| Purpose:Diabetes is a common clinical metabolic disease.Diabetic peripheral neuropathy, which incidence is 47%~91%,is one of common chronic complications of diabetes. Painful diabetes peripheral neuropathy is a early sign of diabetic neuropathy and it’s incidence is 13%~26%。PDPN results in a great pain to patients, even leads to the disability or death. The clinical manifestations of PDPN include numbness, pain, heat, cold, hypoesthesia and aggravated, usually heavier in the afternoon or at night. The characteristics of neuropathy pain include allodynia, spontaneous pain, hyperalgesia. The pathogeny of PDPN is complicated and unclear at present, which may be related to oxidative stress, ion channels, inflammatory reaction, high blood sugar and other unknown factors. Diabetes tertiary prevention purpose is to reduce or delay the occurrence and development of diabetes complications in diabetes prevention and control planning outline of implementation. Modern medicine treatment methods include blood sugar control, tricyclic antidepressants, improving microcirculation, antioxidation, nutritioning nerve, ion channel inhibitors, antiepileptic drugs and opioid. But the pain cannot be relieved obviously used multiple therapy methods mentioned above. So there is no satisfactory treatment plan in western medicine, and western medicine has lots of side effects and adverse reactions. Howeve, traditional Chinese medicine emphasizes on evidence-based medicine, seeking both temporary and permanent solutions, the overall adjustment in order to treat PDPN from multiple perspectives and multiple targets. Previous statistics have shown that TCM has more advantages than western medicine and no obvious side effects.Mu Dan granules was developed by Professor Shijia Yu based on TCM theory and his clinical experience. Mu Dan granules was made from astragalus membranaceus, yanhusuo, notoginseng, red paeony root, salvia miltiorrhiza, rhizoma ligustici wallichii, red flower, wood, caulis spatholobi according to TCM theroy.Therapeutic principles of Mu Dan granules was yiqi huoxue and tongluo to stop pain and there was a possitive therapeutic effect in clinic. Previous researches have shown that mu Dan granules improved Na+-k+-ATP activity and increased NGF m RNA expression in sciatic nerve of diabetic rats. It also improved plasma endorphins level in patients and protected the intact structure and function of peripheral nerve. Above all status mentioned have provided theory basis to investigate the mechanism of painful diabetic peripheral neuropathy。Animal model of the painful diabetes peripheral neuropathy was established and then to investigated the mechanism of Mu Dan granules in PDPN from molecular levels and tissue morphology by morphology, RT-PCR, euzymelinked immunosorbent assay and immune- histochemistry.Material and method:180 male Wistar rats, which weight from 180 g to 220 g, the average age was 8 weeks。After the adaptive feeding one week,30 of 180 rats were chosen randomly as the normal group. The rest rats were established the PDPN model, which induced by streptozotocin(STZ). 12 of PDPN rats were as the PDPN group and other PDPN rats were further divided into two groups according to different drugs treatment including Mu Dan granules and phenytoin sodium. All rats were received intragastric administration once per day for eight weeks. Every 8 rats were measured blood glucose, the dynamic change of nerve electrophysiology, the morphological changes by light microscope, electron microscope of the anesthesia sural, the morphological changes of nerve L4-5 dorsal root ganglion and spinal cord, the N-type Ca2+ channel, Kv7.3 channel expression, tumor necrosis factor alpha and interleukin-6 levels of L4-5 dorsal root ganglia and spinal cord at the end of 2nd, 4th and 8th week after receiving treatment.Results:The first part:Effects of Mu Dan granules on painful diabetic peripheral neuropathy rats in the neural electrophysiological and pathological morphology1. Mu Dan granules significantly improved the symptoms of diabetic rats. Compared with the model group rats, the Mu Dan granules group showed better mental state and decreased eating, drinking and urine volume. During the experiment, the body weight of rats in normal group was obviously increased. But the body weight of the model group was obviously decreased, and there were significant differences between the rats in the model group at the 2nd, 4th and 8th week(P<0.01). The body weight of Mu Dan granules group rats did not change significantly. Compared with the model group, there was no difference; compared with the normal group, there were statistically differences at the 2nd and 4th week(P<0.05) and a significant difference at the 8th weeks(P<0.01).2. During the experiment, the blood glucose levels of the model group increased significantly, and compared with the normal group had significant difference at the 2nd, 4th and 8th week(P<0.01); while the blood glucose of treatment group(Mu Dan granules and phenytoin sodium) did not change significantly, compared with the model group ratio had no significant difference(P>0.05). Compared with the normal group, the blood glucose of treatment group was still higher than normal group, had significant difference the 2nd, 4th and 8th week(P<0.01).3. The mechanical pain threshold of the model group rats was significantly decreased during the experiment, and compared with the normal group had significant difference at the 2nd, 4th and 8th week(P<0.01). Compared with the model group, the mechanical pain threshold of treatment group rats showed a rising trend and had statistical significance at the 2nd week(P<0.05),also there was significant difference at the 4th and 8th week(P<0.01); compared with the normal group, it was statistically significant at the 2nd and 4th week(P<0.05),and no difference at the 8th week(P>0.05).4. During the experiment, the nerve conduction velocity(NCV) of the model group was significantly decreased, compared with the normal group, there were significant differences at the 2nd, 4th and 8th week(P<0.01). NCV of Mu Dan granule group rats was faster than other group, and compared with the model group, there were statistically differences at the 2nd and 4th week(P<0.05) and significant difference at 8th weeks(P<0.01), and compared with the normal group, there were statistically differences at the 2nd,4th and 8th