| 1.BackgroundKnee osteoarthritis is a common orthopedic disease. With the advent of the aging society, the onset of OA rate increased year by year,the incidence rate of OA in 50 years of age or older population is second only to heart disease and ranked second. The knee osteoarthritis is a complex process of articular cartilage damage and repair alternates, also accompanied with inflammation. So, it is a final result caused by the many factors and processes which interact each other, the cartilage destruction and damage is the key link of the pathogenesis of the disease. The pathological process of osteoarthritis is articular cartilage degeneration and its repair capacity decreased. Osteoarthritis is the complex regulation of a variety of factors, age is considered to be the most important risk factors, in the elderly population, not only the rate of osteoarthritis of the knee increases with age, but the degree of joint degeneration and symptoms are increasing, the main reason is the body’s physiological function decreased, organ senescence and the self-repairing capability of articular cartilage decreased with the age increase. So, it is easily development to be knee osteoarthritis if there are other risk factors.Senescence which is considered as the accumulation of damaged and defective cellular components, eventually leading to organ and tissue physiological function decline. Senescence also is the aggregation of biological macromolecules (DNA, proteins and lipids) and damage of organelles. We found the internal environment damaged in the senescence process, and pay more attention to the study the autophagy with senescence.Autophagy is one of the highly conserved cell behavior in organic evolution, and it closely related to cell proliferation and apoptosis. Autophagy play an important role in the damaged organelles clear degradation and intracellular macromolecular material recycling, it also maintain the cell internal environment and promotion of cell survival. When cell is damaged, the autophagy is activated significantly to clean the damaged organelles, inhibit the apoptosis, thus to protect tissues and cells. Apoptosis play a key role in the process of articular cartilage degeneration, the self-repairing capability of articular cartilage is increased when chondrocyte apoptosis is inhibited, slowing the pathological process of osteoarthritis. Autophagy play a critical role in regulation of the apoptosis of cartilage cells, autophagy is the prevention and treatment of knee osteoarthritis potentially one of the most effective ways.2.PurposeEarly use of intra-articular injection of glucocorticoid can relieve pain and good results for the knee osteoarthritis.However, long-term repeated injection of dexamethasone can damage the cartilage cells, promote cell apoptosis and death which leading to further deterioration of chondrocyte and articular cartilage of knee. Therefore, it is important to explore the mechanism of glucocorticoid side effects and to develop coping strategies, to ensure that its efficacy and reduce the side effect. Whether autophagy in glucocorticoid adaptively increased cartilage cells produce a protective effect on cartilage? What is the relationship between autophagy and senescence in glucocorticoid? Still unknown.3.ContentIn vitro:Rat primary knee cartilage cell cultureDexamethasone (DX) concentration:blank group, 0.1mg/L, lmg/L,25mg/L, 50mg/L.3-MA mixed with dexamethasone were divided into 5 groups:control group,10 mmol/L3-MA,10 mmol/L 3-MA+25mg/L DXM,25mg/L DXMAfter stimulation with glucocorticoid, LysoTracker Red staining, MDC staining and Western blot were used to detect the level of autophagy in the chondrocytes.Chondrogenic differentiation of multipotent adult progenitor cell(MAPC)by co-culture with chondrocytesMAPC were cultured and identified by flow cytometry in vitro. The shape and growth condition of MAPC were observed. MAPC was co-cultured with chondrocytes in the experiment group and with culture medium in control group. The rate of MAPC differentiation to chondrocytes was counted.7 days after the co-culture, the aggrecan and collagen type II mRNA expression levels were detected by RT-PCR. All subjects were analyzed by one-way ANOVA.Establish rat knee osteoarthritis models15 SD rats were sacrificed at 2 weeks,4 weeks and 6 weeks after resection of the anterior cruciate