| Objective:To study the regulatory effect of serum containing Yishen Huoxue Tongluo Recipe on NF-kappa B signaling pathway in nucleus pulposus cells under different hydrostatic pressures,so as to explore the possible therapeutic mechanism and target of Yishen Huoxue Tongluo recipe in the treatment of degenerative disc diseases from the perspective of molecular biology.Methods:Nucleus pulposus of thoracic and lumbar segments of New Zealand white rabbits was removed under sterile environment and cultured in vitro to the third generation.Different hydrostatic pressure loading intervention models were set up.They were randomly divided into blank control group,simple pressure intervention group and pressure plus Chinese medicine serum intervention group.The morphology and growth of nucleus pulposus cells of intervertebral discs were observed by inverted phase contrast microscope.Ultrastructural changes and differences of nucleus pulposus cells were observed by fluoroscopy and electron microscopy;apoptotic changes of nucleus pulposus cells were observed by double staining with Annexin V-FITC/Propidium Iodide apoptotic kit;changes of NF-kappa B p65,Collagen? and MMP-13 contents in nucleus pulposus cells were detected by Western Blot method.Results:1.Inverted phase contrast microscopy observation:1.At the same time,the nucleus pulposus cell volume decreased with the increasing hydrostatic pressure,especially at 3MPa hydrostatic pressure.(2)Under the same hydrostatic pressure,the morphological differences of nucleus pulposus cells in different groups were not obvious.(3)Under the same pressure and time,the morphology of nucleus pulposus cells in the serum intervention group was more complete and the growth condition was better than that in the blank control group and the simple pressure intervention group.2.TEM observation:1.At the same time,the ultrastructure of nucleus pulposus cells in the blank control group was intact.The neurite structures of nucleus pulposus cells in the pressure intervention group and the pressure plus Chinese medicine serum intervention group were ruptured in varying degrees.The secondary neurite structures of nucleus pulposus cells could be seen in the hydrostatic pressure group of 0.3 MPa and 1 MPa,but the main neurite structures at both ends of the cell body were intact.The structure of nucleus pulposus cells in the pressure+TCM serum intervention group was more complete than that in the pressure intervention group alone;the main and secondary processes of nucleus pulposus cells were broken under 3 MPa hydrostatic pressure,but the structure of nucleus pulposus cells in the pressure+TCM serum intervention group was not significantly different from that in the pressure intervention group alone.(2)Under the same hydrostatic pressure,there was no significant difference in the ultrastructure of nucleus pulposus cells under transmission electron microscopy at different time.3.Annexin V-FITC/PI method was used to determine the apoptotic rate of nucleus pulposus cells in intervertebral discs under different hydrostatic pressures:1.Under normal pressure(0.1 MPa hydrostatic pressure),there was no significant difference in the apoptotic rate of nucleus pulposus cells at different time of action(P>0.05);the apoptotic rate of nucleus pulposus cells in the pressure plus Chinese medicine serum intervention group was lower than that in the pressure intervention group,with significant difference(P<0.05).)(2)Under the hydrostatic pressure of 0.3 MPa,there was no significant difference in the apoptotic rate of nucleus pulposus cells of intervertebral disc at different time(P>0.05);the apoptotic rate of nucleus pulposus cells in the pressure+Chinese medicine serum intervention group was lower than that in the simple pressure intervention group,the difference was statistically significant(P<0.05).(3)Under 1 MPa hydrostatic pressure,the apoptotic rate of nucleus pulposus cells had significant difference in different time of action(P<0.05);the apoptotic rate of nucleus pulposus cells in pressure+Chinese medicine serum intervention group was lower than that in simple pressure intervention group,the difference was statistically significant(P<0.05).(4)Under 3 MPa hydrostatic pressure,the apoptotic rate of nucleus pulposus cells had significant difference in different time(P<0.05);the apoptotic rate of nucleus pulposus cells in pressure+traditional Chinese medicine serum intervention group was lower than that in simple pressure intervention group,the difference was statistically significant(P<0.05).The apoptotic rate of nucleus pulposus cells in the pressure intervention group was lower than that in the 3MPa hydrostatic pressure group at 2h,4h,6h and 0.3MPa hydrostatic pressure respectively(P<0.05).Under the same hydrostatic pressure and action time,the apoptotic rate of nucleus pulposus cells in the pressure+TCM serum intervention group was lower than that in the simple pressure intervention group(P<0.05).4.Western Blot method was used to detect the expression of related proteins in nucleus pulposus cells:1.Under the same pressure,the expression of Collagen ? decreased and the expression of MMP-13 increased with the increase of time.Compared with blank control group and simple pressure intervention group,the serum intervention group of traditional Chinese medicine could inhibit this trend,and the difference was statistically significant(P<0.05).(2)Under the same pressure,the expression of NF-kappa B p65 in the serum intervention group of traditional Chinese medicine decreased with the increase of time compared with the blank control group and the simple pressure intervention group(P<0.05).(3)Under the same pressure and time,the expression of Collagen ? decreased and MMP-13 increased in the Chinese medicine serum intervention group compared with the blank control group and the simple pressure intervention group,and the difference was statistically significant(P<0.05).(4)Under the same pressure and time,the expression of NF-kappa B p65 in the serum intervention group of traditional Chinese medicine was decreased than that in the blank control group and the simple pressure intervention group(P<0.05).(5)At the same time and under different pressures,the expression of Collagen ? decreased and the expression of MMP-13 increased.Compared with the blank control group and the simple pressure intervention group,the serum intervention group of traditional Chinese medicine could inhibit this trend,and the difference was statistically significant(P<0.05).(6)At the same time and under different pressures,the expression of NF-kappa B p65 in serum intervention group of traditional Chinese medicine was decreased than that in blank control group and simple pressure intervention group(P<0.05).Conclusion:1.There is a "pressure-time" dependent relationship between cultured nucleus pulposus cells in vitro.The prolongation of action time and the increase of pressure can lead to the degeneration of nucleus pulposus cells.2.Within 6 hours,the pressure load factor was the main reason for promoting apoptosis of nucleus pulposus cells.Within 6 hours of low hydrostatic pressure(0.1 MPa,0.3 MPa),the apoptotic changes of nucleus pulposus cells were not obvious.Within 6 hours of high hydrostatic pressure(1 MPa,3 MPa),the apoptotic changes of nucleus pulposus cells were significant.3.Yishen Huoxue Tongluo Decoction can delay the trend of intervertebral disc degeneration.Its therapeutic mechanism may be related to the activation of NF-kappa B signaling pathway in intervertebral disc.4.Yishen Huoxue Tongluo recipe can effectively delay the degeneration of nucleus pulposus cells,but it can not reverse the process of intervertebral disc degeneration. |
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