| Objective:(1)Based on Wnt/β-catenin signal transduction pathway,explore the mechanism of the treatment of disc cartilage in degenerated rats,and provide a new direction for the treatment and research of disc degeneration related diseases;(2)To explore the possible association of Liuwei Dihuang pill with TNF-α inflammatory factors when delaying the mechanism of endplate degeneration of intervertebral disc cartilage.Methods: SPF grade SD rats(250 ± 20g)80 divided into normal control group according to random number table method,sham group,model group,six,each group of 20,model group,six group in the rat tail vertebra Co5 / 6 intervertebral disc cartilage end plate(CEP)under bone injection of alcohol to create cartilage final degeneration model,sham group only acupuncture,blank group rats do not do any treatment.Drug intervention immediately after successful mold building,The control,sham and model group received equal amount of saline gavage,Liuwei Dihuang pill group to give Liuwei Dihuang pill gastric gavage,Rats were killed in batches at2,4,6,and 8 weeks of gavage,Co5 / 6(together with upper and lower vertebrae),The degeneration and scoring,Matrix class 4 degenerative changes in annulus fibrosus structure,nucleus pulposus,cells and nucleus pulposus were observed and scored,Scocore from 4 for normal discs(1 for each category)to 12 for severely degenerated discs(3 for each category),For the statistical analysis of the scoring results,To evaluate the therapeutic effect of Liuwei Dihuang pill on rats with lumbar intervertebral disc degeneration;And TNF-α expression of β-catenin was detected by immunohistochemical staining,The positive cell rate was calculated under light microscopy,And the statistical analysis of the results;The protein expression of β-catenin and TNF-α were detected by Western Blot immunoblotting and used for statistical analysis.Results :(1)HE staining results showed that in normal control and sham group,The nucleus pulposus matrix is normal gelgel,Structural structure of the cartilage endplate,The boundaries are clear,The layered structure of the fiber ring is clear and complete,No obvious fracture,Carondrocytes are evenly distributed;Compared with the normal controls,The degeneration score(P <0.01),A marked reduction of nucleus pulposus cells was visible by light microscopy,Moderate to severe condensation of the nucleus pulposus matrix,The layered structure of the fiber ring is unclear,Structural disorder,And had a significant fracture,The structure and boundary of the cartilage endplate,Chondrocytes are hypertrophic and widely distributed;After the pharmacological intervention,Compared to the model group,The degeneration score of the intervertebral disc cartilage was significantly decreased in rats(P <0.01),Under the microscope,the overall structure of the intervertebral disc tissue is relatively complete,Nuclear pulposus cells are more abundant,Mild condensation of the nucleus pulposus matrix,Fiber annulus lamina has a clear structure,Small amount of local fissure,The cartilage endplate structure is clear and complete,Hypertrophic chondrocytes were significantly reduced;And changes with treatment time,Compared with the four groups of rats,The trend of degeneration score of rats in model group(P <0.05),The decrease of rats(P <0.05),The degeneration score of intervertebral disc cartilage endplate in normal control and sham-operated rats did not change significantly over time(P> 0.05).(2)Immunohistochemistry results showed that: compared with the normal controls,The significant increase of β-catenin and TNF-α protein positive cells in the model group(P <0.01),After drug intervention with Liuwei Dihuang Pill,Compared to the model group,The rate of β-catenin and TNF-α protein positive cells in the cartilage endplate of Liuwei Dihuang pill group was significantly reduced(P <0.01);And with the extension of the treatment time,Comparing the four groups of rats,The rate of β-catenin and TNF-α protein positive cells decreased(P<0.05),The β-catenin and TNF-α protein-positive cell rate increased in the model group(P<0.05);The rate of β-catenin and TNF-α protein positive cells in normal control and sham group did not change significantly over time(P> 0.05).(3)Western blot The results showed that,Compared with the normal controls,The expression level of β-catenin and TNF-α in the model group(P <0.01),After drug intervention with Liuwei Dihuang Pill,Compared to the model group,The expression levels of β-catenin and TNF-α in the cartilage endplate of rats was significantly reduced(P <0.01);With treatment time,Compared with the four groups of rats,The expression level of β-catenin and TNF-α increased with time(P <0.05);The expression levels of β-catenin and TNF-α in Liuwei decreased with time(P <0.05);The protein expression of β-catenin and TNF-α in normal control and sham group did not change significantly over time(P> 0.05).Conclusion:(1)Liuwei Dihuang pill may delay the degeneration of the TNF-α of the intervertebral disc cartilage;(2)Liuwei Dihuang pill may delay the degeneration of the activation of Wnt / β-catenin signaling pathway;(3)Liuwei Dihuang pill may delay the activation of Wnt / β-catenin signaling pathway by affecting the expression of TNF-α by downregulating the degeneration of the end plate in rats. |
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