Polydatin inhibited TNF-α-induced apoptosis of skeletal muscle cells through AKT-mediated p38 MAPK and NF-κB pathways.

Skeletal muscle atrophy severely impacts one's quality of life. The effects and mechanism of polydatin on skeletal muscle atrophy are unclear. This study investigated the effects and mechanism of polydatin on TNF-α-induced skeletal muscle cells. The skeletal muscle cell atrophy model was established by inducing C2C12 cells with TNF-α. Cell viability, IL-1ß levels and cell apoptosis were assessed. The mRNA and protein expression levels of apoptosis-related proteins were measured. Meanwhile, the binding of polydatin to AKT was analyzed by molecular docking. TNF-α reduced cell fusion and viability while up-regulated IL-1ß level and promoted cell apoptosis. TNF-α activated AKT, NF-κB, and p38 MAPK signaling pathways. Polydatin reversed these effects induced by TNF-α, with a low concentration being more effective. Polydatin was predicted to bind to GLY162, PHE161, GLU198, THR195 and GLU191 sites of AKT protein through van der Waals force and conventional hydrogen bonds. Overexpression of AKT led to increased phosphorylation levels of AKT, p38, and p65 proteins, as well as IL-1ß levels and cell apoptosis. Polydatin inhibited TNF-α-induced apoptosis of C2C12 cells by regulating NF-κB and p38 MAPK signaling pathways through AKT. This suggests that polydatin shows promise as a new drug for the treatment of skeletal muscle atrophy.

[Proliferation effect of ligamentum flavum cells induced by transforming growth factor ß 1 and its effect on connective tissue growth factor].

OBJECTIVE: To investigate the effect of transforming growth factor ß 1 (TGF-ß 1) induced proliferation of ligamentum flavum cells and ligamentum flavum hypertrophy and its effect on connective tissue growth factor (CTGF) expression. METHODS: The ligamentum flavum tissue in lumbar intervertebral disc herniation was extracted and the ligamentum flavum cells were isolated and cultured by collagenase pre-digestion method. Morphological observation, immunofluorescence staining observation, and MTT assay were used for cell identification. The 3rd generation ligamentum flavum cells were divided into 5 groups. The cells of groups A, B, C, and D were respectively sealed with 3 ng/mL TGF-ß 1, 50 ng/mL CTGF, 3 ng/mL TGF-ß 1+CTGF neutralizing antibody, and 50 ng/mL CTGF+CTGF neutralizing antibody. Serum free DMEM was added to group E as the control. MTT assay was used to detect the effects of TGF-ß 1 and CTGF on the proliferation of ligamentum flavum cells. Western blot was used to detect the expression of CTGF protein. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of collagen type Ⅰ, collagen type Ⅲ, and CTGF genes. RESULTS: The morphological diversity of cultured ligamentum flavum cells showed typical phenotype of ligamentum flavum fibroblasts; all cells expressed collagen type Ⅰ and vimentin, and some cells expressed collagen type Ⅲ; MTT identification showed that with the prolongation of culture time, the absorbance ( A) value of each generation of cells increased gradually, and the A value of the same generation of cells at each time point was significantly different ( P<0.05), there was no significant difference in A value between the cells of each generation at the same time point ( P>0.05). After cultured for 24 hours, MTT assay showed that the A value of cells in groups A and B was significantly higher than that of group E ( P<0.05). After adding CTGF neutralizing antibody, the A value of cells in groups C and D decreased, but it was still higher than that of group E ( P<0.05). There were also significant differences among groups A, C and groups B, D ( P<0.05). Western blot analysis showed that the relative expression of CTGF protein in groups A and B was significantly higher than that in group E ( P<0.05), while the relative expression of CTGF protein in groups C and D was significantly lower than that in group E ( P<0.05), and the difference between groups A, C and groups B, D was also significant ( P<0.05). qRT-PCR detection showed that the mRNA relative expression of CTGF, collagen type Ⅰ, and collagen type Ⅲ in group A was significantly higher than that in group E ( P<0.05). After adding neutralizing antibody, the mRNA relative expression of genes in group C was inhibited and were significantly lower than that in group A, but still significantly higher than that in group E ( P<0.05). The mRNA relative expressions of collagen type Ⅰ and collagen type Ⅲ in group B was significantly higher than that in group E ( P<0.05), but the mRNA relative expression of CTGF was not significantly different from that in group E ( P>0.05); after neutralizing antibody was added, the mRNA relative expression of collagen type Ⅰ and collagen type Ⅲ in group D was inhibited and was significantly lower than that in group B, but still significantly higher than that in group E ( P<0.05); there was no significant difference in the mRNA relative expression of CTGF between group D and groups B, E ( P>0.05). CONCLUSION: TGF-ß 1 can promote CTGF, collagen typeⅠ, collagen type Ⅲ gene level and protein expression in ligamentum flavum cells, and TGF-ß 1 can synergistically promote proliferation of ligamentum flavum cells through CTGF.

