Study On The Neural Repair Effect Of Achyranthes Bidentata Polypeptide On Traumatic Brain Injury Model

Background:Traumatic brain injury（TBI）is a kind of traumatic brain injury or peak fracture in Chinese medicine,with high incidence,high disability rate and high mortality.At present,the therapeutic effect of TBI is not ideal in clinical work,and more drug research and development is urgently needed.TCM and its extracts have great research and application prospect in TBI.According to the theory of traditional Chinese medicine,TBI belongs to the acute deficiency syndrome,which should not only supplement the deficiency and damage,but also remove the evil substance.No matter what causes brain injury,blood stasis is the main cause."The Famous Doctor","The Classics of Materia Medica"and"Materia Medica Justice"and other ancient books found that the traditional Chinese medicine Radix ubiosa Radix can"cure the injury in less qi","fill the bone marrow,remove the pain in the brain and lumbar spine pain","walk and replenish,the sex is good down","Qu and can reach,take good care of,the evil person,Gu Yi as the king,the healthy person,also rely on assistance".Recent studies have also found that Achyranthes bidentata polypeptide（ABPP）,an extract of achyranthes Bidentata,has neuroprotective and immunological effects in the central nervous system,and ABPP may play a good role in the treatment of TBI.Purpose:The microstructure and physical and chemical properties of ABPP were analyzed,and the role and possible mechanism of ABPP in neural function repair after brain injury in rats were explored through its intervention in the in vitro and in vivo models of TBI rats,providing theoretical support for the clinical treatment of TBI by ABPP.Method:1.Structure analysis and physicochemical properties of Achyranthes bidentata polypeptide:Scanning electron microscopy was used to observe the microstructure of achyranthes Bidentata polypeptide,X-ray diffractometer and infrared spectrometer were used to detect the crystallinity,absorption spectrum and other properties of achyranthes Bidentata polypeptide,and detect and analyze the purity of ABPP through liquid chromatography.2.Targeted differentiation of bone marrow mesenchymal stem cells and mechanical injury model of neuron-like cells（NLCs）were established:SD rats aged 4-5 weeks were extracted from femur and tibia and cultured with whole-bone marrow adhesion method.By the third generation,BMSCs were identified by flow cytometry using fluorescence-labeled antibodies CD90,CD105,CD73,and CD45.Induction of BMSCs into neuron-like cells by basic fibroblast growth factor（b FGF）25ng/ml,the induced cells were identified by immunofluorescence using TUJ1,Nestin and GFAP antibodies.The neuron-like cells were scratched by 3mm×3mm to make cell damage model.3.Study on the effect of ABPP on the repair of damaged neuron-like cells:experimental groups were divided into blank control group（conventional cell culture）,cell damage group（only damage）,and cell damage ABPP intervention group（damage+0.5,1.0,2.0,4.0,6.0,10.0μg/ml ABPP）.At 24h after injury,CCK-8 was used to observe the effect of different ABPP doses on cell viability.The effects of lactic dehydrogenase（LDH）were measured to evaluate the effects of LDH.The effect of ABPP on the growth and apoptosis of NLCs was determined by western blot（WB）analysis of total protein on phosphase and tension homolog（PTEN）,phosphorylated protein kinase B（p-Akt）,B-cell lymphoma/Leukemia-2-associated X protein（Bax）.4.Study on the neural repair effect of ABPP on TBI in SD rats:30 8-week-old SD rats were randomly divided into 5 groups:Sham operation（Sham）group,model（TBI）group,ABPP low-dose（TBI+0.1mg/kg,L-ABPP）group,ABPP medium-dose（TBI+0.2mg/kg,M-ABPP）group,ABPP high-dose（TBI+1.0mg/kg,H-ABPP）group,6 patients in each group;TBI models were established with electric controlled cortical impactor（e CCI）.ABPP was administered once a day for 3 days.The modified neurological severity scores（m NSS）were evaluated 1 day and 3 days after modeling.3 days after modeling,the nerve repair effect of ABPP on TBI rats was evaluated by brain tissue water content,HE,and