| Objective To understand the effects of different intensities of LIPUS on condylar chondrocytes in an inflammatory state.Preliminary investigation of the effect of LIPUS on the expression of PI3K/Akt pathway-related factors p53,Bax,Akt and p Akt in condylar chondrocytes.Methods(1)The chondrocytes of the condylar process of the temporomandibular joint of 3-4week old male SD rats were extracted and cultured by mechanical separation combined with secondary enzymatic digestion,and identified by cytomorphological methods,toluidine blue staining and type II collagen immunocytochemistry.(2)To establish an in vitro SD rat condylar chondrocyte inflammatory state model:SD rat condylar chondrocytes of the temporomandibular joint were treated with 10ng/ml IL-1βfor 24 hours to induce an inflammatory state.The inflammatory state(OA)condylar chondrocytes were randomly divided into 3 groups and the chondrocytes were stimulated using LIPUS at intensities of 0m W/cm~2,30 m W/cm~2and 45 m W/cm~2,respectively.LIPUS was performed at an ultrasound frequency of 1.5 MHz with a pulse frequency of 1000 Hz and an on/off ratio of 20%for 20min/time,once/day,for 6 days of continuous intervention.The expression of Col-2,MMP-13in each group was detected using immunocytochemistry.(3)The 2nd generation condylar chondrocytes were randomly divided into the following 3 groups:(i)normal control group(NC group),(ii)inflammation model group(OA group),and(iii)inflammation model+LIPUS intervention group(OA+LIPUS group).LIPUS was stimulated once daily at a fixed time for 20 min/time for 6 days using the intensity with the best effect in part II.Immunocytochemical staining was performed on each group after 6 days to detect Expression of p53,Bax,Akt,p Akt.Results(1)Rat condylar chondrocytes were polygonal in shape.The morphology of condylar chondrocytes in the 4th generation and later changed from polygonal to long spindle-shaped and their growth rate became slow.Toluidine blue staining showed blue-purple nuclei and blue heterochromatic granules in the cytoplasm.Immunocytochemical staining for type II collagen showed positive results,i.e.brownish-yellow granules were visible in the cytoplasm and the nucleus was largely uncoloured.Both methods confirmed the successful isolation and culture of rat condylar chondrocytes.(2)Comparison of the mean integrated optical density of MMP-13 and Col-2 in different intensity groups:the mean integrated optical density of MMP-13 in the 30 m W/cm~2and 45 m W/cm~2groups was lower than that in the 0 m W/cm~2group(0.014365±0.003474,P<0.05),and the lowest in the 45m W/cm~2group(0.009425±0.001453,P<0.05).The mean integrated optical density of Col-2in both the 30m W/cm~2and 45m W/cm~2groups was higher than that in the 0m W/cm~2group(0.011390±0.003109,P<0.05),and the highest in the 45m W/cm~2group(0.024278±0.005715,P<0.05).(3)Expression of each factor in the NC,OA and OA+LIPUS groups:the mean integrated optical density of p53 in the NC and OA+LIPUS groups was 0.020324±0.003829 and 0.02506±0.002971,respectively,which were lower than that in the OA group(0.030953±0.006027,P<0.05),and the NC group was lower than that in the OA+The mean integrated optical density of Bax was 0.014761±0.001941 and 0.017425±0.00278 in the NC and OA+LIPUS groups,respectively,both lower than that of 0.020169±0.003893 in the OA group(P<0.05),and lower in the NC group than in the OA+LIPUS group(P<0.05).The mean integrated optical density of p Akt was 0.018716±0.008238 and 0.007406±0.00299 in the NC and OA+LIPUS groups,respectively,which were both higher than that of 0.002064±0.00075 in the OA group(P<0.05),and higher in the NC group(P<0.05).The difference in the mean integral optical density of Akt between the groups was not statistically significant(P>0.05).Conclusion(1)Rat temporomandibular joint condylar chondrocytes could be successfully isolated and cultured using mechanical separation combined with secondary enzyme digestion.(2)LIPUS enhanced the expression of Col-2 and inhibited the expression of MMP-13 in inflammatory state condylar chondrocytes,indicating that LIPUS had a protective effect on inflammatory state condylar chondrocytes,and the protective effect of 45 m W/cm~2intensity was better than that of 0 m W/cm~2and 30 m W/cm~2intensity.(3)LIPUS enhanced the expression of p Akt,a factor downstream of the PI3K/Akt pathway,and decreased the expression of p53 and Bax,pro-apoptotic factors,in inflammatory state condylar chondrocytes,suggesting that LIPUS may inhibit apoptosis in condylar chondrocytes by activating the PI3K/Akt pathway. |
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