Protective Effects Of Jinmaitong Against Oxidative Stress Induced Disturbed Nerve Regeneration In Experimental Diabetic Neuropathy

[Objective]To investigate protective effects of Jinmaitong (JMT) against oxidative stress induced disturbed nerve regeneration in rats with diabetic neuropathy and dorsal root ganglion (DRG) neurons cultured in high glucose.[Methods]1. In vivo experiment:The STZ-induced diabetic rats (single intraperitoneal injection,60mg/kg) were randomly divided into 4 groups including normal control group (CON), Diabetes (DM) group, JMT group (treated with JMT similar to the fifteen-fold dose of adult recommended dosage) and Taurine group (treated with Taurine similar to the fifteen-fold dose of adult recommended dosage). Ten rats were in each group. All rats were given intragastric administration for 16 weeks (the CON and DM groups were treated with distilled water at dose of 1 mL/100g/d) and then killed. Body weight and blood glucose were detected before and at the 4th,8th,12th, 16th week after treatment. The pain threshold to mechanical stimulation with Von Frey filament were carried out before death. Morphological changes in DRG were observed through transmission electron microscopy (TEM). The apoptotic cells were detected by TUNEL assay with fluorescence staining. The expressions of UCP3 and IGF-1 were detected by immunohistochemical and western blot analyses. The target genes were validated by qRT-PCR.2. In vitro experiment:DRG neurons were isolated from embryonic day 15 SD rats, then dissociated in 0.25% trypsin, purified by density gradient centrifugation and differential attachment technique. Neurons were cultured in Neurobasal medium supplemented with B-27 minus AO additives and nerve growth factor. The immunofluorescence method with anti-MAP-2 was used to identify the purity of the DRGn.DRG neurons were cultured respectively in following conditions including Neurobasal medium supplemented with 20% rat serum, high glucose (45 mM) media supplemented with 20% rat serum,45mM glucose media containing medicated serum of JMT at a dilution of 1:1 and Taurine at a dilution of 1:1,1:2,1:4. After 12 h,24 h and 48 h cultures, CCK-8 assay was used to determine the optimal time of drugs and concentration of Taurine.DRGn were randomly divided into control group (cultured with Neurobasal medium containing blank serum), high glucose group (cultured with 45 mM glucose medium), JMT group (cultured with JMT containing serum at a dilution of 1:1) and Taurine group (cultured with Taurine containing serum at a dilution of 1:1). After 4 h cultures, The levels of ROS were determined by fluorescent probe. After 24 h cultures, Apoptosis was detected by the TUNEL assay with fluorescence staining. The expressions of UCP3 and IGF-1 were detected by fluorescence-immunocytochemistry, Western blot analysis and qRT-PCR.[Results]1. In vivo experiment:(1) Blood glucose and body weight:The blood glucose levels of STZ-DM rats were much 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). And it got the same result when concerning about body weight no matter how the rats were dealt with(P>0.05).(2) Pain threshold to mechanical stimulation with Von Frey filament: Compared with normal group, the pain thresholds in both sides of diabetes (DM) model group decreased extremely (P<0.01). Compared with DM group, the threshold values of JMT group and Taurine group raised significance (left, P＜0.05; right P<0.01). There was no significant difference between JMT group and Taurine group (P>0.05).(3) Transmission electron microscopy:DM model group:Mitochondria were highly expanded. Loss of the mitochondrial cristae and mitochondria vacuolar degeneration was found in DRG neurons. JMT and Taurine treatment group:The ultrastructures of dorsal root ganglia neurons were both better than that of rats in DM model group.(4) Apoptosis:The apoptosis percentage in DM group was much higher than that in control group (P＜0.01). Compared with DM group, the apoptosis percentage in treatment groups significantly decreased (P＜0.01). There was no significant difference between two treatment groups (P＜0.01).(5) Indicators of oxidative stress in DRG of rats:① Immunohistochemistry:The average optical density (AOD) of UCP3 expression in STZ-DM rats was much lower than the normal (P<0.01). And the AOD of UCP3 in JMT and Taurine treated groups increased notably compared with DM model group (P＜0.01). There were no significant differences between the two treated groups (P>0.05).② Western blot analysis:The protein level of UCP3 in DM group was much lower than that in control group (P＜0.01). Compared with DM group, the protein level of UCP3 in both treatment groups significantly increased (P<0.01). There were no significant differences between JMT group and Taurine group (P>0.05).③ qRT-PCR analysis:The levels of UCP3 mRNA expression in STZ-DM rats were much lower than those of the normal rats (P＜0.01). Compared with DM model group, UCP3 mRNA expressions of JMT group up-regulated noticeably (P<0.01), but Taurine group showed no significant changes (P> 0.05). The levels of UCP3 mRNA expression in JMT group were much higher than those in Taurine group (P＜0.01).(6) Indicators of neurotrophic factors in DRG of rats:① Immunohistochemistry:The average optical density (AOD) of IGF-1 expression in STZ-DM rats was much lower than the normal (P<0.01). And the AOD of IGF-1 in JMT and Taurine treated groups increased notably compared with DM model group (P<0.01). There were no significant differences between the two treated groups (P>0.05).