Experimental Study On The Effect Of Yanghe Decoction On Chondrocyte Apoptosis In SD Rats In Vitro Based On Mitochondrial Apoptosis Pathway

ObjectiveThe study was conducted to observe the effects of serum containing Yanghe Decoction on the expression of chondrocyte apoptosis factor Bax,Caspase-9,Caspase-3 and anti-apoptotic factor Bcl-2 in apoptosis model induced by sodium nitroprusside,and to explore the effect of Yanghe Decoction-containing serumon chondrocyte apoptosis.Methods1.Forty healthy SPF-grade SD rats of 2 months old were divided unlimited between male and female.And the daily dosage of rats was calculated according to the "equivalent dose rate table of human and animal body surface area converted".The rats were given Yanghe decoction gavage 2 times a day(once every morning and evening)for 7 consecutive days.Two consecutive gavage on the last day.After an interval of 2 hours,the blood was taken from the abdominal aorta after anesthesia with 10%chloral hydrate,and drug-containing serum was prepared after high-speed centrifugation at low temperature.2.Mechanical-enzyme digestion was used to digest cartilage tissue from 4-week-old SD rats'knee joints and femoral head cartilage.And the chondrocytes were separated from the cartilage.An in vitro culture system was established with toluidine blue staining and type ?collagenase identifying.The growth of chondrocytes was observed under the inverted phase contrast microscope.3.The 1mm SNP was used to induce second generation chondrocytes to produce a degenerative chondrocyte model.The morphological changes of the cells were observed under the inverted phase contrast microscope for identification.4.The study used MTT to detect the optimal intervention time and intervention concentration for the inhibition of apoptosis of degenerative chondrocytes in serum of Yanghe decoction,flow cytometry to detect the rate of apoptosis,the Q-PCR method to detect the genes expression of Bax,Bcl-2,Caspase-9 and Caspase-3,Western Blot to detect the protein expression of Bax,Bcl-2,Caspase-9 and Caspase-3.Results1.The second-generation chondrocytes have the same morphology,abundant cytoplasm,clear nuclei,and good growth state.After staining with toluidine blue,the nuclei were stained dark blue,and both the interstitial and cytoplasm were stained light blue.After staining with type ? collagen,the cytoplasmic area of the positive control group was brownish yellow.Cartilage cells induced by 1 mm SNP showed irregular morphology,large nucleus,unclear cytoplasmic cytoplasm,and some cells suspended in culture medium.2.MTT results showed that Yanghe Decoction-containing serum had a relationship to the inhibition of apoptosis of degenerative chondrocytes.The optimal intervention concentration and intervention time were 10%drug-containing serum and 48 hours,respectively.Flow cytometry analysis showed that Yanghe Decoction containing serum could significantly reduce the apoptosis rate of degenerative chondrocytes.The results of Q-PCR and Western-Blot showed that Yanghe Decoction-containing serum group could significantly reduce the expression of Bax,Caspase-9 and Caspase-3 genes and proteins,and increase the expression of anti apoptosis factor Bcl-2 gene and protein.ConclusionsBying reduce the expression of chondrocyte apoptosis factor Bax,Caspase-9,Caspase-3 and increase the expression of anti apoptosis factor Bcl-2 in apoptosis model induced by SNP,Yanghe Decoction serum can effectively regulate the mitochondrial apoptosis pathway,thus effectively inhibit chondrocyte apoptosis,reduce cartilage matrix degradation,and help repair cartilage.Therefore,our study can provide theoretical basis for Yang he decoction to prevent and delay OA.