Protective Effects Of Polygala On Sciatic Nerve And Correlated Neuronal Soma Of Rats With Diabetic Peripheral Neuropathy

Diabetic peripheral neuropathy (DPN) is a very common chronic complication of diabetes mellitus (DM), whose incidence rate is higher than47%～91%, and it is also one of the main reasons of mutilation and death for DM patients.Owing to high morbidity and mortality of DPN, how to prevent and treat DPN is becoming a momentous topic in DM research. However, the pathogenesis of DPN is not clear, so it has vital significance to further explore pathogenesis of DPN for preventing and treating DPN.In recent years, using Traditional Chinese Medicine to treat diseases in multitarget points attracts more and more attention. It is confirmed that Traditional Chinese Medicine-Polygala has protective effects on central nervous system in previous studies, but there has seldom reports about effects of Polygala on peripheral nerveous system.In this study, DPN rat model was established by intraperitoneal injection of streptozotocin (STZ) to explore the protective effects of Polygala on sciatic nerve and correlated neuronal soma of DPN rats, and provide theoretical and experimental bases for preventing and treating DPN.Part I Protective effects of Polygala on sciatic nerve of DPN ratsObjective:The DPN rat model was established by intraperitoneal injection of STZ in this study. To investigate the protective effects of Polygala on sciatic nerve of DPN rats by observing changes of morphology and functoin of sciatic nerve, and to further explore pathogenesis of DPN, providing theoretical and experimental bases for preventing and treating DPN. Methods:Forty-eight male Wistar rats were randomly divided into4groups (n=12):normal control group, DPN model group, Polygala treatment group and Polygala prevention group. The rats in DPN model group, Polygala treatment group and Polygala prevention group were all made DM model by injecting STZ intraperitoneally. After the DM model was established, the rats in Polygala prevention group were lavaged with Polygala (2.7g·kg-1·d-1) for6weeks. The rats in DPN model group and Polygala treatment group were made DPN model by taking caudal sense nerve conduction velocity (SNCV)<30m/s as standard; then the rats in Polygala treatment group were lavaged with Polygala (2.7g·kg-1·d-1) for6weeks.The blood glucose (BG) and caudal SNCV of rats in each group were respectively detected; Ultraviolet spectrophotometry was used to detect aldose reductase (AR) activity in sciatic nerve; Light microscope and electron microscope to observe the morphology of sciatic nerve; Immunohistochemical staining to observe the expression of neurofilament protein (NFP) in sciatic nerve.Results:1The BG of ratsThe BG of rats in DPN model group was (25.40±4.22) mmol/L, which increased obviously compared with rats in normal control group (P<0.01). The BG of rats in Polygala treatment group and Polygala prevention group were (15.80±5.60) mmol/L and (17.20±4.56) mmol/L respectively, which were both obviously lower than that of DPN model rats (P<0.01).2The caudal SNCV of ratsThe caudal SNCV of rats in DPN model group was (27.45±1.16) m/s, which decreased significantly compared with rats in normal control group (P<0.01). The caudal SNCV of rats in Polygala treatment group and Polygala prevention group were (33.01±1.98) m/s and (33.20±1.09) m/s respectively, which were both significantly higher than that of DPN model rats(P<0.01). 3Morphology of sciatic nerveLight microscope:Normal control group:The myelin sheath of sciatic nerve fibers was intact and neuraxon was clear. DPN model group:The myelin sheath of sciatic nerve fibers was undefined, neuraxon shrinked and degenerated, part of the nerve fibers in transverse section became vacuolization. Polygala treatment group and Polygala prevention group:The structures of sciatic nerve fibers were both better than that of rats in DPN model group.Electron microscope:DPN model group:The myelin sheath of sciatic nerve fibers was in greate disorder, layers separated; microtubule and micro filament in part of the nerve fibers broken and dissolved; mitochondria in neuraxon were expanded and degenerated, the cristae broken. Polygala treatment group and Polygala prevention group:The ultrastructures of sciatic nerve fibers were both better than that of rats in DPN model group.4AR activity of sciatic nerveThe AR activity of sciatic nerve of rats in DPN model group was (1.0695±0.1811) U/mgprot, which increased markedly compared with rats in normal control group (P<0.01). The AR activity of sciatic nerve of rats in Polygala treatment group and Polygala prevention group were (0.6266±0.1633) U/mgprot and (0.3475±0.1068) U/mgprot respectively, which were both markedly lower than that of DPN model rats (P<0.01).5NFP expression in sciatic nerveThe NFP immuno-positive products were buffy exquisite granulas and located at neuraxon of sciatic nerve fibers. Compared with normal control rats, the NFP expression in sciatic nerve of rats in DPN model group decreased remarkably (P<0.01). The NFP expression in sciatic nerve of rats in Polygala treatment group and Polygala prevention group were both remarkably higher than that of DPN model rats (P<0.01).Conclusions:1Polygala can decrease BG, increase caudal SNCV and improve morphology of sciatic nerve of DPN rats. Polygala can also prevent decreasing of caudal SNCV and delay injury of the morphology of sciatic nerve