Roles Of Inflammatory Factors On The Regeneration Of Rats Diabetic Peripheral Nerve And Effects Of The Traditional Chinese Medicine Jinmaitong

[Objective] To study the effects of Jinmaitong capsule on oxidative stress and cell apoptosis of dorsal root ganglion in diabetic rats.[Methods] Male SD rats were randomly divided into normal group and model group. Streptozotocin (STZ) method (60mg/kg of intraperitoneal injection) was used to manufacture model of diabetic rats, then randomly divided them into model group, the small、middle and the high dose group of Jinmaitong and vitamin C group. All experimental rats were sacrificed at 16W and then isolated dorsal root ganglion.Using the Nissl’s staining to observe the morphological changes of DRG, immunohistochemistry and qRT-PCR assay for detection NADPH oxidase subunit p22-phox, Cyt, C, Bcl-2 and Caspase-3 of DRG in rats.Cell apoptosis was detected by TUNEL.[Results] Compared with model group, the expression of NADPH oxidase subunit p22-phox protein、Cyt expression of C protein, Caspase-3 protein and mRNA cell apoptosis rate of each treatment group decreased significantly, while the expression of Bcl-2 mRNA and protein was increased,which were both statistical significant different(P< 0.05 or P< 0.01). The high dose of Jinmaitong capsule group had the best effect and there is significant difference compared with the vitamin C group(P<0.01).[Conclusion] Jinmaitong capsule has the effects to prevent the nerve injury in rats with DPN,which are based on inhibiting oxidative stress and decreasing the apoptosis to some extent. The high dose of Jinmaitong capsule group had the best effect and was much better than vitamin C groups.[Objection]Study the roles of inflammatory factors on the regeneration of diabetic peripheral nerve and effects of the traditional Chinese medicine Jinmaitong from the whole, cell and molecular levels,and to find out whether JMT can reduce the nerve tissue injury, promote nerve regeneration and reduce the incidence of DPN from the perspective of inflammatory cytokines.[Methods]1.In vivo experimentMale SD rats were randomly divided into normal group and model group, diabetic rat model was established by intraperitoneal streptozotocin injection(60mg/kg). After that, they were randomly divided into three groups:DM group, JMT group and Ntp group, with 10 rats in each. Then came to the intragastric administration, JMT group and Ntp group were respectively by 15 times adult dosage of the drug, Con group and DM group were given equal volume of distilled water. Continuous intragastric administration lasted for 16 weeks. Observing the expression of inflammatory factors such as IL-1β、IL-6、 IL-10、 TNF-a and neurotrophic factor NGF, NT-3 in sciatic nerve tissue of diabetic rats and the effects of JMT on it.2.1n vitro experimentSCs were primary cultivated and were identified with S-100 protein antibody. The cells were cultured respectively in following conditions including normal control group(25mmol/L glucose) and 50,75 and 100mmol/L of different concentrations of glucose medium. The most suitable glucose concentration was explored by MTT method. Then the SCs was cultured in DMEM, high glucose(50mmol) media supplemented with 20% rat serum, 50mmol glucose media containing medicated serum of JMT and Ntp. DMEM served as negative control. The effects of Jinmaitong medicated serum on the activity of SCs under high glucose condition was found out by CCK-8 colorimetric assay. The content of IL-6, TNF-a, NGF in the SCs supernatant under the conditions of different intervention was detected by ELISA method; The expression of IL-1β、IL-6、IL-10、TNF-α、NGF and NT-3 protein was tested by Western Blot.[Results]1.In vivo experiment(1) Blood glucose and body weight:The blood glucose levels of STZ-DM rats were higher than those of normal rats (P<0.05). In all the treated groups, there were no significant differences among them compared each other or compared with model group (P >0.05). After modeling, the body weight of rats between each group had no significant difference (P> 0.05). Compared with Con group, the body weight of DM group decreased after 4 weeks (P<0.05), each model group decreased (P<0.05) after 8、12、16 weeks with no significant difference between each other (P>0.05).