Attenuation of Oxidative Stress-Induced Cell Apoptosis and Pyroptosis in RSC96 Cells by Salvianolic Acid B.

OBJECTIVE: To determine whether salvianolic acid B (Sal B) exerts protective effects on diabetic peripheral neuropathy by attenuating apoptosis and pyroptosis. METHODS: RSC96 cells were primarily cultured with DMEM (5.6 mmol/L glucose), hyperglycemia (HG, 125 mmol/L glucose) and Sal B (0.1, 1, and 10 µ mol/L). Cells proliferation was measured by 3-(4, 5-cimethylthiazol-2-yl)-2, 5-dilphenyltetrazolium bromide assay. Reactive oxygen species (ROS) generation and apoptosis rate were detected by flow cytometry analysis. Western blot was performed to analyze the expressions of poly ADP-ribose polymerase (PARP), cleaved-caspase 3, cleaved-caspase 9, Bcl-2, Bax, NLRP3, ASC, and interleukin (IL)-1ß. RESULTS: Treatment with HG at a concentration of 125 mmol/L attenuated cellular proliferation, while Sal B alleviated this injury (P<0.05). In addition, Sal B inhibited HG-induced ROS production and apoptosis rate (P<0.05). Furthermore, treatment with Sal B down-regulated HG-induced PARP, cleaved-caspase 3, cleaved-caspase 9, Bax, NLRP3, ASC, and IL-1ß expression, but mitigated HG-mediated down-regulation of Bcl-2 expression (P<0.05). CONCLUSION: Sal B may protect RSC96 cells against HG-induced cellular injury via the inhibition of apoptosis and pyroptosis activated by ROS.

Puerarin may protect against Schwann cell damage induced by glucose fluctuation.

Puerarin is one of the major active ingredients in Gegen, a traditional Chinese herb that has been reported to have a wide variety of beneficial pharmacology functions. Previous studies have implicated that the damaging effects of hyperglycemia resulting from oxidative stress and glucose fluctuation may be more dangerous than constant high glucose in the development of diabetes-related complications. The present study focuses on the effects of puerarin on glucose fluctuation-induced oxidative stress-induced Schwann cell (SC) apoptosis in vitro. Primarily cultured SCs were exposed to different conditions and the effect of puerarin on cell viability was determined by MTT assays. Intracellular reactive oxygen species (ROS) generation and mitochondrial transmembrane potential were detected by flow cytometry analysis. Apoptosis was confirmed by the Annexin V-FITC/PI and TUNEL method. Quantitative real-time reverse transcriptase polymerase chain reaction was performed to analyze the expression levels of bax and bcl-2. Western blot was performed to analyze the expression levels of some important transcription factors and proteins. The results showed that incubating SCs with intermittent high glucose for 48 h decreased cell viability and increased the number of apoptotic cells whereas treating with puerarin protected SCs against glucose fluctuation-induced cell damage. Further study demonstrated that puerarin suppressed activation of apoptosis-related proteins including PARP and caspase-3, downregulation of bcl-2, and upregulation of intracellular distribution of bax from cytosol to mitochondria, which was induced by glucose fluctuation. Moreover, puerarin inhibited the elevation of intracellular ROS and mitochondrial depolarization induced by glucose fluctuation. These results suggest that puerarin may protect SCs against glucose fluctuation-induced cell injury through inhibiting apoptosis as well as oxidative stress.

Jinmaitong alleviates the diabetic peripheral neuropathy by inducing autophagy.

OBJECTIVE: To observe the deregulation of autophagy in diabetic peripheral neuropathy (DPN) and investigate whether Jinmaitong ( JMT) alleviates DPN by inducing autophagy. METHODS: DPN models were established by streptozotocin-induced diabetic rats and Schwann cells (SCs) cultured in high glucose medium. The pathological morphology was observed by the improved Bielschowsky's nerve fiber axonal staining and the Luxol fast blue-neutral red myelin staining. The ultrastructure was observed by the transmission electron microscopy. Beclin1 level was detected by immunohistochemistry and Western blot. The proliferation of cultured SCs was detected by methylthiazolyldiphenyl-tetrazolium bromide. RESULTS: Diabetic peripheral nerve tissues demonstrated pathological morphology and reduced autophagic structure, accompanied with down-regulation of Beclin1. JMT apparently alleviated the pathological morphology change and increased the autophagy [in vivo, Beclin1 integral optical density (IOD) value of the control group 86.6±17.7, DM 43.9±8.8, JMT 73.3 ±17.8, P<0.01 or P<0.05, in vitro Beclin1 IOD value of the glucose group 0.47±0.25 vs the control group 0.88±0.29, P<0.05]. Consequently, inhibition of autophagy by 3-methyladenine resulted in a time- and concentration-dependent decrease of the proliferation of SCs (P<0.05, P<0.01). CONCLUSIONS: Down-regulation of autophagy in SCs might contribute to the pathogenesis of DPN. JMT alleviates diabetic peripheral nerve injury at least in part by inducing autophagy.