② Western blot analysis:The protein level of IGF-1 in DM group was much lower than that in control group (P＜0.01). Compared with DM group, the protein level of IGF-1 in both treatment groups significantly increased (P<0.01). There were no significant differences between JMT group and Taurine group (P>0.05).③ qRT-PCR analysis:The levels of IGF-1 mRNA expression in STZ-DM rats were much lower than those of the normal rats (P<0.01). Compared with DM model group, IGF-1 mRNA expressions of JMT and Taurine group up-regulated noticeably (P<0.01). The two treatment groups showed no significant differences (P>0.05).2. In vitro experiment:(1) CCK-8 assay: ①The cell viabilities in high glucose groups significantly decreased at 24 h and 48 h compared with 12h (P＜0.01) Furthermore, the cell viabilities in high glucose groups decreased at 48 h compared with 24 h (P＜0.05). The cell viabilities of DRGn at 24 h in 45mM glucose were suppressed significantly (P<0.01).② Cultured for 24 h in different medium:Compared with HG group, the cell viability levels in JMT group and Tau1:1 group increased significantly (P＜0.01), however there was no change in Tau1:2 and Tau1:4 treatment groups (P>0.05). There was no difference of cell viability between JMT group and Tau1:1 group (P>0.05). The cell viability levels in JMT group were much higher than those in Tau1:2 group and Taul:4 group (P＜0.01). Therefore, Tau1:1 was applied in the following study as the positive control group.(2) Apoptosis:The apoptosis percentage in HG group was much higher than that in control group (P<0.01). Compared with HG group, the apoptosis percentage in JMT and Tau1:1 treatment groups significantly decreased (P<0.01). There was no significant difference between two treatment groups (P＜001).(3) Indicators of oxidative stress in DRGn:① ROS levels in DRGn:Measurement of intracellular ROS levels with the DCFH-DA assay revealed that cells in HG group produced a marked increase in intracellular ROS levels, compared to cells in CON group. JMT and Tau1:1 groups blocked this increase in intracellular ROS (P<0.01).② Fluorescence-immunocytochemistry:The integrated optical density (IOD) of UCP3 expression in HG group was much lower than that in CON group (P<0.05). And the IOD of UCP3 in JMT and Taurinel:1 groups increased notably compared with HG group (P<0.01). There were no significant differences between the two treated groups (P>0.05).③ Western blot analysis:The protein level of UCP3 in HG group was much lower than that in CON group (P＜0.01). Compared with HG group, the protein level of UCP3 in JMT group significantly increased (P＜0.05), however there was no significant change in Tau1:1 group (P>0.05).④ qRT-PCR analysis:The levels of UCP3 mRNA expression in HG group were much lower than those in CON group (P＜0.01). Compared with HG group, UCP3 mRNA expressions of JMT and Tau:1 group up-regulated noticeably (P＜0.01). The levels of UCP3 mRNA expression in Tau1:1 group were much higher than those in JMT group (P＜0.01).(4) Indicators of neurotrophic factors in DRGn:① Fluorescence-immunocytochemistry:The integrated optical density (IOD) of IGF-1 expression in HG group was much lower than that in CON group (P＜0.01). And the IOD of IGF-1 in JMT and Taurine1:1 groups increased notably compared with HG group (P<0.01). There were no significant differences between the two treated groups (P>0.05).② Western blot analysis:The protein level of IGF-1 in HG group was much lower than that in control group (P＜0.01). Compared with HG group, the protein level of IGF-1 in both treatment groups significantly increased (P＜0.01). There were no significant differences between JMT and Tau1:1 group (P> 0.05).③ qRT-PCR analysis:The levels of IGF-1 mRNA expression in HG group were much lower than those in CON group (P＜0.01). Compared with HG group, IGF-1 mRNA expressions of JMT and Tau:l group up-regulated noticeably (P＜0.01). The levels of UCP3 mRNA expression in Tau1:1 group were much higher than those in JMT group (P<0.01).[Conclusion]1. In vivo experiment:① STZ-induced diabetic rats had hyperalgia at 16w, which demonstrated the sensory nerve fibers were injured and the DPN models were established.② Ultrastructural pathological injury in DRG of DPN rats and up-regulated apoptosis indicated serious damages caused by oxidative stress. Down-regulated the expression of UCP3 and IGF-1 manifested the weak abilities to repair and regenerate peripheral nerves.③ JMT treatment significantly ameliorated hyperalgesia in DPN rats. Besides, it could also improve the oxidative stress-induced pathological morphology of mitochondria and inhibit apoptosis of DRGn. In addition, JMT stimulated neuroprotection as well as neuroregeneration through up-regulating the expression of UCP3 and IGF-1 in DRG.2. In vitro experiment:① High glucose remarkably inhibited the cell viability levels of DRGn and the levels of ROS and apoptosis of DRGn cultured in high glucose medium significantly increased compared to CON group. The reduced levels of UCP3 and IGF-1 showed the weak anti-oxicative and neuroprotective abilities in DPN rats.② These results suggest that JMT containing serum significantly ameliorated oxidative stress-induced disturbed nerve regeneration in DRGn cultured in high glucose medium by up-regulating UCP3 as a ROS scavenger and IGF-1 as a neuroprotectant.