(2) Pain threshold to mechanical stimulation with Von Frey filament:Compared with Con group, the mechanical pain threshold in DM group and each treatment group were all reduced (P<0.05). Which means that the DPN model was successfully established.(3) ELISA method was used to detect the expression of IL-6, TNF-α, NGF in DPN rats serum:①IL-6:Compared with Con group, IL-6、TNF-α in each group were increased (P<0.05);compared with DM group, IL-6、TNF-α in the JMT group were reduced(P<0.05), while the Ntp group has no significant difference (P> 0.05); compared with Ntp group, IL-6、TNF-a in the JMT group were decreased(P<0.05).②NGF:Compared with Con group, NGF in both DM group and JMT group was reduced (P<0.01, P<0.05); compared with DM group, each treatment group was significantly increased (P<0.01);compared with DM group, the JMT group was decreased(P<0.01).(4)Immunohistochemical method to detect the NT-3 expression of sciatic nerve in DPN rats:Compared with Con group,NT-3 in DM group was reduced(P<0.05), JMT and Ntp groups had no significant difference(P>0.05); compared with DM group, NT-3 in both JMT and Ntp groups were increased (P<0.05); there was no significant difference between the JMT group and Ntp group (P>0.05).(5) The expression of IL-1β, IL-6, IL-10, TNF-α, NGF, NT-3 protein in sciatic nerve:① IL-1β、IL-10:Compared with Con group, IL-1β and IL-10 protein expression in DM group was increased (P<0.05); compared with DM group, IL-1β and IL-10 protein expression in JMT group was reduced (P< 0.05); there was no significant difference (P>0.05) between JMT and Ntp groups.② IL-6:There was no significant difference (P>0.05) among the Con group, DM group, JMT group and Ntp group.③TNF-α:Compared with Con group, TNF-α protein expression in DM group was increased (P<0.05); compared with DM group, TNF-α protein expression was decreased both in JMT and Ntp groups (P< 0.05); there was no significant difference between JMT and Ntp group (P>0.05).④NGF:Compared with Con group, NGF protein expression in DM group was decreased(P< 0.05); compared with DM group, the expression was increased in JMT group (P<0.05); NGF protein expression increased in JMT group when compared with Ntp group (P<0.05).⑤NT-3:Compared with Con group, NT-3 protein expression in DM group was decreased(P< 0.05);compared with DM group, the expression was increased in JMT group (P< 0.05); there was no significant difference between JMT and Ntp groups.2.1n vitro experiment(1) Schwann cells were successfully cultured, and the purity of which is 93.1+2.1%.(2)Cell activity was detected by CCK-8 colorimetric method:① Glucose concentration:With the prolongation of the culture time, the earliest inhibition to SCs activity was at time of 48h among different glucose concentration groups. Therefore, the time of 48h and 50 mMGlu were chose as the best action time and the most suitable sugar concentration.①Effects of Jinmaitong medicated serum on the activity of SCs under the condition of high glucose:compared with the normal control group, there was no significant difference in both normal rat serum group and JMT group (P> 0.05), While the activity of SCs in 50mM cell Glu group decreased significantly (P<0.01) and the Ntp group decreased too(P< 0.05); compared with the 50mM group Glu, the relative cell activity of JMT group was significantly increased (P< 0.01); compared with Ntp group, the JMT group was increased significantly (P< 0.01).(3) ELISA method was used to detect the expression of IL-6, TNF-a, NGF in the SCs supernatant:① IL-6:There was no significant difference among the Con, Glu, JMT and Ntp groups (P> 0.05).② TNF-α:Compared with Con group, TNF-α in Glu group was higher (P<0.05); compared with Glu group, TNF-α in the JMT group was reduced(P<0.05), while the Ntp group has no significant difference (P> 0.05); TNF-a in the JMT group was lower than Ntp group (P<0.05).