Jinmaitong decreases sciatic nerve DNA oxidative damage and apoptosis in a streptozotocin-induced diabetic rat model.

Diabetic peripheral neuropathy (DPN) is a common chronic complication of diabetes. Jinmaitong (JMT), a Traditional Chinese Medicine, improves certain symptoms of DPN, such as limb pain and numbness. The aim of the present study was to investigate the effects of JMT on DNA oxidative damage and apoptosis in the sciatic nerve of diabetic rats. The rats were divided into a normal and a diabetic group. Diabetes was induced using streptozotocin (60 mg/kg). The diabetic model (DM) rats received vitamin C (0.05 g/kg/day) or JMT [low-dosage (L), 0.44 g/kg/day; medium-dosage (M), 0.88 g/kg/day or high-dosage (H), 1.75 g/kg/day]. After 16 weeks, the mechanical pain threshold of the rats was evaluated. The expression of 8-hydroxy-deoxyguanosine (8-OHdG), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase p22phox, B-cell lymphoma 2 (Bcl-2), caspase 3 and cleaved-poly(ADP-ribose) polymerase 1 (PARP-1) in the sciatic nerve tissues was measured using the reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and western blotting. JMT had no effect on body weight and fasting blood glucose levels. Following treatment, the rats in the JMT groups had an improved pain threshold compared with the DM controls (JMT-L, 52.9±6.5 g; JMT-M, 74.7±9.3 g; and JMT-H, 61.7±2.0 g vs. DM control, 35.32±12.06 g; all P<0.01), while the threshold in the JMT-M rats was similar to that of normal controls (P>0.05). 8-OHdG and NADPH oxidase p22phox expression was significantly decreased in the three JMT groups compared with that in the DM controls (all P<0.05). Following JMT treatment, Bcl-2 levels were increased, while caspase 3 and cleaved-PARP-1 levels were decreased compared with those in the DM controls (all P<0.01). In conclusion, JMT may reduce DNA oxidative damage to the sciatic nerve in diabetic rats, as well as regulate genes involved in peripheral neuronal cell apoptosis, suggesting that JMT could be used to prevent or treat DPN in diabetic patients.

Effects of Jinmaitong Capsule () on ciliary neurotrophic factor in sciatic nerves of diabetes mellitus rats.

OBJECTIVE: To study the effects of the Chinese medicine Jinmaitong Capsule (, JMT) on the pathomorphology of sciatic nerves, ciliary neurotrophic factor (CNTF), and the mRNA expressions of CNTF in rats with streptozotocin-induced diabetes mellitus (STZ-DM). METHODS: The animal model was established by one time intraperitoneal injection of streptozotocin. The rats were simply divided by random into 5 groups including model group, low-dose JMT group (JL), medium-dose JMT group (JM), high-dose JMT group (JH) and neurotropin group. For each of the above 5 groups, a group of 10 normal Wistar rats matched in body weight, age and gender were set as normal group. Intragastric administrations were started after the animal model established. The JL group were administered with five times the JMT dose recommended for a human adult; the JM group were administered with ten times the JMT dose recommended for a human adult; the JH group were administered with twenty times the JMT dose recommended for a human adult. The neurotropin group was administered with ten times the neurotropin dose recommended for a human adult. All rats were given intragastric administration for 16 weeks and then killed. In the 4th, 8th, 12th, 16th week, body weight and blood glucose level were detected before and after the intervention. The morphologic changes of the sciatic nerves were observed by optical microscope and transmission electron microscope. The CNTFmRNA expressions were detected by real-time fluorescent quantitative polymerase chain protein, and the CNTF protein expressions were detected by immunohistochemical method. RESULTS: The blood glucose levels of the STZ-DM rats were much higher than normal group (P<0.01), and there was no apparent difference between any treatment groups and the model group (P>0.05). Before and after the intervention in the 4th, 8th, 12th, 16th week, there were no significant differences in the body weight among all the groups (P>0.05). The sciatic nerves of STZ-DM rats might have pathomorphological changes in axons, myelin sheaths, and interstitium. The levels of CNTF and CNTF-mRNA expressions in the STZ-DM rats were both significantly decreased (P<0.01). The sciatic nerves of STZ-DM rats might have pathomorphological changes in axons, myelin sheaths, and interstitium. CONCLUSION: JMT could improve the pathomorphology of sciatic nerves by increasing CNTF's and CNTF-mRNA expressions in sciatic nerve tissues, and promote the repair and regeneration of damaged nerve fibers.

Inhibitory effects of salvianolic acid B on apoptosis of Schwann cells and its mechanism induced by intermittent high glucose.

