Corresponding Changes of Autophagy-Related Genes and Proteins in Different Stages of Knee Osteoarthritis: An Animal Model Study.

OBJECTIVE: To investigate the effect of autophagy expression levels of different weight-bearing states and different stages of osteoarthritis in animal models, as well as the corresponding mechanisms. METHODS: We used the male Sprague-Dawley (SD) rats (12-week-old, SPF) to establish the OA animal models by modified Hulth method, and grouped animal models according to the length of time after surgery and different weight-bearing areas. RT-qPCR was carried out for detection of autophagy-related genes such as Atg7, Atg12, P62, etc. Western blot analysis was used to detect the expression levels of corresponding autophagy-related proteins such as LC3B, P62, etc. T test was performed for statistical analysis to compare different groups, while the differences were deemed statistically significant with P < 0.05. Transmission electron microscopy was used to observe the autophagosome to demonstrate the level of autophagy expression and the status of the chondrocytes. RESULTS: The results of the RT-qPCR testing showed that when the weight-bearing cartilage of the 4-week group (relatively mild) was compared with that of the 10-week group (relatively severe), there were statistically significant differences in all the genes tested, in detail: Atg3 (P < 0.01), Atg7 (P < 0.01), Atg12 (P < 0.01), P62 (P < 0.0001). The expression of autophagy-related mRNA in the 4-week group is increased compared with that of the 10-week group. As for the expression of proteins, Western blotting showed that in the comparison between the 4- and the 10-week groups, statistically significant results include Atg12 (P < 0.01) in the non-weight-bearing area, with decreased autophagy in the 10-week group compared with that of the 4-week group, while expression of LC3B (P < 0.05) protein was significantly higher in the 4-week group than in the control in the non-weight-bearing area. The expression of LC3B (P < 0.0001) and P62 (P < 0.05) in the 10-week group were higher than that of the control. Transmission electron microscope showed that autophagy in the weight-bearing area is stronger than that in the non-weight-bearing area, and autophagy in the 4-week group is stronger than in the 10-week group for the weight-bearing area. CONCLUSIONS: The expression of autophagy varies during different stages of osteoarthritis, in which the autophagy is stronger in the early stage of osteoarthritis, and gradually decreases with the progression of the disease. Autophagy in different weight-bearing areas may also be different.

Torsional Fatigue Life Prediction of 30CrMnSiNi2A Based on Meso-Inhomogeneous Deformation.

In this paper, torsional fatigue failure of 30CrMnSiNi2A steel which exhibited non-Masing behavior was studied under different constant shear strain amplitudes, using thin-walled tubular specimens. The relationship between shear fatigue and the evolution of meso-deformation inhomogeneity and the prediction method of the torsional fatigue life curve were investigated. Shear fatigue of the material under constant amplitude was researched by numerical simulation with reference to tests, by using crystal plasticity of polycrystalline representative volume element (RVE) as the material model. Considering the non-Masing behavior of material, when determining the parameter values of the crystal plasticity model the correlation between these parameters and strain amplitude was taken into account. The meso-deformation inhomogeneity with increments in the number of cycles was characterized by using the statistical shear strain standard deviation of RVE as the basic parameter. Considering the effect of strain amplitude on fatigue damage, ratio cycle peak stress/yield stress was taken as the weight to measure the torsional fatigue damage and an improved fatigue indicator parameter (FIP) to measure the inhomogeneous deformation of the material was proposed. The torsional fatigue life curve of 30CrMnSiNi2A steel was predicted by the critical value of the FIP and then the result was confirmed.

Effects of Tumor Necrosis Factor Alpha on the Expression of Programmed Cell Death Factor 5 in Arthritis.

