To Investigate The Effect Of TGF-β1 On Olf In Elderly With Liver And Kidney Deficiency Based On Cell Level

Objective:Based on the basic theories of traditional Chinese medicine such as "the main tendons of the liver,the main bones of the kidney" and "the homology of the liver and kidney",the human lumbar vertebra flavum cells(LFCs)were taken as the subjects to observe and compare the differences in the morphology and fibrosis related indexes of LFCs in the elderly people with liver and kidney deficiency and the young people without liver and kidney deficiency.Moreover,the differences of cell morphology,growth state and fibrosis related indexes after the intervention of different concentrations of TGF-β1 in LFCs of the elderly with liver and kidney deficiency were observed,so as to explore the influence of TGF-β1 on fibrosis of the yellow ligament in the elderly with liver and kidney deficiency,providing new ideas and reference for the treatment and prevention of degenerative lumbar spinal stenosis.Methods: 1.Strictly according to the inclusion and exclusion criteria of cases,the patients who were hospitalized in the Spinal Surgery Ward of Ruikang Hospital Affiliated to Guangxi University of Traditional Chinese Medicine from January 2022 to November 2022 and underwent surgical treatment(posterior lumbar laminectomy with enlarged spinal canal decompression,posterior lumbar laminectomy with nucleus pulpotomy,lumbar intervertebral disc with nucleus pulpotomy under spinal endoscope,etc.)were selected.LFCs were extracted,subcultured and cultured in the cell laboratory.2.The LFCs were identified by immunofluorescence staining and flow surface antigen identification.3.The basic morphology of LFCs in hepato-renal deficiency group and non-hepato-renal deficiency group was observed under inverted optical microscope.4.The expressions of TGF-β1 and fibrosis-related collagen type I,collagen type II and collagen type III in LFCs of hepato-kidney deficiency group and non-hepato-kidney deficiency group were detected by Western Blot.5.Different concentrations of TGF-β1(0,5,20ng/ml)were used to intervene LFCs in patients with liver and kidney deficiency.6.The basic morphology and growth state of cells in blank control group,TGF-β1 low concentration group(5ng/ml)and TGF-β1 high concentration group(20ng/ml)were observed under inverted optical microscope.7.Expression of TGF-β1 and collagen type I,collagen type II and collagen type III of fibrosis-related proteins in blank control group,TGF-β1low concentration(5ng/ml)group and TGF-β1 high concentration(20ng/ml)group were detected by Western Blot.Results:1.Collagen type I,collagen type III and Vimentin were stained by immunofluorescence staining.Both collagen type I and Vimentin proteins showed positive expression with red fluorescence staining.collagen type III fluorescence staining was also red and weakly positive,consistent with the fibroblastic phenotype.The results of flow cytometry showed that CD73,CD90 and CD105 were strongly positive,and STRO-1 was negative,which was consistent with the dry nature of ligamenta flava cells.In summary,the two identification results indicate that the cells obtained from primary extraction and culture in this experiment are LFCs and can be used for subsequent related experiments.2.The morphological differences of LFCs between the non-hepatorenal deficiency group and the hepatorenal deficiency group were observed under an inverted optical microscope.The LFCs in the non-hepatorenal deficiency group were in good growth state,uniform in shape,basically spindle-shaped and long spindle-shaped,with large number,compact arrangement,high density,full cells and no obvious atrophy.The LFCs in the deficiency group of liver and kidney were varied in form,most of which were spindle shaped or long spindle shaped,and some cells were polygonal,triangular and oval with small volume,fewer cells,sparse arrangement and lower cell density.3.Western Blot method was used to detect the expressions of TGF-β1and fibrosis related proteins collagen type I,collagen type II and III in LFCs of hepato-kidney deficiency group and non-hepato-kidney deficiency group.The result is: The protein expressions of TGF-β1,collagen type I,collagen type II and III in hepato-kidney deficiency group were higher than those in non-hepato-kidney deficiency group.There were statistically significant differences in TGF-β1,collagen type II and III(P<0.01),and statistically significant differences in collagen type I(P<0.001).4.The LFCs of liver and kidney deficiency group were observed under inverted optical microscope after the intervention of different concentrations of TGF-β1.In the blank control group,the cells grew well,the cells were closely arranged,the density was the highest,and the cells were relatively full.TGF-β1 low concentration group followed;The cells in the high concentration group of TGF-β1 were sparsely arranged and had the lowest density.The morphology of the three groups of cells were mainly spindle-shaped or long spindle-shaped,and some cells were triangular,irregular polygon,oval,and so on.5.Western Blot method was used to detect the expressions of TGF-β1and fibrosis related proteins collagen type I,collagen type II and collagen type III in LFCs of liver and kidney deficiency group after intervention with different concentrations of TGF-β1.The results showed that compared with blank control group,the protein expressions of TGF-β1 and collagen type I,collagen type II and fibrosis related proteins in TGF-β1 low concentration group were significantly increased,with statistically significant differences(P<0.001).Similarly,compared with blank control group,the protein expressions of TGF-β1 and fibrosis-related collagen type I,collagen type II and collagen type III in TGF-β1 high concentration group were also significantly increased,with statistically significant differences(P<0.001).In addition,compared with the low concentration group of TGF-β1 and high concentration group,the expression of TGF-β1 protein in the high concentration group of TGF-β1 was significantly higher than that in the low concentration group,the difference was statistically significant(P<0.001);The protein expressions of collagen type II and collagen type III associated with fibrosis in TGF-β1 high concentration group were higher than those in low concentration group,and the difference was statistically significant(P<0.05),collagen type III expression levels were statistically significant(P<0.001);There was no significant difference in the protein expression of collagen type I between the two groups(P>0.05).Conclusion:1.The morphology of LFCs in the lumbar spine was observed under an inverted optical microscope.The morphology of LFCs in the non-hepatorenal deficiency group was uniform,while the morphology of LFCs in the hepatorenal deficiency group was diverse,that is,the morphology of non-degenerative LFCs in the lumbar spine was uniform,but the morphology of degenerative LFCs was diverse,and there were some differences between the two.2.TGF-β1 can inhibit the proliferation of LFCs in the lumbar spine of the elderly with liver and kidney deficiency.3.The expression of TGF-β1 was positively correlated with the deficiency syndrome of liver and kidney.4.The degree of fibrosis of ligamenta flava was positively correlated with the expressions of collagen type I,collagen type II and III.5.In the process of fibrosis of the ligamenta flavum in the elderly with liver and kidney deficiency,TGF-β1 played its role in promoting fibrosis of the ligamenta flavum by promoting the expression of collagen type I,collagen type II and collagen type III,and TGF-β1 is a key cytokine in the pathogenesis of ligamentum flavum fibrosis.