Mechanism Of Hypertrophic Differentiation Of Rat Chondrocytes Regulated By The Guilu Erxian Gum Drug-containing Serum Through IHH-Gli Signaling Pathway

ObjectiveBased on the IHH-Gli signaling pathway,to explore the mechanism of action of Guilu Erxian Gum drug-containing serum on the hypertrophic changes of chondrocytes induced by interleukin-1βin vitro,and to verify whether the inhibition of this pathway can improve the hypertrophic changes of chondrocytes and protect articular cartilage.Further elucidates the action and partial mechanism of Guilu Erxian gum on OA from the cellular level,so as to provide evidence support for clinical promotion and application of Guilu Erxian gum and subsequent scientific research.Methods1.Preparation of blank serum and medicated serum of Guilu Erxian gumTwenty SPF SD rats aged 2 months were divided into blank group and Guilu Erxian Gum group with 10 rats each by random number table method.The blank group was given 10m L/(kg·d),0.9%normal saline by gavage.Guilu Erxian Gum group was intragastric with 1g/m L Guilu Erxian Gum liquid at 2.7m L/(kg·d).The two groups were given gavage continuously for 1 week,twice on the 7th day,with an interval of 2 h between the two gavages,and fasting for 12 h before blood collection.2 h after the last intragastric administration,abdominal aorta blood collection was performed after abdominal anesthesia with 10%pentobarbital sodium injection.The blood stood at room temperature for 2 h,was centrifuged at 3000 r/min for 15 min,and the upper serum was absorbed into a water bath in a centrifuge tube of 15 m L for inactivation.The water bath condition was 56℃for 30 min.After inactivation,0.22μm filter membrane was used to filter and remove bacteria.Blank rat serum was obtained from the blank group,and medicated rat serum was obtained from the Guilu Erxian Gum group,which were stored in the refrigerator at-80℃for future use.2.Chondrocyte extraction,identification,and determination of the optimal intervention concentration and optimal intervention time of drug-containing serumTwenty healthy 4-week-old clean SD rats were collected from knee cartilage and cultured chondrocytes in vitro.Chondrocytes of the first generation were inoculated in96-well plates with 1.0×10~4/well,cultured in DMEM medium containing 10%FBS for 24h,and then cultured in DMEM medium without FBS for 24h to synchronize chondrocytes.The activity of chondrocytes was detected by cc K-8 method after culture for 24h,48h and 72h with the addition of drug-containing serum and blank serum containing 5%,10%,15%and20%,respectively.3.Observation and detection of hypertrophic chondrocytes after intervention with medicated serumHypertrophic chondrocyte models were prepared by 10 ng/m L interleukin-1βintervention in the third generation of chondrocytes,and morphological changes of the cells were observed by inverted phase contrast microscope.The experimental design was divided into 4 groups,including blank group,model group,Guilu Erxian Gum drug-containing serum group(lower Guilu group)and pathway inhibitor group(lower inhibitor group).After 72h of intervention,morphological changes of chondrocytes were observed under light microscope,and cell activity in each group was detected by CCK-8 method.The m RNA expressions of IHH,Smo,Gli,Runx2,COL10A1 and MMP-13 in chondrocytes were detected by q RT-PCR.Western Blot was used to detect the expression of IHH,Smo,Gli,Runx2,COL10A1 and MMP-13 in chondrocytes after intervention with drug-containing serum.Immunofluorescence staining was used to detect Gli entry into chondrocytes after the intervention of drug-containing serum.ResultsCompared with the blank group,the cell morphology of the model group increased,the cell space became larger and less,and the cell debris and suspended dead cells increased;compared with the model group,the cell morphology of the guilu group and inhibitor group decreased,the cell space became smaller and increased,and the cell debris and suspended dead cells decreased.Compared with the blank group,the activity of the other cells was decreased to a certain extent;compared with the model group,the activity of the guilu group and the inhibitor group increased significantly.Compared with the blank group,the relative expression of IHH,Smo,Gli,Runx 2,COL10A1,MMP-13 m RNA and protein increased to some extent in the remaining group;Compared with the model group,the relative expression of IHH,Smo,Gli,Runx 2,COL10A1,MMP-13 m RNA and protein were significantly down-regulated in the guilu group and the inhibitor group.ConclusionsGuilu Erxian Gum drug-containing serum improves Interleukin-1βInduced hypertrophic status of rat chondrocytes in vitro,and the mechanism is closely related to the IHH-Gli signaling pathway.