immunohistochemical staining of brain tissue with GFAP,Iba-1,Nestin and TUJ1 antibodies.Results:1.The results of structural analysis and physicochemical properties of ABPP showed that:scanning electron microscopy showed that ABPP presented different morphologies,mostly circular,with uneven surface and diameter of 3.6-34.5μm;X-ray diffraction showed that the scanning Angle increased rapidly from 0°to 20°,and a sharp diffraction peak appeared at 20°.After that,the crystallinity of the sample decreased with the increase of the scanning Angle.The infrared spectrum shows that there is an obvious peak value at 2399.37 cm-1,2399.60 cm-1,1666.13 cm-1,1635.42cm-1,1000-1600cm^-1.Liquid chromatography shows that ABPP exhibits peaks at multiple time points.2.Study on the identification of BMSC,directional induction and differentiation,and establishment of NLCs damage model:The cells extracted from the primary generation were cultured to the third generation,and flow cytometry showed CD90（+）,CD105（+）,CD73（+）,CD45（-）.After 5 days of b FGF induction,the cell body was enlarged,the protrusions grew and connected with each other,forming a network structure.Immunofluorescence staining showed strong positive expression of TUJ1 and Nestin antibody,but not obvious expression of GFAP antibody.After the scratch injury of neuron-like cells,the cells showed a"well"shape distribution under the inverted microscope,and the cells disappeared or the process broke at the scratch.3.Study on the effect of ABPP on the repair of damaged neuron-like cells:CCK-8experiment showed that different concentrations of ABPP improved the viability of damaged neuron-like cells to varying degrees,among which 4μg/ml ABPP alleviated the decline of viability of damaged neuron-like cells most obviously.The LDH release experiment showed that ABPP could reduce the LDH release in injured neuron-like cells.WB assay showed that ABPP decreased the expression of PTEN and Bax protein and increased the expression of P-Akt protein in the total protein of damaged neuron-like cells.4.Study on the repair effect of ABPP on TBI in SD rats:1 day after TBI,m NSS evaluation showed that the behavioral score of TBI rats was significantly decreased in M-ABPP group;3 day after TBI,the behavioral score of TBI rats was significantly decreased in all ABPP dosage groups,with the most significant reduction effect in M-ABPP group.After3 days of TBI,the percentage of water content in brain tissue of TBI rats was decreased in ABPP and M-ABPP and H-ABPP groups.HE staining microscope showed that the arrangement of nerve cells was disordered,interstitial edema,cell edema,degeneration and severe necrosis were observed in the TBI group,and the reduction of cell and interstitial edema and cell necrosis were observed in ABPP groups.Immunohistochemical staining showed that GFAP and Iba-1 antibodies were significantly expressed after brain injury in SD rats.The expression of GFAP in ABPP groups was increased compared with that in TBI group,and the increase was more obvious in H-ABPP group.Compared with TBI group,Iba-1expression in ABPP dose groups was decreased,and the decrease was more significant in H-ABPP group.Conclusion:1.ABPP is an irregularly shaped complex with low crystallinity and more water-soluble and fat-soluble-CH,-CO,-NH2 and-CN chemical bonds that are beneficial to cell growth.2.BMSCs were extracted successfully.b FGF induced differentiation of BMSF into CNS-like cells,and the mechanical damage model of CNS-like cells was established.3.ABPP has a certain protective effect on injured CNS-like cells,and the protective effect of 4μg/ml ABPP is more obvious.The protective mechanism may be related to the regulation of the expression of PTEN,p-Akt and Bax proteins.4.Different doses of ABPP have certain neurological repair effects and significantly improve behavioral scores in the early stage of TBI in rats,and M-ABPP group has the best improvement effect.The improvement of neurological function may be closely related to reducing neuroinflammation and brain tissue edema,the reduction of brain tissue edema may be a manifestation of ABPP promoting blood circulation and promoting diuresis.