| Background: Osteoarthritis caused by articular cartilage defects is one of the main reasons for the disability of the middle-aged and the elderly.The method of promoting cartilage repair is an urgent issue requires immediate resolution.It is shown in previous studies that cartilage repair is the result of the synergy of multiple signaling pathways which Notch and p38 MAPK are involved as important signaling pathways.Previous studies have found that simply inhibiting the Notch signaling pathway or the p38 MAPK signaling pathway can promote chondrocyte differentiation,but there is no report of the combined regulation of the two signaling pathways.Objective: To explore the effect and mechanism of the combined regulation of Notch and p38 MAPK signaling pathways in articular cartilage defects’ repairs.Method: To select a total number of 24 healthy adult male rabbits with weights ranging from 1.5kg to 2kg and to divide those rabbits on a random basis into the blank group,the cartilage defect group,and the combined inhibition group,with 8 rabbits in each group.The control group was the healthy group,without constructing cartilage defect model,and feeding normally.Rabbits in the cartilage defect group were constructed into rabbit knee-joint articular cartilage defect models by drilling a small hole with a diameter of about 2.5 mm in the center of the medial condyle of the femur,then no intervention was applied to allow it to heal naturally.In addition,rabbits in the combined inhibition group were given intervention treatments with Notch signaling pathway inhibitor(DAPT)combined with p38 MAPK signaling pathway inhibitor(SB203580).The Notch signaling pathway inhibitor was γ-secretase inhibitor DAPT.The dosage of DAPT was 10 mg/kg,which was given through intraperitoneal injections per day for 7consecutive days.The p38 MAPK signaling pathway inhibitor was SB203580 and the dosage of SB203580 was 50 mg/kg,which was given through intraperitoneal injections per day for 3 consecutive weeks.At the 3rd and the 8th week of the experiment,rabbits were put to deaths through air embolization.The cartilage tissue samples from the defect repair area were taken to observe and to test the chondrocyte activity.(1)Experiment in vivo:(1)Changes in expression of p38 and Notch signaling pathway: RNA kit was used to extract RNA for the detection of p38 MAPK and Notch signaling pathway m RNA expression.2)Pathological features:(1)General observation: The body weight of each rabbit was measured once a week,and the mental conditions and activities of all rabbits were observed and recorded.The texture of the cartilage was observed to judge whether there is degeneration and still a gap in the cartilage defect site.(2)Tissue samples test and observation: The articular cartilage tissues of rabbits in each experiment group were histologically stained with Hematoxylin-Eosin(HE)and Masson.The samples were placed under an optical microscope and an electron microscope to observe changes in chondrocytes.(3)The degree of cartilage repair within each group was evaluated using the Mankin evaluation system.3)The cartilage was sliced under light microscope to calculate empty bone lacuna’s ratios and to compare between the ratios afterwards.(2)Experiments in vitro: Observation on the proliferation of chondrocytes.The CCK-8method was utilized to detect the effects on the chondrocyte proliferation after articular cartilage defects in rabbits.Results:(1)Experiment in vivo1)Real-time PCR Test Result: The m RNA expression of p38 and Notch in the 3rd week after model construction was significantly higher than in normal cartilage tissues(P<0.05),and the m RNA expression of p38 and Notch was lower than it(P<0.05)after combined inhibition interventions.In the 8th week after model construction,the p38 and Notch m RNA expression in the combined inhibition group was closer to the normal level compared with the cartilage defect group2)Pathological features:(1)General observation: The cartilage repair in the combined inhibition group was better than that in the cartilage defect group in week 3,the defect area was filled more fully after growing,the repaired cartilage showed a smoother surface,and the color was closer to normal cartilage.After the 8th week of modeling,in the defect area,the cartilage in the combined inhibition group was growing fuller than the cartilage defect group,and the boundary between the repaired cartilage and the normal cartilage was blurred.However,the repaired cartilage was more transparent and harder in texture.(2)Observation results on articular cartilage tissues: After combined inhibition interventions,in contrast to the rabbits in the cartilage defect group,the general conditions and the chondrocyte recovering situations of rabbits in the combined inhibition group were obviously improved.In the combined inhibition group,the arrangement of bone trabecula was more regular than that of the cartilage defect group,a large number of hematopoietic cells exist in the bone medullary cavity,there was less obvious proliferation of adipocytes,and there are less empty bone lacunae existing compared with those of the cartilage defect group.(3)Mankin evaluation system: After construction,the Markin score is signifcantly higher than that of the control group(P<0.05).In the 3rd and 8th week after model construction,compared with that of the model group,the Mankin score of the combined inhibition group was lower(P<0.05).3)Results of empty bone lacuna: In the 3rdweek after model construction,there is a significant increase in rates of empty bone space,and the difference was statistically significant(P <0.05)comparing the model group with the combined inhibition group.In the 8th weeks after model construction,the articular cartilage empty bone lacuna rate of rabbits in the combined inhibition group was(13.01 ± 1.80)%,which was obviously lower than the synchronous(19.54 ± 1.48)% articular cartilage empty bone lacuna rate of rabbits in the cartilage defect group.This difference is statistically significant(P <0.05).(2)Experiments in vitro:Chondrocyte proliferation conditions: Compared with rabbits in the blank group,the absorbance values of chondrocytes in the cartilage defect group and the combined inhibition group were obviously decreased,the OD values were respectively 0.10±0.020 and 0.34±0.015,and the differences were statistically significant(P<0.05).Compared with rabbits in the cartilage defect group,there is a statistically significant difference(P<0.05)between the OD value of the combined inhibition group and that of the cartilage defect group with the combined inhibition group showing a much higher OD value.Conclusion: Combined inhibition interventions of Notch and p38 MAPK signaling pathway can improve the repairing effectiveness of rabbit cartilage defects by improving the active levels of chondrocytes.The combined intervention of Notch and p38 MAPK is expected to provide a novel strategy for the clinical treatment of cartilage defects in the future. |
PDF Full Text Request