ligament. At the different time points in rat knee joint tissue by HE staining, Masson staining and toluidine blue staining, according to Mankin score of grading and assessment.30 SD rats divided into three groups,10 rats in each group, group A only skin incision 6 weeks after the establishment of model operation in the knee joint injection of dexamethasone, group B only anterior cruciate ligament cutting 6 weeks after modeling in knee joint cavity injection of dexamethasone and group C simple anterior cruciate ligament cut 6 weeks after modeling in knee joint cavity injection of dexamethasone. Each group was divided into 2 groups (i.e. A1, A2, B1, B2, C1, C2); Al, B1 and C1 groups only be injected one time of dexamethasone, A2, B2 and C2 groups are injected two times of dexamethasone. Then use the HE staining, Masson staining, Safranin O-fast green staining and immunohistochemistry to detect the expression of SA-beta-gal and LC3 in the cartilage specimens respectively.4.Method:4.1 Effect of dexamethasone on autophagy and senescence of chondrocytes4.1.1 Rat primary knee cartilage cell culture:Thirty-four 3-month-old Sprague-Dawley male rats, ranging in weight from 300 to 340 g, were obtained from the laboratory animal center of Southern Medical University. These rats were killed, and we separated articular cartilage from femoral condyles and tibial plateaus under a microscope. Cartilage slices were incubated with trypsin (0.5 mg/ml) (Sigma-Aldrich, St. USA) for 30 mins at 37℃. After trypsin was removed, the cartilage slices were incubated with 0.1% Ⅱ collagenase (Sigma-Aldrich, St. USA) in Dulbecco s modified Eagles medium (DMEM, Life Technologies, NY, USA) with 5% fetal calf serum (FCS, Life Technologies) for 4 hours at 37℃ with shaking. The isolated chondrocytes were recovered and plated in DMEM supplemented with 10% calf serum and antibiotics. The chondrocytes were incubated at 37℃ in a humidified gas mixture containing 5% CO2 balanced with air.4.1.2 Group:Dexamethasone was added to the chondrocytes at difference concentrations (0 ug/mL,0.1 ug/mL,1 ug/mL,25 ug/mL and 50 ug/mL) 3-MA and DXM (DX) were added to chondrocytes at difference concentrations (0 ug/mL,10 mmol/L 3-MA,10 mmol/L 3-MA+25 μg/mL DX and 25 ug/mL DX).4.1.3 Western blotChondrocytes incubated with various concentrations of dexamethasone were washed with PBS. Total protein was isolated and it was mixed with a standard protein solution from a BCA kit and became the BCA working fluid. Each well was filled with the working fluid (200 ul) for 15 minutes at room temperature. The protein concentration was determined according to the measured OD values. According to the protein concentration of each group (the sample volume was 30 μg, and the total volume was 20 uL), the total amount of protein in each group was calculated. After heating with buffer and PBS at 100℃ for 5 minutes, protein samples were prepared. Equal quantities (30 μg) of protein from each sample were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto polyvinylidene difluoride membranes. After blocking with 5% nonfat milk, the membranes were incubated with the following rabbit anti-rat primary antibodies overnight. Next, the membranes were incubated with HRP-conjugated secondary antibodies. The first antibodies were diluted 1:1000, and the second antibodies were diluted 1:5000. Finally, bands were detected using ECL plus reagent on an enhanced chemiluminescence detection system. In addition, the intensity of the bands was quantified using Alpha Ease FC 4.0 software.4.1.4 LysoTracker Red stainingChondrocytes (3x105 cells/well) were fixed with various concentrations of DXM for 4 days at 37℃ in 24-well plates and rinsed with DMEM three times. To each well of the plate, we added 66 mM Lyso-Tracker Red culture medium (1 ml) and then cultured the cells for 30 minutes at 37℃. After the plates were washed with PBS three times, we observed chondrocytes using an inverted fluorescence microscope.4.1.5 MDC stainingChondrocytes (3×106 cells/well) were fixed at different concentrations of DXM for 4 days at 37℃ in 6-well plates and rinsed with PBS three times. Then, chondrocytes were incubated at 37℃ in 0.05 mM MDC for 30 minutes. After incubation, chondrocytes were washed three times with PBS at 37℃ and fixed for 30 minutes in 4% paraformaldehyde. After fixation, chondrocytes were washed four