Protective Effects Of Polygala On Sciatic Nerve Correlated Neuronal Soma And Hippocampus Neurons Of DPN Rats

Diabetic neurologic complication is one of the most common chroniccomplications of diabetes mellitus (DM). Diabetic peripheral neuropathy(DPN) is one of the main reasons of mutilation and death for DM patients,whose incidence rate reaches up to47%91%. In addition, the incidence ofdiabetic encephalopathy (DE) which is characterized by cognitivedysfunction is increasing. Because it significantly impacts on life quality ofDM patients, diabetic neurologic complication has become an importantsubject of DM research. However, its mechanism still is not very clear, sofurther discuss the pathogenesis has extremely vital significance for clinicalprevention and treatment on diabetic neurologic complication.In recent years, using Traditional Chinese Medicine to treat diseases inmultitarget points attracts more and more attention. It is confirmed thatTraditional Chinese Medicine-Polygala has protective effects on centralnervous system of animal models such as dementia and aging, but there areseldom reports about effects of Polygala on peripheral nervous system andDM hippocampal injury.In this study, DPN rat model was established by intraperitonealinjection of streptozotocin (STZ) to explore the protective effects of Polygalaon sciatic nerve correlated neuronal soma and hippocampus neurons of DPNrats, so to provide theoretical and experimental bases for preventing andtreating diabetic neurologic complication.Part Ⅰ Protective effects of Polygala on sciatic nerve correlatedneuronal soma of DPN ratsObjective:The DPN rat model was established by intraperitoneal injection of STZ in this study. Changes of morphology and apoptosis correlated indices ofdorsal root ganglia neurons and anterior horn motor neurons of spinal cordwere observed to further explore pathogenesis of DPN, and to investigate theprotective effects of Polygala on sciatic nerve correlated neuronal soma ofDPN rats, provide theoretical and experimental bases for preventing andtreating DPN.Methods:48male Wistar rats were randomly divided into4groups (n=12):normal control group, DPN model group, Polygala treatment group andPolygala prevention group. The rats in DPN model group, Polygala treatmentgroup and Polygala prevention group were all made DM model by injectingSTZ intraperitoneally. After the DM model was established, the rats inPolygala prevention group were lavaged with Polygala (2.7g·kg-1·d-1) for6weeks. The rats in DPN model group and Polygala treatment group weremade DPN model by taking caudal sense nerve conduction velocity (SNCV)＜30m/s as standard. Then the rats in Polygala treatment group were lavagedwith Polygala (2.7g·kg-1·d-1) for6weeks.The blood glucose (BG) and caudal SNCV of rats in each group wererespectively detected. Nissl’s staining was used to observe the morphology ofdorsal root ganglia neurons and anterior horn motor neurons of spinal cordrespectively. Terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) was used to detect apoptotic index (AI) of dorsal rootganglia neurons and anterior horn motor neurons of spinal cord.Immunohistochemical staining and Western blotting were used to observeBcl-2and Bax protein expression of dorsal root ganglia.Results:1BGThe BG of rats in DPN model group was (25.40±4.22) mmol/L, whichincreased obviously compared with rats in normal control group (P<0.01).The BG of rats in Polygala treatment group and Polygala prevention groupwere (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).2SNCVThe 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 andPolygala prevention group were (33.01±1.98) m/s and (33.20±1.09) m/srespectively, which were both significantly higher than that of DPN modelrats (P＜0.01).3The morphology of dorsal root ganglia neuronsNormal control group: Neuronal soma was intact, nuclei rounded andsituated in the middle of soma, Nissl body in small plaque shape. DPNmodel group: Neuronal soma shrinked obviously, neuronal pool enlargedsignificantly, Nissl body dissolved, part of the neuronal somas degenerated,nuclei dissolved or disappeared. Polygala treatment group and Polygalaprevention group: The structures of dorsal root ganglia neurons were bothsimilar with that of normal control rats and better than that of rats in DPNmodel group.4AI of dorsal root ganglia neuronsThe AI of dorsal root ganglia neurons in DPN model group was(40.22±2.79)%, which increased significantly compared with rats in normalcontrol group (P＜0.01). The AI of dorsal root ganglia neurons in Polygalatreatment group and Polygala prevention group were (22.87±2.50)%and(15.45±2.24)%respectively, which were both significantly lower than that ofDPN model rats (P＜0.01).5Bcl-2and Bax protein expression in dorsal root ganglia neuronsImmunohistostaining: The Bcl-2and Bax immuno-positive productswere buffy and located at the cytoplasm and nuclear membrane of dorsal rootganglia neurons, mainly at cytoplasm. Compared with normal control rats,the number of Bcl-2immuno-positive cells in dorsal root ganglia neurons ofrats in DPN model group decreased obviously (P＜0.01), while that of Baxincreased significantly (P＜0.01). Compared with DPN model rats, the number of