week(P<0.05). Compared with the normal group, the phenytoinum natricum group had significant differences at the 2nd, 4th and 8th week(P<0.01), and compared with the model group, there is no statistical difference(P>0.05).5. From pathological tissue morphological observation, myelin layer plate structure of sural nerve fiber of the normal group rats was clear, dense, uniform, regular, and the axon was no swelling and shrinking, and nerve membrane structure was intact. The myelin structure of sural nerve fiber of the model group rats was fuzzy and loose. The axon filaments and microtubules were fracture and dissolved and had phenomenons of myelinoclasis and plate separation. Schwann cells appeared irregular nucleus, and mitochondrial vacuole were appeared. The rats in the model group were significantly more severe with the time, while the damagements of the treatment group rats were significantly alleviated, and the effect was better than the model group.The second part: Effects of Mu Dan granules on painful diabetic peripheral neuropathy rats in calcium ion channel and potassium channels Kv7.31.The effects of Mu Dan granules on the calcium ion channel expression in painful diabetic peripheral neuropathy rats.Results of RT-PCR:With the extended time, N-type Ca2+ channel m RNA expression level of the L4-5 dorsal root ganglia in the model group presented a rising trend. Compared with the normal group, there were statistical differences(P<0.05). While m RNA expression level of the treatment group showed a gradual falling trend. There were statistical differences at the 4th and 8th week(P<0.05) compared with the model group and at the 2nd week compared with the normal group; there is no difference between the Mu Dan granules group and phenytoin sodium group in m RNA expression level(P>0.05).Results of immunohistochemistry:Cell positive expression of the normal group was rare,but the model group showed gradually increased expression at various time points(2nd,4th and 8th week). There were a large amount of brown N-type Ca2+ channel positive cells in the cytoplasm of the spinal cord cells, it was statistical differences compared with the normal group(P<0.05). Cell positive expressions of the Mu Dan granules group and the phenytoinum natricum group showed a weaker trend compared with the model group, but there was no statistical difference at the 2nd week(P>0.05). However, cell positive expressions of the Mu Dan granules group and the phenytoinum natricum group significantly decreased, compared with the model group at 4th week(P<0.05) and at 8th week(P<0.01). Compared with the normal group, cell positive expressions of the Mu Dan granules group and the phenytoin sodium group were significantly higher at 2nd week(P<0.05), But there were no difference at 4th week and 8th week(all P>0.05). At all time points, cell positive expression of the spinal cord dorsal was higher than it in root ganglion.2. Effects of Mu Dan granules on the potassium channels Kv7.3 expression in painful diabetic peripheral neuropathy rats.Results of RT-PCR:Compared with the normal group, the m RNA expression of potassium channels Kv7.3 in L4-5 dorsal root ganglia of the model group significantly decreased at 2nd week(P<0.05) 4th week(P<0.01) and 8th week(P<0.01). But compared with the model group, the m RNA expressions of Mu Dan granules group and the phenytoin sodium group were increased from 2nd week(P>0.05). Specially, it increased significantly at 4th week(P<0.05) and 8th week(P<0.01). The m RNA expressions of Mu Dan granules group and the phenytoinum natricum group were statistical lower than the normal group, at 2nd week(P<0.05), but there were no differences at 4th week and 8th week(all P>0.05).Results of immunohistochemistry:There were a large amount of positive cells with brown cytoplasm in the spinal cord of the normal rats, and the expression of positive cell was higher. The positive expressions of the model group at each time point(2nd week, 4th week, 8th weeks) were gradually weakened, significantly lower than normal group. Compared with the normal group at 2nd week, 4th week and 8th week(P<0.05, P<0.01, P<0.01). Positive cells expression of the Mu Dan granules group and the phenytoinum natricum group showed increasing trend, had statistical significance at 4th week(P<0.05) compared with the model group. And compared with normal group, the expressions of positive cells were slightly weaker, had statistical significant at 2nd week(P<0.05), but no statistical significance at 4th week and 8th week(all P>0.05). At all time points, positive cell expressions of the spinal cord dorsal were higher than it in root ganglion.The third part:Effects of Mu Dan granules on proinflammatory cytokines in painful diabetic peripheral neuropathy rats.The levels of TNF-α and IL-6 in spinal cord of the model group were significantly higher than the normal group, and it was positive relationship with the time(P<0.05 at 2nd week, P<0.01 at 4th and 8th week). Mu Dan granule can markedly decrease the level of TNF- alpha and IL-6 compared with the model group(P<0.05 at 2nd week and 4th, P<0.01 at 8th week).Conclusion:The first part:Effects of Mu Dan granules on the neural electro-physiological and pathological morphology in painful diabetic peripheral neuropathy rats.1. It was feasible that the model established method of painful diabetic peripheral neuropathy rat by single intraperitoneal injecting 53 mg/Kg urea.2. Mu Dan granules had only inhibited the blood glucose levels of PDPN rats raising.3. Mu Dan granules increased mechanical pain threshold in diabetic neuropathy rats.4. Mu Dan granules accelerated sciatic nerve conduction velocity in diabetic neuropathy rats.5. Mu Dan granules had protected the sural nerve and dorsal root ganglion tissue which were injuried.The second part:Effects of Mu Dan granules on calcium ion channel and potassium channels Kv7.3 in painful diabetic peripheral neuropathy rats.Mu Dan granules reduced pain and other symptoms which were caused by PDPN, through down-regulating gene expressions of N-type calcium channel and through up-regulating gene expressions of potassium ion channel Kv7.3.The third part:Effects of Mu Dan granules on proinflammatory cytokines in painful diabetic peripheral neuropathy rats.Mu Dan granules had inhibited the inflammation of PDPN rats, through down-regulating levels of TNF-αand IL-6.
|
PDF Full Text Request