of DM rats.2Polygala can decrease AR activity and up-regulate NFP expression in sciatic nerve of DPN rats. Polygala can also prevent increasing of AR activity and down-regulation of NFP expressoin in sciatic nerve of DM rats.3Polygala has preventive and protective effects on sciatic nerve injury of DPN rats.Part Ⅱ Protective effects of Polygala on sciatic nerve correlated neuronal soma of DPN ratsObjective:The DPN rat model was established by intraperitoneal injection of STZ. To investigate the protective effects of Polygala on sciatic nerve correlated neuronal soma of DPN rats by observing changes of morphology and NGF protein expression of dorsal root ganglia neurons and anterior horn motor neurons of spinal cord, providing theoretical and experimental bases for preventing and treating DPN.Methods:Forty-eight male Wistar rats were randomly divided into4groups (n=12):normal control group, DPN model group, Polygala treatment group and Polygala prevention group. The rats in DPN model group, Polygala treatment group and Polygala prevention group were made DM model by injecting STZ intraperitoneally. After the DM model was established, the rats in Polygala prevention group were lavaged with Polygala (2.7g·kg-1·d-1) for6weeks. The rats in DPN model group and Polygala treatment group were made DPN model by taking caudal SNCV<30m/s as standard; then the rats in Polygala treatment group were lavaged with Polygala (2.7g·kg-1·d-1) for6weeks.Light microscope and electron microscope were used to observe the morphology of dorsal root ganglia neurons and anterior horn motor neurons of spinal cord respectively, immunohistochemical staining to observe NGF protein expression in dorsal root ganglia neurons and anterior horn motor neurons of spinal cord.Results:1The morphology of dorsal root ganglia neuronsLight microscope:Normal control group:neuronal soma structure was intact, nuceli rounded and situated in the middle of soma, tigroid body in granular shape. DPN model group:Neuronal soma shrinked obviously, neuronal pool enlarged obviously, tigroid body dissolved; part of the neuronal soma degenerated, nuclei dissolved or disappeared. Polygala treatment group and Polygala prevention group:The structures of dorsal root ganglia neurons were both similar with that of normal control rats and better than that of rats in DPN model group.Electron microscope:DPN model group:Mitochondria were highly expanded and vacuolizated, lysosome increased obviously; rough endoplasmic reticulum ruptured and ribosome mobilized in endochylema; nuclear membrane depressed and folded, chromatin marginated; nuclei changed obviously and in pyknosis, nuclear membrane dissolved; nuclear debris and many dense masses were found in endochylema. Polygala treatment group and Polygala prevention group:The ultrastructures of dorsal root ganglia neurons were both better than that of rats in DPN model group.2NGF protein expression in dorsal root ganglia neuronsNGF protein immuno-positive products were buffy exquisite granulas and located at the cytoplasm and nucleus of dorsal root ganglia neurons, mainly at cytoplasm. Compared with normal control rats, the NGF expression in dorsal root ganglia neurons of rats in DPN model group decreased obviously (P<0.01). The NGF expression in dorsal root ganglia neurons of rats in Polygala treatment group and Polygala prevention group were both obviously higher than that of DPN model rats (P<0.01).3The morphology of anterior horn motor neurons of spinal cordLight microscope:Normal control group:Neuronal soma was intact, apophysis were clear, tigroid body in plaque shape. DPN model group: Neuronal soma shrinked, neuronal pool enlarged, tigroid body was not observed obvious changes. Polygala treatment group and Polygala prevention group:The structures of anterior horn motor meurons of spinal cord were both similar with that of normal control rats.Electron microscope:DPN model group:The ultrastructures of anterior horn motor meurons of spinal cord were not found obvious changes, only individual tigroid body dissolved.Polygala treatment group and Polygala prevention group:The ultrastructures of anterior horn motor meurons of spinal cord were not found obvious changes.4NGF protein expression in anterior horn motor meurons of spinal cordNGF protein immuno-positive products were buffy exquisite granulas and located at the cytoplasm and nucleus of anterior horn motor neurons of spinal cord, mainly at cytoplasm. Compared with normal control rats, NGF expression in anterior horn motor neurons of spinal cord of rats in DPN model group decreased obviously (P<0.01). NGF expression in anterior horn motor neurons of spinal cord of rats in Polygala treatment group and Polygala prevention group were both obviously higher than that of DPN model rats (P<0.01).Conclusions:1Polygala can improve the morphology of dorsal root ganglia neuronal soma and anterior horn motor meuronal soma of spinal cord of DPN rats and prevent injury of dorsal boot ganglia neuronal soma and anterior horn motor meuronal soma of spinal cord of DM rats.2Polygala can up-regulate NGF protein expression in dorsal root ganglia neurons and anterior horn motor neurons of spinal cord of DPN rats and prevent down-regulation of NGF protein expression in dorsal root ganglia neurons and anterior horn motor neurons of spinal cord of DM rats.3Polygala has preventive and protective effects on sciatic nerve correlated neuronal soma injury of DPN.