increased(P < 0.05);compared with NC group, the expression of p62 in mi R mimics group decreased,the expression of beclin-1increased(P < 0.05);compared with NC group,the expression of p62 in mi R inhibitor group increased,and the table of beclin-1 increased It was decreased(P < 0.05).Flow cytometry showed that the total apoptosis rate of SCS in high glucose group was significantly higher than that in normal group(P < 0.05);compared with high glucose group,the apoptosis rate of Qi GUI Tang Tong Ning group was lower(P < 0.05);compared with NC group,the apoptosis rate of mi R mimics group was lower(P < 0.05);the apoptosis rate of mi R inhibitor group was higher(P < 0.05).Experiment 3: 1.The rescue experiment of autophagy mechanism: Compared with high glucose group,the expression of beclin-1 decreased,p-Akt / Akt,p-m TOR / m TOR increased in miRNA-155 inhibited expression group(P < 0.05,P < 0.01),beclin-1increased,p-Akt / Akt,p-m TOR / m TOR decreased in Qigui tangtongning group(P <0.01);p-Akt / Akt,p-m TOR / m TOR increased and beclin-1 decreased in Qigui tangtongning + mi R-155 inhibited expression group compared with Qigui tangtongning group The expression decreased(P < 0.05,P < 0.01).2.The rescue experiment of apoptosis mechanism: Compared with the high glucose group,the apoptosis rate of the high glucose + 3m A group increased(P < 0.05),the apoptosis rate of the Qigui tangtongning group decreased(P < 0.05).The apoptosis rate of the Qigui tangtongning+ 3m A group increased significantly compared with the Qigui tangtongning group(P <0.05).Conclusions1.Qigui Tangtongning Granule can alleviate the demyelination of sciatic nerve in DPN rats,promote the secretion of neurotrophic factors and promote the neurophysiological function recovery.2.In Schwann cells,miRNA-155-5p can inhibit the activation of PI3 K / Akt / m TOR signaling pathway by inhibiting the target gene pi3 kca.3.Qigui Tangtongning Granule can improve the Schwann cells proliferation in high glucose environment.And it can inhibit the PI3 K / Akt / m TOR signal pathway by up regulating the expression of miRNA-155-5p,and then promote the autophagy of Schwann cells.miRNA-155-5p is an important target for Qigui tangtongning to regulate autophagy.4.Qigui Tangtongning Granule can reduce the apoptosis of Schwann cells in high glucose environment.Up regulation of autophagy is an important way for this effection.