times with PBS and observed under a fluorescence microscope. To observe the rate of autophagy, stained chondrocytes were tested using flow cytometry.4.1.6 β-galactosidase stainingChondrocytes were treated with 3-MA and DXM (DX) at various concentrations (0 ug/mL,10 mmol/L 3-MA,10 mmol/L 3-MA+25 ng/mL DX and 25 μg/mL DX) for 24 hours,48 hours and 72 hours; then, chondrocytes were fixed using a β-galactosidase kit for 15 minutes at room temperature. Chondrocytes were stained with β-galactosidase dye for 12 hours and then washed three times. The stained cells were observed using a phase-contrast microscope to determine the percentage of positive cells out of the total chondrocytes under 200x amplification.4.1.7GFP-RFP-LC3 determinationBefore glucocorticoid treatment, chondrocytes were transfected with mRFP-GFP-LC3 when the confluence was 50-70%. The MOI was reach to 100 and the adenoviral vectors with mRFP-GFP-LC3 were obtained from HanBio Technology Co. Ltd. The chondrocytes were incubated with the adenovirus in the half no-serum medium for 2 hours at the 37℃. The transfected chondrocytes were incubated with 10% FBS DMEM overnight before the glucocorticoid treatment to eliminate the effect of starvation on autophagic level. Then, after different does glucocorticoid treatments for 4 days, autophagosome and autolysome in chondrocytes were observed under confocal microscopy.4.1.8 StatisticsStatistical analyses were performed by the SPSS 19 (SPSS Inc., USA). Analysis of variance was used to analyze the difference between groups. Tukey’s significance test was used to detect differences between two groups. P<0.05 was considered to indicate a statistically significant difference.4.2 Adult progenitor cells (MAPC) were co cultured with chondrocytes to induce their differentiation into chondrocytes.4.2.1 Primary culture and identification of MAPCExtraction of bone marrow 5 ml into the culture bottle. After 1 days of culture medium was changed to remove non adherent cells was changed every 3 days for 1 time. Cell were washed with PBS twice and trypsin digestion for 1 minute, with a straw wind and percussion to single cell suspension, centrifugal 10 minutes later abandoned to the supernatant by inoculated in the culture bottle. Inverted phase contrast microscope was used to observe the growth of cells. The number of living cells was counted and the average number of cells was counted. Digestion of the second generation of MAPC, adding fluorescent labeled antibodies, washed away excess antibodies with PBS, flow cytometry analysis of positive cell expression.4.2.2Primary culture and identification of chondrocytesCut SD rat limb articular cartilage into about 2 mm3 placed in steriled test tube, PBS rinse after 3 minutes under the centrifuge. Successive with trypsin and collagenase digestion were placed inside. The supernatant was collected after centrifugation for 3 minutes, and the supernatant was inoculated with 1* 105/ml in the bottle containing DMEM medium. Primary cell wall fusion after cultured for 2 days was changed once. Observation of cultured cartilage cell morphology and toluidine blue staining.4.2.3 Co-culture of MAPC and chondrocytesCollection is the second generation of cartilage cells dissociated into single cell suspension inoculation in the lower part of the Transwell 6 hole plate, the experimental group will the second generation of MAPC cells in the same inoculum density in the upper, middle septal pore size of 0.3 m polycarbonate membrane, control group of upper placed medium. Observation of cell morphological changes,2 days for liquid 1 times.4.2.4 ComparisonAfter 4 days co-culture, cells were removed to the upper, cells in the experimental group and the control group were placed in 5% fetal bovine serum culture medium, the medium was changed every 2 days time. On the 7th day, the end of culture, we used anti type II collagen fluorescence labeled antibody cartilage cells.10 field of views were selected from experimental group and the control group, the double blind method were counted two groups of positive cells by the fluorescence microscope.4.2.5 RT-PCR to detect the content of mRNA in the accumulation of protein and type Ⅱ collagenAfter 14 days of co-culture. The absorbance of RNA at 260 nm and 280 nm was determined, and the purity and concentration of RNA were calculated. RT-PCR method for detection