OBJECTIVE: To investigate the effect of tumor necrosis factor alpha (TNF-α) on the proliferation of fibroblast-like synoviocytes (FLS) and the expression of programmed cell death factor 5 (PDCD5) in an inflammatory microenvironment, for the further understanding of the mechanism of action of TNF-α in promoting the proliferation of synovial cells and the apoptosis of the chondrocytes. METHODS: Articular carriage specimens were obtained from 21 cases with osteoarthritis and 12 cases with femoral neck fractures as healthy controls during arthroplasties. The expression of PDCD5 was evaluated by immunofluorescence analyzed by mean option density (MOD) detected using the software ImagePro Plus. Real-time PCR was performed to evaluate the transcriptions of PDCD5 and TNF-α in synovium. FLS cells derived from rheumatoid arthritis patients were cultured in vitro and incubated with different concentrations of TNF-α. The effects of TNF-α at different concentrations on the proliferation of FLS cells were detected by Cell Counting Kit-8 (CCK-8) assay to evaluate the cell proliferation rate. After incubation with the absence or presence of recombinant human TNF-α at different concentrations, the FLS cells were isolated for detection of PDCD5 protein and PDCD5 gene. The expression of PDCD5 protein was detected by western-blot and the transcription of PDCD5 gene from the cells was detected by real-time quantitative PCR. RESULTS: The MOD of PDCD5 as well as TNF-α of osteoarthritis cartilage sections were significantly increased compared with those of the controls, and in synovium there was a positive correlation between transcriptions of their mRNA. When the concentration of TNF-α was 1 ng/mL, the cell proliferation rate was not significantly different from that of the control group (P = 0.592), while the proliferation of FLS cells was significantly promoted when the concentration of TNF-α was 5, 10, 15, or 20 ng/mL, and the proliferation-promoting rates were 35.64% ± 6.96%, 48.72% ± 7.69%, 45.60% ± 8.85%, and 39.32% ± 6.18%, respectively (P < 0.01). The transcription of PDCD5 gene was significantly downregulated, which was 80.44% ± 4.07% and 84.30% ± 5.48%, respectively (P < 0.05), in the FLS cells incubated with TNF-α at the concentration of 10 and 15 ng/mL for 24 h. When the concentration of TNF-α was 1, 5, or 20 ng/mL, the transcription of PDCD5 mRNA in FLS cells was not significantly different from that in the control group (P > 0.05). The expression of PDCD5 protein was only significantly downregulated when the concentration of TNF-α was 10 ng/mL (P < 0.01), while the expression of PDCD5 protein in FLS cells was not significantly different from that in the control group (P > 0.05). CONCLUSION: The expression of PDCD5 as well as TNF-α in osteoarthritis cartilage and synovium was significantly higher than in healthy tissues, and TNF-α can promote the proliferation of FLS cells in patients with rheumatoid arthritis, and inhibit the expression of PDCD5. PDCD5 may be involved in the abnormal proliferation of synoviocytes and the degeneration of chondrocytes stimulated by TNF-α.

Remaining Life Assessment for Steel After Low-Cycle Fatigue by Surface Crack Image.

After pre-fatigue cycles at different strain amplitudes with different N/Nf values (33.3%, 50%, and 75%), specimens of HRB335 steel were subjected to uniaxial tension until failure. By this method the mechanical properties of the specimens after pre-fatigue testing were measured, and the fracture morphology and microscopic morphology in the vicinity of the specimen's neck surface near the fracture were observed. The verification of the characteristics to be used to estimate the damage caused during the loading cycles was conducted. By observing optical microscope images of the surface area near the neck of the specimens, it was found that the images of surface cracks were significantly different and strongly depended on the number of pre-fatigue cycles the specimen had undergone. In response to this phenomenon, both the microscopic images taken directly from the photos of the surface crack distribution and the binary images based on them were statistically analyzed, and then a parameter, S, denoted as the "unit crack area", characterizing the cumulative fatigue damage was suggested. Furthermore, the test procedure and the calculation formula for determining the image parameters were summarized, and a method for evaluating the remaining life of steel after low-cycles of reversed tension and compression was proposed.

Application of Differential Entropy in Characterizing the Deformation Inhomogeneity and Life Prediction of Low-Cycle Fatigue of Metals.

The relation between deformation inhomogeneity and low-cycle-fatigue failure of T2 pure copper and the nickel-based superalloy GH4169 under symmetric tension-compression cyclic strain loading is investigated by using a polycrystal representative volume element (RVE) as the material model. The anisotropic behavior of grains and the strain fields are calculated by crystal plasticity, taking the Bauschinger effect into account to track the process of strain cycles of metals, and the Shannon's differential entropies of both distributions of the strain in the loading direction and the first principal strain are employed at the tension peak of the cycles as measuring parameters of strain inhomogeneity. Both parameters are found to increase in value with increments in the number of cycles and they have critical values for predicting the material's fatigue failure. Compared to the fatigue test data, it is verified that both parameters measured by Shannon's differential entropies can be used as fatigue indicating parameters (FIPs) to predict the low cycle fatigue life of metal.

Experimental Investigations on Subsequent Yield Surface of Pure Copper by Single-Sample and Multi-Sample Methods under Various Pre-Deformation.

Using copper thin-walled tubular specimens, the subsequent yield surfaces under pre-tension, pre-torsion and pre-combined tension-torsion are measured, where the single-sample and multi-sample methods are applied respectively to determine the yield stresses at specified offset strain. The rule and characteristics of the evolution of the subsequent yield surface are investigated. Under the conditions of different pre-strains, the influence of test point number, test sequence and specified offset strain on the measurement of subsequent yield surface and the concave phenomenon for measured yield surface are studied. Moreover, the feasibility and validity of the two methods are compared. The main conclusions are drawn as follows: (1) For the single or multi-sample method, the measured subsequent yield surfaces are remarkably different from cylindrical yield surfaces proposed by the classical plasticity theory; (2) there are apparent differences between the test results from the two kinds of methods: the multi-sample method is not influenced by the number of test points, test order and the cumulative effect of residual plastic strain resulting from the other test point, while those are very influential in the single-sample method; and (3) the measured subsequent yield surface may appear concave, which can be transformed to convex for single-sample method by changing the test sequence. However, for the multiple-sample method, the concave phenomenon will disappear when a larger offset strain is specified.