AIMS: To investigate protective effects of Salvianolic acid B (Sal B) on the intermittent high glucose (IHG)-induced oxidative stress, mitochondrial pathway activation and Schwann cell (SC) apoptosis in vitro. MAIN METHODS: SCs were primarily cultured and exposed to the different conditions. Apoptosis was confirmed by the Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and concentration of 8-hydroxy-2-deoxy Guanosine (8-OHdG) was detected by Elisa. Intracellular ROS generation and mitochondrial transmembrane potential (ΔΨm) were detected by flow cytometry analysis. Quantitative real-time reverse transcriptase PCR was performed to analyze the expression levels of Bax and BcL-2. Western blot was performed to analyze the expression levels of some important transcription factors and proteins. KEY FINDINGS: Treatment with Sal B inhibited the IHG-induced oxidative stress by reducing ROS production and 8-OHdG levels, mitochondrial depolarization and apoptosis in SCs in a dose-dependent manner. Furthermore, treatment with Sal B down-regulated the IHG-induced release of cytochrome c, AIF nuclear translocation and Bax expression, but mitigated the IHG-mediated down-regulation of BcL-2 expression in SCs. In addition, treatment with Sal B attenuated the IHG-induced activation of caspase-9 and caspase-3 and minimized the cleavage of PARP in SCs. SIGNIFICANCE: Our results indicated that IHG induced SC apoptosis in both caspase-dependent and caspase-independent pathways by activating the mitochondrial pathway. Sal B inhibited the IHG-induced oxidative stress, activation of the mitochondrial pathway and apoptosis in SCs.

Effect of Jinmaitong serum on the proliferation of rat Schwann cells cultured in high glucose medium.

OBJECTIVE: To investigate the effect of Jinmaitong (JMT) serum on the proliferation of rat Schwann cells (SCs) primarily cultured in high glucose medium. METHOD: SCs were primarily cultured in Dulbecco's minmum essential medium (DMEM control), 50 mmol/L glucose medium (50 mmol/L Glu), 75 mmol/L glucose medium (75 mmol/L Glu), as well as 50 mmol/L glucose medium, with different concentrations of JMT serum (undiluted, 1:2 diluted and 1:8 diluted) and Neurotropin (Ntp), respectively. The proliferation of SCs under different conditions was detected by MTT. RESULT: SCs grew exuberantly in DMEM within 24-72 h, but slowed down at 96 h. The proliferation of SCs was inhibited in 50 mmol/L Glu and 75 mmol/L Glu after cultures of 48, 72 and 96 h, which showed that both were significantly different compared to the control group (P<0.01). The inhibition was more significant in 75 mmol/L Glu than in 50 mmol/L Glu (P<0.05). Spearman's rho analysis revealed that the proliferation of SCs had a negative correlation with the concentration of glucose (r=-0.471, P<0.01). Excluding the time factor, partial correlation showed similar results (r=-0.679, P<0.01). After 48 h, the proliferation of SCs increased significantly in JMT1:2 and Ntp compared with 50 mmol/L Glu (control 0.437+or-0.019, 50 mmol/L Glu 0.367+or-0.035, JMT1:2 0.426+or-0.024, Ntp 0.422+or-0.013; P<0.01), and there were no statistically significant differences among the JMT groups, the Ntp group and the control group (P>0.05). CONCLUSIONS: The proliferation of SCs was inhibited in high glucose medium, and the inhibition was reduced by different concentrations of JMT serum, especially at JMT1:2.

[Relationship between syndrome differentiation of traditional Chinese medicine and vascular endothelial function in patients with diabetic arterial occlusion of lower extremities].

OBJECTIVE: To investigate the relationship between syndrome differentiation of traditional Chinese medicine (TCM) and characteristic changes of vascular endothelial function in patients with diabetic arterial occlusion (DAO) of lower extremities. METHODS: Forty patients with DAO were selected as trial group. Twenty patients among them were attributed to blood stasis syndrome (group A1), and the others were attributed to syndrome of pathogenic dampness-heat attacking the lower limb (group A2) according to syndrome differentiation type of TCM. Patients with diabetes (group B), arteriosclerosis obliterans (group C) and healthy people (group D) were observed as the control groups, respectively. There were 20 cases in each group. Endothelium-dependent dilation (EDD) and endothelium-independent dilation (EID) were measured by high resolution ultrasound in the 100 subjects and the changes of vascular tension factors were also studied. RESULTS: The results showed that EDD in group A was reduced significantly as compared with that in the groups B, C and D. The levels of vascular contractile factors such as endothelin-1 (ET-1) and thromboxane B2 (TXB2) in group A were higher than those in the groups B, C and D, while the levels of vascular dilatory factors such as nitric oxide (NO) and 6-keto-prostaglandin F1alpha(6-Keto-PGF1alpha) were declined significantly as compared with those in the groups B and D. Linear correlation analysis showed that EDD was correlated positively with the levels of NO and 6-Keto-PGF1alpha, while the levels of ET-1 and TXB2 had negative correlation with EDD. EDD and EID in group A2 were declined significantly as compared with those in group A1. CONCLUSION: Our findings indicate that endothelial dysfunction may play an important role in the pathogenesis of DAO and may be associated with syndrome differentiation of TCM.