[Mechanism of p38 mitogen activated protein kinase signaling pathway on promoting the hypertrophy of human lumbar ligamentum flavum via transforming growth factor ß 1/connective tissue growth factor].

OBJECTIVE: To investigate the mechanism of p38 mitogen activated protein kinase (MAPK) signaling pathway in regulating the hyperplasia and hypertrophy of human lumbar ligamentum flavum via transforming growth factor ß 1 (TGF-ß 1)/connective tissue growth factor (CTGF). METHODS: The lumbar ligamentum flavum tissue taken from patient with lumbar intervertebral disc herniation was isolated by collagenase-predigested explant cultures. The ligamentum flavum cells were treated with the extracellular regulated protein kinase pathway blocker PD98059, c-Jun N-terminal kinase pathway blocker SP600125, and p38 pathway blocker SB203580, and then the mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescence quantitative PCR (qRT-PCR). The ligamentum flavum cells were divided into 4 groups, and transfected with small interfering RNA (siRNA), p38 siRNA, siRNA+3 ng/mL TGF-ß 1, and p38 siRNA+3 ng/mL TGF-ß 1 in groups A, B, C, and D, respectively. After 24 hours of transfection, immunofluorescence staining was performed to observe the expressions of p38 and phosphorylation p38 (p-p38); the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ in each group were detected by qRT-PCR; the protein expression of CTGF in each group was detected by Western blot. RESULTS: p38 pathway blocker SB203580 could significantly reduce the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ ( P<0.05). After 24 hours of transfection, immunofluorescence staining showed positive staining with p38 and p-p38 expressions in groups A, C, and D and negative staining in group B. Compared with group A, the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ and relative protein expression of CTGF in group B decreased significantly ( P<0.05), while those in groups C and D increased significantly ( P<0.05); and those indicators significantly increased in group C than in group D ( P<0.05). CONCLUSION: TGF-ß 1/CTGF based on the p38 MAPK signaling pathway play an important role in the occurance and development of hypertrophy of human lumbar ligamentum flavum.

The effect of intra-articular injection of different concentrations of ozone on the level of TNF-α, TNF-R1, and TNF-R2 in rats with rheumatoid arthritis.