③ NGF:Compared with Con group, the NGF in Glu group was reduced (P<0.05); compared with Glu group, the treatment groups were both increased (P<0.05).(4) The expression of IL-1β, IL-6, IL-10, TNF-a, NGF, NT-3 protein in SCs:① IL-1β:Compared with Con group, IL-1β protein expression in Glu group was increased (P< 0.05); compared with Glu group, IL-10 protein expression in JMT group was significantly decreased (P< 0.01); the protein expression was decreased in JMT group when compared to Ntp group (P< 0.05).② IL-6:There was no significant difference among the Con group, Glu group, JMT group and Ntp group (P> 0.05).③ IL-10:Compared with Con group, IL-10 protein expression in Glu group and Ntp group was increased (P<0.01, P<0.05); compared with Glu group, IL-10 protein expression in JMT group was significantly reduced (P < 0.01); the protein expression was significantly decreased in JMT group when compared to Ntp group (P< 0.01).④ TNF-α:Compared with Con group, TNF-a protein expression in Glu group was increased (P< 0.05); compared with Glu group, TNF-α protein expression was decreased in JMT group (P<0.05); there was no significant difference between JMT and Ntp group (P> 0.05).⑤NGF:Compared with Con group, NGF protein expression in both Glu group and Ntp group was significantly decreased(P< 0.01); compared with Glu group, the expression was increased in JMT group (P< 0.05); NGF protein expression increased in JMT group when compared to Ntp group (P< 0.05).⑥NT-3:Compared with Con group, NT-3 protein expression in Glu group was decreased(P < 0.05):compared with Glu group, the expression was increased in JMT group (P< 0.05); there was no significant difference between JMT and Ntp groups(P> 0.05).[Conclusion]1. In vivo experiment(1)SZT-induced diabetic rats (single intraperitoneal injection, 60mg/kg) had hyperalgia at 16w, which demonstrated the sensory nerve fibers were injured and the DPN models were established. JMT can alleviate hyperalgia strikingly. (2) The expression of IL-1β, IL-10, TNF-a protein in sciatic nerve tissue and IL-6, TNF-a in blood serum of DPN rats were significantly increased which suggested that inflammation and cytokines were involved in the occurrence and development of DPN; (3) Chinese medicine Jinmaitong can significantly reduce the IL-1 β, IL-10, TNF-a protein expression in sciatic nerve tissue and IL-6, TNF-a in blood serum of DPN rats, which means it can alleviate inflammatory reaction; At the same time it could significantly increase the expression of NGF and NT-3 in sciatic nerve tissue. (4)Jinmaitong can play an important role in the prevention and treatment of DPN injury in rats by intervention on inflammatory reaction and promoting the secretion of neurotrophic factors.2. In vitro experiment(1)SCs were successfully cultured by tissue explant method and reached a purity of more than 90%. (2) SCs cultured in 50mmol/L glucose after 48h had obvious inhibitory effects on its activity. Jinmaitong medicated serum could obviously improve the activity of SCs under high glucose condition which was better than Ntp group. (3) The IL-1β, IL-10, TNF-a protein expression in SCs and TNF-α in cell supernatant was increased, which showed that high glucose stimulated SCs to produce inflammation and cytokines; (4) Chinese medicine Jinmaitong can reduce IL-1β, IL-10, TNF-α protein expression in SCs and TNF-a in cell supernatant. At the same time it can significantly increase the expression of NGF, NT-3, which was basically consistent with the vivo experiment results.[Innovation]At present, the specific role of inflammatory cytokines in diabetic peripheral neuropathy is still unclear. Our study aimed to find out the role of inflammatory factors in the peripheral nerve regeneration and the effects of Chinese medicine Jinmaitong in the way of inflammatory cytokines from the whole, cellular and molecular levels, which has certain innovation.