| Objective(1)To preliminarily explore the pathogenesis of CSM,the differential proteins between patients with cervical spondylotic myelopathy(CSM)and asymptomatic cervical spinal cord compression patients and healthy individuals were screened out by proteomics,and verified by mass spectrometry multiple reaction monitoring(MRM)and clinical practice(2)To explore the effect of Bushen Huoxue recipe through HIF-1 α in vitro/Protective effect and mechanism of Notch signaling pathway on PC 12 cell injury induced by oxygen glucose deprivation(OGD).To investigate the effect and mechanism of Bushen Huoxue Recipe on spinal cord injury through HIF-1α/Notch signaling pathway in vivo.Methods1.Clinical research(1)Differential proteins of CSM were screened by proteomics(2)The clinical scores and differential protein changes of CSM patients before and after operation were observed.In strict accordance with the inclusion and exclusion criteria,30 patients with CSM were selected to observe the changes of clinical symptoms and differential proteins before and after operation.2.In vitro experimental study(1)Preparation of medicated serum of Bushen Huoxue Recipe.(2)Oxygen glucose deprivation--establishment of OGD model.(3)CCK-8 method was used to detect the toxicity of serum containing Bushen Huoxue Recipe and its protective effect on PC 12 cell injury induced by OGD.(4)Western blot was used to detect the effect of serum containing Bushen Huoxue Recipe on the expression of endoplasmic reticulum stress-related protein GRP78/Chop/Ccaspase12,apoptosis related protein Bcl-2/Bax,Notch signal pathway protein Notch-1 and NICD induced by OGD.(5)The effect of serum containing Bushen Huoxue Recipe on the expression of GFAP and NeuN in PC 12 cell injury model induced by OGD was detected by cellular immunofluorescence.(6)Cells transfected with sihif-1 a,Sihif-1 was detected by qPCR Silence efficiency.(7)Sihif-1 was detected by Western blot a The expression of apoptosis proteins Bax,Bcl-2 and Notch signal pathway related proteins Notch-1 and NICD in each group after transfection.3.In vivo experimental study(1)Preparation of Bushen Huoxue Recipe:buy pieces of traditional Chinese medicine and prepare Bushen Huoxue recipe according to the decocting method and the dose required by gavage(2)Animal grouping,modeling,intervention and material selectionThe modified Allen’s strike method was used to model spinal cord injury,and Bushen Huoxue Formula was used for intragastric treatment.The corresponding experiments were carried out at 7 days.(3)Behavioral evaluationThe behavior of mice with spinal cord injury was evaluated by BBB behavior score and footprint test(4)Histological evaluationIncluding HE staining,Nissl staining and TUNEL experiment(5)MBP/MAP-2/GFAP/gap43/Arg-1/iNOS/IBA-1/HIF-1 was detected by Western blot α/Expression of Bcl-2/Bax/Notch-1/NICD and other proteins(6)TNF was detected by ELISA-α And IL-6 expression(7)The expression of GFAP/MBP/CD163/CD68/NeuN was detected by tissue immunofluorescence stainingResults1.Clinical research(1)Differential protein expression in patients with CSM:HIF-1α was found in proteomic screening.It is the differential protein.(2)he clinical symptoms and scores of CSM patients were significantly improved after operation(P<0.05),and the differential protein HIF-1α was found.It also decreased significantly(P<0.05).2.In vitro experiment(1)CCK-8 cytotoxicity test showed that the serum containing Bushen Huoxue Recipe had no significant toxic effect on PC12 cells(P>0.05).The serum containing Bushen Huoxue recipe could reduce the effect of OGD on PC 12 cytotoxicity and protect cells(P<0.05).(2)After the intervention of serum containing Bushen Huoxue recipe,compared with OGD group,the protein expression of GRP78/Chop/C-caspase12/Bax decreased significantly(P<0.05),and the protein expression of Bcl-2/Notch-1/NICD increased significantly(P<0.05).Compared with the serum containing Bushen Huoxue Recipe group,the HIF-1α of Bushen Huoxue Recipe+RO4929079(Notch pathway inhibitor)group/The protein expression of Bax/Bcl-2/Notch-1/NICD showed an opposite trend(P<0.05).It is suggested that the serum containing Bushen Huoxue recipe may reduce the damage of PC 12 cells induced by OGD by enhancing Notch signal pathway.