of collagen type Ⅱ mRNA and the accumulation of protein polysaccharide content.4.2.6 StatisticsSPSS 19 for statistical analysis, P< 0.05 that the difference was statistically significant.4.3 Establishment of SD rat model of knee osteoarthritis by cut the anterior cruciate ligament4.3.1 OperationAfter anesthesia, we take knee joint medial incision into the joint cavity, pull open patella, try to knee flexion to expose the anterior cruciate ligament, and cut anterior cruciate ligament.4.3.2 Postoperative management200 thousand units of penicillin were injected for 3 day to prevent infection. Rats were killed at 2 weeks,4 weeks and 6 weeks respectively. Articular cartilage histology observation in 10% formalin fixed tissue.4.3.3 EvaluationThe knee joint cartilage by HE staining, Masson staining and toluidine blue staining. Mankin score according to different time points.4.4 In vivo4.4.1 Establishment of rat model of knee osteoarthritisSD rats with anesthetized by medial longitudinal incision, cut off the anterior cruciate ligament, pay attention not to damage the articular cartilage surface and close the wound. Establishment of knee osteoarthritis model in 6 weeks after operation.Dexamethasone solution configuration:a dose is 0.125g (5ml), calculated in accordance with the adult weight of 60kg, human routine dosage for 0.42mg/kg. According to the human and rat dose conversion formula is:Rats dose (mg/kg)=6.25* 0.42mg/kg. Each rat (270g) was injected 0.709mg (0.028ml), which is the normal dose of the human body.4.4.2 Group:30 SD rats divided into three groups,10 rats in each group, group A only skin incision 6 weeks after the establishment of model operation in the knee joint injection of dexamethasone, group B only anterior cruciate ligament cutting 6 weeks after modeling in knee joint cavity injection of dexamethasone and group C simple anterior cruciate ligament cut 6 weeks after modeling in knee joint cavity injection of dexamethasone. Each group was divided into 2 groups (i.e. A1, A2, B1, B2, C1, C2); A1, B1 and C1 groups only be injected one time of dexamethasone, A2, B2 and C2 groups are injected two times of dexamethasone. Then use the HE staining, Masson staining, Safranin O-fast green staining and immunohistochemistry to detect the expression of SA—beta-gal and LC3 in the cartilage specimens respectively.5 Results5.1 Effect of dexamethasone on autophagy and senescence of chondrocytesCompared to the control group, only a small number of chondrocytes displayed Lyso-Tracker-positive staining. We found that the intensity of Lyso-Tracker positive cells increased drastically with increasing concentrations of dexamethasone. Most chondrocytes incubated with 50 ug/ml dexamethasone for 4 days were positively stained with Lyso-Tracker. Therefore, DXM can promote autophagous vesicles in chondrocytes.In the control group, only a few cells were stained positive for MDC and the intensity of MDC staining in the cells increased drastically with increasing concentrations of dexamethasone. The MDC-stained chondrocytes were observed by flow cytometry, and it was demonstrated that cells that were incubated with different concentrations of dexamethasone had a higher incidence of autophagy than the control group after 4 days (P<0.05). In the 50 ug/ml group, autophagy occurred with an average incidence of 56.74%.In vitro, autophagic flux can be determined by the transfection of adenovirus harboring mRFP-GFP-LC3. After transfection, the autophagosome in cells is shown as yellow dots (the combination of red and green fluorescence), and the autolysosome is shown as red dots (the extinction of GFP in the acid enviroment of lysosomes). After culturing for 4 days, ADSCs enhanced the yellow autophagosomes in the cytoplasm of dexamethasone-treated chondrocytes with does of dexamethasomes increased, so as the red autolysosomes were distributed across the DXM-treated chondrocytes cocultured with ADSCs, indicating the conversion from autophagosomes to auolysosomes and the high level of autophagic flux. Also,the number of yellow autophagosomes was significantly raised by the ADSCs in the dexamethasone-treated chondrocytes with does of dexamethasomes increased, suggesting the later formation of autophagosome and later autophagic flux in DXM-treated chondrocytes.When chondrocytes were incubated with different concentrations of