Bcl-2immuno-positive cells in dorsal root ganglia neurons of ratsin Polygala treatment group and Polygala prevention group were bothincreased obviously (P＜0.01), while that of Bax decreased significantly(P＜0.01).Western Blotting: Compared with normal control rats, the Bcl-2expression in dorsal root ganglia neurons of rats in DPN model groupdecreased obviously (P＜0.01), while that of Bax increased significantly(P＜0.01). Compared with DPN model rats, the Bcl-2expression in dorsalroot ganglia neurons of rats in Polygala treatment group and Polygalaprevention group were both increased obviously (P＜0.01), while that of Baxdecreased significantly (P＜0.01).6The morphology of anterior horn motor neurons of spinal cordNormal control group: Neuronal soma was intact, apophysis clear,nuclei rounded and situated in the middle of soma, nucleolus clear, Nisslbody in plaque shape. DPN model group: Neuronal somas were colouredwith defferent depth, somas swellen, Nissl body dissolved, nuclei displaced,part of the nuclei dissolved or disappeared. Polygala treatment group andPolygala prevention group: The structures of anterior horn motor neurons ofspinal cord were both similar with that of normal control rats and better thanthat of rats in DPN model group.7AI of anterior horn motor neurons of spinal cordThe AI of anterior horn motor neurons of spinal cord in DPN modelgroup was (45.25±3.67)%, which increased significantly compared with ratsin normal control group (P＜0.01). The AI of anterior horn motor neurons ofspinal cord in Polygala treatment group and Polygala prevention group were(27.84±3.91)%and (11.95±1.44)%respectively, which were bothsignificantly lower than that of DPN model rats (P＜0.01).Conclusions:1Polygala can decrease BG and increase caudal SNCV of DPN rats.2Polygala can improve pathological changes of sciatic nerve correlatedneuronal soma. It also can up-regulate Bcl-2and down-regulate Bax protein expression of dorsal root ganglia neurons and decrease apoptosis of dorsalroot ganglia neurons and anterior horn motor neurons of spinal cord of DPNrats.3Polygala has preventive and protective effects on sciatic nervecorrelated neuronal soma injury of DPN.Part Ⅱ Protective effects of Polygala on hippocampus neurons of DPNratsObjective:The DPN rat model was established by intraperitoneal injection of STZ.Changes of morphology, quantity, AI and expression of apoptosis correlatedproteins such as Bcl-2and Bax of hippocampal neurons were observed toinvestigate the protective effects of Polygala on hippocampus neurons ofDPN rats.Methods:48male Wistar rats were randomly divided into4groups (n=12):normal control group, DPN model group, Polygala treatment group andPolygala prevention group. The rats in DPN model group, Polygala treatmentgroup and Polygala prevention group were all made DM model by injectingSTZ intraperitoneally. After the DM model was established, the rats inPolygala prevention group were lavaged with Polygala (2.7g·kg-1·d-1) for6weeks. The rats in DPN model group and Polygala treatment group weremade DPN model by taking caudal SNCV＜30m/s as standard. Then the ratsin Polygala treatment group were lavaged with Polygala (2.7g·kg-1·d-1) for6weeks.Nissl’s staining was used to observe the morphology of hippocampalCA1area and to measure the number of neurons respectively. AI of neuronsin hippocampal CA1area was observed by TUNEL. Bcl-2and Bax proteinexpression of hippocampus were assayed by Western blotting.Results:1The morphology and quantity of neurons in hippocampal CA1area Normal control group: Neurons arranged closely and neat, nuclearmembrane intact, nucleus clear, Nissl body intensive and situated aroundnuclear. DPN model group: Neurons arranged sparsely, the number ofsurvival neurons decreased obviously, nucleus of part neurons dissolved ordisappeared, Nissl body dissolved, glial cell proliferated. Polygala treatmentgroup and Polygala prevention group: Morphology of neurons was similarwith normal rats, only a few cells lost.2AI of neurons in hippocampal CA1areaThe AI of neurons in hippocampal CA1area of DPN model group was(64.42±4.73)%, which increased significantly compared with rats in normalcontrol group (P＜0.01). The AI of neurons in hippocampal CA1area ofPolygala treatment group and Polygala prevention group were(42.68±2.38)%and (31.68±1.48)%respectively, which were bothsignificantly lower than that of DPN model rats (P＜0.01).3Bcl-2and Bax protein expression in hippocampusCompared with normal control rats, the Bcl-2protein expression inhippocampus of DPN model rats decreased obviously (P＜0.01), while thatof Bax increased significantly (P＜0.01). Compared with DPN model rats,the Bcl-2protein expression in hippocampus of rats in Polygala treatmentgroup and Polygala prevention group were both increased obviously(P＜0.01), while that of Bax decreased significantly (P＜0.01).Conclusions:1Polygala can improve pathological changes of neurons inhippocampal CA1area of DPN rats.2Polygala can up-regulate Bcl-2protein expression and down-regulateBax protein expression of hippocampus of DPN rats. It also can decrease AIand reduce apoptosis of hippocampal CA1area of DPN rats.3Polygala has preventive and protective effects on hippocampusneurons injury of DPN rats.