| Objective: Spinal cord injury(SCI)is a central nervous system disease with a very high disability rate.Once sick,it will cause sensory and motor dysfunction and bring huge economic and psychological burdens to patients and their families.It has become a major disease problem that the medical community urgently needs to overcome.Therefore,continuing to explore effective methods or drugs for the treatment of SCI is of great research significance.After a long-term screening,we found that Total Flavonoids of Hawthorn Leaves(TFHL)have a potential therapeutic effect on SCI.In our previous research,we found that TFHL can obviously inhibit the release of inflammatory factors and have a protective effect on SCI.This study will further explore the potential of TFHL in the treatment of SCI,and provide a strong theoretical basis for the value development of Chinese medicine and the treatment of SCI.Methods: 1.The mass spectrometry data of the TFHL was collected by Q-Orbitrap high-resolution LC-MS technology.According to chromatographic retention time,primary and secondary mass spectrometry information,after processing by metabolomics data analysis software CD2.1,search and compare with the database(mz Cloud,mz Vault,Chem Spider)to obtain the corresponding substance composition.2.Adult Sprague-Dawley(SD)rats were randomly divided into Sham group,SCI group and TFHL group.Construct SCI model through Allen’s strike method.After TFHL treatment,the BBB score to evaluate the motor recovery function of each group of rats,HE staining to observe the changes of spinal cord tissue in each group,transmission electron microscopy to observe the ultrastructural changes of spinal cord tissue in each group,TUNEL staining to observe the apoptosis of nerve cells in each group,Nissl staining to observe the changes of neuron Nissl bodies of spinal cord tissue in each group,immunohistochemistry to observe the autophagy of spinal cord tissue in each group,Western Blot to detect the expression of the autophagy-related protein LC3,P62,Beclin-1 and the nerve growth-related protein SCG10 of spinal cord tissue in each group.3.The spinal cord anterior horn motor neurons were extracted and cultured in vitro,and they were randomly divided into Control group,Hurt group,Astrocyte group,Direct drug group and Co-culture group.After glutamate damages the motor neurons of the anterior horn of the spinal cord,a co-culture model of astrocytes and injured neurons is constructed.After TFHL treatment,the morphological changes of neurons in each group were observed by optical microscope.The changes in ultrastructure of neurons in each group were observed by transmission electron microscope.The regeneration of neuronal axons in each group was observed by MAP2 immunofluorescence.The autophagy of neurons in each group was observed by LC3-Ⅱimmunofluorescence.The secretion of brain-derived neurotrophic factor(DBNF),glial cell-derived neurotrophic factor(GDNF),nerve growth factor(NGF)and ciliary neurotrophic factor(CNTF)in the culture medium of each group were detected by ELISA.The expression of the autophagy related protein LC3,P62,Beclin-1 and the nerve growth-related protein SCG10 of neurons in each group were detected by Western Blot.Results: 1.TFHL were detected by Q-Orbitrap high-resolution LC-MS technology and found that the extract matched a total of 856 compounds in mz Cloud.In the mz Cloud best match,there are 304 compounds with a comprehensive score greater than 70,of which 19 compounds have a mz Cloud score greater than 80.The mass spectrometry analysis of the compounds revealed that the 19 compounds with an mz Cloud score greater than 80 were quercetin 3β-D-glucoside,rutin,catechin,quercetin,isorhamnetin,genistein,Astragalus,luteolin,(-)-epicatechin,3-methoxy-5,7,3’,4’-tetrahydroxy-flavonoids,etc.2.The BBB score results showed that compared with the Sham group,the BBB score of the SCI group was significantly decreased,and after TFHL treatment,the BBB score of the TFHL group was increased(p<0.05).The results of HE staining and transmission electron microscopy showed that the morphology of neurons in the spinal cord tissue of the SCI group was disordered,the nucleus was retracted,and the morphology of neurons could not be distinguished,accompanied by an amount of inflammatory cell infiltration.There were phagesomes in neurons of the SCI group.The spinal cord tissue and neurons in the TFHL group were improved significantly,and a larger number of autophagosomes appeared in neurons.The results of TUNEL staining showed that a large number of apoptosis occurred in the spinal cord tissue of the SCI group.Compared with the SCI group,the apoptosis of the TFHL group was significantly reduced(p<0.05).The results of Nissl staining showed that Nissl bodies in the neurons of the SCI group atrophied and the number decreased,while the tabby-like Nissl bodies appeared in the neurons of the THFL group and the number increased(p<0.05).The results of immunohistochemistry showed that the positive intensity of LC3 in the SCI group was significantly enhanced,compared with the SCI group,the positive intensity of LC3 in the TFHL group was more significant(p<0.05).The results of Western Blot showed that compared with the sham group,the protein expression levels of LC3-Ⅱ,Beclin-1 and LC3-Ⅱ/LC3-Ⅰin the spinal cord tissue of the SCI group increased,while the protein expression levels of SCG10 and P62decreased(p<0.05).Compared with the SCI group,the protein expression levels of P62 and SCG10 decreased,but the protein expression levels of LC3-Ⅱ,Beclin-1 and LC3-Ⅱ/LC3-Ⅰincreased in the THFL group(p<0.05).3.The results of optical microscope showed that the cell bodies of the neurons in the Hurt group were significantly retracted,and the axons and dendrites were broken.Compared with the Hurt group,the state of neurons in the Co-culture group recovered best.The results of transmission electron microscopy showed that the nucleus of neurons retracted,the nucleus edges were irregular,and there were working autophagosomes in the cytoplasm in the Hurt group.After TFHL treatment,the neuronal morphology of the Co-culture group recovered the best and the number of autophagosomes in the cytoplasm was the largest.The results of MAP2 immunofluorescence showed that the green fluorescent neuron cell bodies were seen in the Hurt group,but its axons and dendrites were difficult to see.There were some axons regenerated in the Astrocyte group,the Direct drug group and the Co-culture group,and the neuron axons in the Co-culture group grew significantly and the length was the longest(p<0.05).The results of LC3-Ⅱ immunofluorescence showed that positive LC3-Ⅱ with red fluorescence were observed in the Hurt group,Astrocyte group,Direct drug group and Co-culture group.Comparing the average optical density of LC3-Ⅱ,it was found that the average optical density value of LC3-Ⅱ in the Co-culture group was the highest(p<0.05).The test results of ELISA showed that the neurotrophic factors such as DBNF,GDNF,NGF,CNTF,etc.were completely detected in the Co-culture group,and the contents of them were the most(p<0.05).The results of Western Blot showed that the protein expression levels of LC3-Ⅱ,Beclin-1 and LC3-Ⅱ/LC3-Ⅰ increased(p<0.05),the protein expression levels of P62 decreased(p<0.01),while the protein expression of SCG10 was not statistically significant in Hurt group(p>0.05).Compared with the Hurt group,the protein expression levels of P62 and SCG10 in the Astrocyte group and Direct drug group decreased,while the protein expression levels of LC3-Ⅱ/LC3-Ⅰ,Beclin-1 increased(p<0.05).There was no significant difference between the Astrocyte group and the Direct drug group(p>0.05).The Co-culture group was significantly different from the first three groups(p<0.05).Conclusion: TFHL have abundant flavonoids,which can make the spinal cord microenvironment better,reduce neuronal apoptosis,improve cell autophagy and neuron functional status,promote the regeneration of spinal motor neuron axons to play a neuroprotective effect,and ultimately effectively alleviate the sensory and motor dysfunction of the spinal cord. |
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