dexamethasone for 2 days, different expression levels of LC3-Ⅱ/beta-actin in different groups was not apparent; however, after 4 days, the expression levels of LC3-Ⅱ/beta-actin increased significantly with increasing concentrations of dexamethasone, and after six days, the expression levels of LC3-Ⅱ/beta-actin decreased at the highest concentration of dexamethasone. Changes in the expression levels of P62 and Beclin-1 were also similar, but interestingly, the expression level of P62 decreased with increasing concentrations of dexamethasone after two days.In the 1 ug/ml and 25 ug/ml treatment groups, the expression levels of P-p70S6K and 4EBP1 decreased in comparison to the control group and the difference was found to be statistically significant, which shows that dexamethasone inhibit autophagy through an mTOR-dependent pathway.The proportion of β-Gal positive chondrocytes significantly increased with time upon treatment with 25 ug/ml glucocorticoid and was highest after 72 hours. However, the proportion of P-Gal positive chondrocytes increased significantly when 3-MA, which can inhibit autophagy, was added in comparison to the dexamethasone group alone after 72 hours. Furthermore, 3-MA alone cannot increase the proportion of β-Gal positive chondrocytes.5.2 Adult progenitor cells (MAPC) were co-cultured with chondrocytes to induce their differentiation into chondrocytes.From the growth curve can be seen, MAPC in 1 days after inoculation began to proliferate, the number of cells reached 1.32×106/ml in sixth day.Chondrocytes were stained with toluidine blue. The chondrocytes’ extracellular matrices were stained blue and the nuclei were stained dark blue, which is consistent with typical chondrocyte characteristics.Flow cytometry results showed that the percentages of SSEA-1 and CD 13 positive cells were 21.47% and 14.31%, respectively, which indicated that MAPC accounted for most of the cells.The results of RT-PCR showed that the experimental group and the control group gathered mRNA content respectively is 1.46+0.51 and 6.61+0.34, Ⅱ collagen mRNA content are 2.14+0.48 and 8.37+0.79, the difference is significant (P< 0.05).5.3 Rat knee osteoarthritis modelAfter 2 weeks, HE staining showed cartilage surface appear minority scattered in the irregular fracture; toluidine blue colored dark blue, evenly stained; Masson staining of cartilage matrix was stained blue, were evenly stained cells arranged regularly.After 4 weeks, HE staining showed mild proliferation of cartilage cells, cells arranged in disorder began to appear, cartilage shallow cracks, surface layer and middle layer cell shrinkage; toluidine blue stain less uniform; Masson staining of cartilage matrix was stained red and blue and white colors.After 6 weeks, HE staining showed that the cartilage layer to thin, surface fibrosis, a little cartilage cells necrosis, arrangement disorder; toluidine blue staining showed that most of the layers of significantly lighter. Masson staining showed more red and less blue.Mankins score of 2 weeks after resection of anterior cruciate ligament was 1.6 points,4 weeks after operation, Mankins score was 4.8 points, and 6 weeks after operation, the Mankins score was 7.2 points.5.4 The effect of dexamethasone on the animal model of knee osteoarthritisLC3 immunohistochemistry:Al and B1 were significantly different, A2 and B2 were significantly different, B2 and C2 were significantly different.Bl and C1 has no significant difference, B2 and C2 has no significant difference, and dexamethasone did not induce aging; however autophagy is induced by dexamethasone. Cartilage autophagy induced by dexamethasone also inhibited the aging, so it can explanation many patients felt no pain with local injection of dexamethasone.6. Conclusion6.1 Autophagy induced by dexamethasone protects chondrocytes from senescence. The mTOR pathway may be involved in dexamethasone-induced autophagy activation.6.2 MAPCs can be induced into chondrocytes by co-culture with chondrocytes.6.3 Resection of anterior cruciate ligament can cause knee osteoarthritis model in rats, Mid-osteoarthritis pathological changes can be observed in 6 weeks after operation.6.4 For the patients of mid-knee osteoarthritis, injection of dexamethasone does not aggravate cartilage aging, but autophagy may be obviously induced. |
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