The objectives of this study were to observe the therapeutic effect of ozone (O3) of different concentrations on rat with rheumatoid arthritis (RA), and to investigate the role of O3 in regulating the level of TNF-α (tumor necrosis factor), TNF-R1 (tumor necrosis factor receptor 1), and TNF-R2. Forty-eight Wistar rats were randomly divided into eight groups. There are five O3 groups which were marked by 10, 20, 30, 40, and 50 µg/mL, respectively, control group, oxygen group, and RA model group. RA was induced in all rats by hypodermic injection of collagen II and complete Freund's adjuvant except that in the control group. At 21 days after modeling, the rats in oxygen group were given an injection of oxygen in the knee joint weekly for 3 weeks, and the rats in O3 groups were injected the concentration of O3 as they marked weekly for 3 weeks. The thickness of hind paw, as well as the serum and synovial levels of TNF-α, TNF-R1, and TNF-R2 was observed. At the end of treatments, the thickness of the hind paws in O3-40 group is much less compared to RA group (P < 0.01). The serum levels of TNF-α, TNF-R1, and TNF-R2 showed no significant difference among all the groups (P > 0.05). However, the synovial levels of TNF-α and TNF-R2 in O3-40 and O3-50 groups are lower than those in RA group (P < 0.01). The synovial level of TNF-R1 in O3-40 group is higher than that in RA group (P < 0.05). In conclusion, intra-articular injection of O3 at 40 µg/mL can effectively suppress the joint swelling caused by RA. This mechanism is probably mediated by down-regulating synovial TNF-α and TNF-R2 and up-regulating TNF-R1 in the joint.

[Effect of intra-articular ozone injection on serum and synovial TNF-α, TNFR I, and TNFR II contents in rats with rheumatoid arthritis].

OBJECTIVE: To observe the effects of intra-articular ozone injection at different concentrations on the contents of tumor necrosis factor-α (TNF-α), TNF receptor I (TNFR I), and TNFR II in the serum and synovium of rats with rheumatoid arthritis (RA) and explore the therapeutic mechanism of ozone in RA treatment. METHODS: Forty-eight Wistar rats were randomized into 8 groups, including 5 ozone groups receiving intra-articular injection of 10, 20, 30, 40 or 50 µg/ml ozone, a blank control group, an oxygen group and a RA model group. All the rats, except for those in the blank control group, were subjected to hypodermic injection of bovine collagen II and complete Freunds adjuvant to induce RA. Ozone treatment was administered once weekly for 3 weeks starting at 21 days after the modeling. The swelling and thickness of the hind paws were observed, and the serum and synovial contents of TNF-α, TNFR I, and TNFR II were detected. RESULTS: At the end of treatment, the paw thickness was reduced significantly in rats with 40 µg/ml ozone injection compared with that in the model RA group (P<0.01). The serum contents of TNF-α, TNFR I and TNFR II showed no significant difference between the RA model group, oxygen group and the ozone groups, but their synovial contents showed significant reductions in rats with 40 and 50 µg/ml ozone injection (P<0.01); the synovial TNFR I was significantly higher in 40 µg/ml ozone group than in the model group (P<0.05). CONCLUSION: Intra-articular injection of 40 µg/ml ozone can attenuate synovitis in rats with RA, the mechanism of which may involve the inhibition of TNF-α and TNFR II activity and enhancement of TNFR I activity in the synovium.

[Establishment of a three-dimensional finite element model and stress analysis of the talus during normal gait].

OBJECTIVE: To develop a three-dimensional (3D) finite element model of the human ankle with fine details and analyze the stress distribution on the talus during different gait phases. METHODS: Mimics13.0 and Geomagic10.0 software were used for geometric reconstruction of the ankle based on the 3D CT data of the foot. The model was meshed and assigned with the material properties in Hypermesh10.0 software. The model was then imported to Abaqus6.9, and the stress condition of the talus during the 3 phases (heel-strike, midstance, push-off) of normal gait was simulated to calculate the stress distribution within the bone. RESULTS: The three-dimensional finite element model of the ankle established consisted of 21 865 nodes and 73 440 elements. The stress distribution within the bone in 3 phases of normal gait differed significantly. The peak von Mises stress on the talus dome, from the heel-strike to push-off phases, was 3.0 MPa, 4.3 MPa and 4.8 MPa, as compared to 1.3 MPa, 1.9 MPa and 2.8 MPa on the talar neck, 2.8 MPa, 3.0 MPa, and 3.4 MPa on the talonavicular joint surface, and 2.2 MPa, 1.8 MPa and 1.5 MPa on the subtalar joint, respectively. CONCLUSION: The finite element model of the talus shows a good response against the experimental data and can be used to simulate the biomechanic experiment of the talus.