(3)Immunofluorescence test showed that compared with OGD group,the HIF-1 of BSHX group α/The expression of GFAP decreased significantly(P<0.05),and the expression of NeuN increased significantly(P<0.05)(4)The validation results of RT qPCR showed sihif-1 of hifla-rat-837α The construction effect is the best.The results of Western blot showed that the validation in OGD model also met the experimental requirements.(5)The results of Western blot showed that BSHX group and BSHX+sihif-1αCompared with the corresponding OGD group and OGD+sihif-la Group HIF-1α/The expression of Bax decreased significantly(P<0.05),and the protein expression of Bcl-2/Notch-1/NICD increased(P<0.05).In vivo experiments:(1)The score of the spinal cord injury group was significantly higher than that of the model group on day 7(P<0.05),and the score of the model group was also significantly higher than that of the model group on day 7(P<0.05).It is suggested that Bushen Huoxue recipe can improve the motor function of mice after spinal cord injury.(2)After the intervention of Bushen Huoxue recipe,compared with SCI group,the TUNEL positive cells in the corresponding days decreased significantly(P<0.05),suggesting that Bushen Huoxue recipe may improve the spinal cord function by reducing the apoptosis of tissue cells in the early stage.The results of HE staining and Nissl staining showed that Bushen Huoxue recipe could reduce the injury of spinal cord cells,promote the recovery of spinal cord function in mice.(3)After the intervention of Bushen Huoxue recipe,compared with SCI group,the protein expression of MAP-2 and MBP in BSHX group increased significantly(P<0.05),the protein expression of GFAP and GAP-43 decreased significantly(P<0.05),the protein expression of iNOS/IBA-1 in BSHX group decreased compared with SCI group(P<0.05),and the protein expression of Arg-1 increased significantly(P<0.05);ELISA results showed that after BSHX intervention,TNF-α The expression levels of and IL-6 were lower than those in SCI group(P<0.05).It suggests that Bushen Huoxue recipe may promote the recovery of spinal cord function by reducing the activation of macrophages and microglia after spinal cord injury,transforming pro-inflammatory M1 cells into anti-inflammatory M2 cells,improving inflammatory response.(4)The results of immunofluorescence showed that the expression of CD68 fluorescent cells in BSHX group was significantly lower than that in SCI group(P<0.05),and the expression of NeuN was significantly higher than that in SCI group(P<0.05).(5)The results of Western blot showed that BSHX group was higher than SCI group and BSHX+2-meoe2(HIF-1α The expression of Bcl-2 in inhibitor group was significantly higher than that in SCI+2-meoe2 group(P<0.05),and the expression of Bax was significantly lower(P<0.05),while the expression of Notch-1/NICD protein was higher than that in model group(P<0.05),and the trend of BSHX+2-meoe2 group was more obvious than that in BSHX group,suggesting that Bushen Huoxue recipe may inhibit HIF-1α,Enhance the activation of Notch pathway and reduce the apoptosis of tissue cells after spinal cord injury in mice,so as to improve the function of spinal cord,Conclusion(1)Clinical research:HIF-1 was found through proteomic screening and clinical verification a It plays an important role in the pathogenesis of CSM and is a potential target for clinical prevention and treatment of CSM.(2)In vitro experimental study:Bushen Huoxue recipe can improve the injury of PC 12 cells induced by OGD by activating Notch signal pathway.Its possible mechanism is to improve the hypoxia state of cells,inhibit the stress response of endoplasmic reticulum,reduce apoptosis,and then improve the functional state of cells.(3)In vivo experimental study:Bushen Huoxue recipe may improve the hypoxia state in vivo after spinal cord injury,increase the number of neurons in spinal cord tissue,reduce the activation of early astrocytes,inhibit inflammatory response and apoptosis,and promote the recovery of motor function by activating Notch signal pathway. |
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