| Objective:Through the study of Modified Duhuo Jisheng mixture containingserum on rabbit articular cartilage cells,cartilage degeneration andbone marrow cell model the effect of mesenchymal stem cells inducedthe proliferation and apoptosis of cartilage cells,Wnt signaling pathwayfrom the point of view,clarify the mechanism of Modified Duhuo Jishengmixture protection of rabbit articular cartilage.Methods:Using high performance liquid chromatography-four bar-time offlight mass spectrometry(UPLC-Q-TOF)analysis of main components ofModified Duhuojisheng mixture and serum;cultured chondrocytes andmesenchymal stem cells in vitro cell culture;cell cycle and apoptosisdetection flow cytometry detection conditions,cell activity and drugtoxicity MTT method,Tunel method to detect cell apoptosis,Western-Blot,gene and protein expression of Real time-PCR andimmunohistochemical method to detect the Wnt signaling pathway.Results:(1)Modified Duhuojisheng mixture UPLC-Q-TOF analysisidentified 47 common peaks,through the analysis comparison includinglinolenic acid methy lester,adenosine,phthalate buty lester,hexahydrofarnesyl acetone,twelve 47 compounds such as acid glyceride,mainly living by flavored parasitic mixture of prescription: living alone,mulberry parasitism,eucommia bark,radix gentianae macrophyllae,twotooth achyranthes root,tuckahoe,windproof,rhizoma ligusticiwallichii,dangshen,licorice,Chinese angelica,radix paeoniae alba,radix rehmanniae,cinnamon,rhizoma arisaematis,radix scutellariae,papaya,clematis root,the analysis of the condition of current has notyet been detected asarum pharmaceutical ingredients.(2)Living by flavored parasitic mixture containing medicineserum UPLC-Q-TOF analysis identified 33 common peaks,through theanalysis comparison mainly include phthalate butyl ester,hexahydrofarnesyl acetone,twelve acid glyceride,phthalic acid butyloctyl ester,peach leaf coral glycosides,such as 33 compounds,mainlyliving by flavored parasitic mixture of prescription: living alone,mulberry parasitism,eucommia bark,poria cocos,windproof,rhizomaligustici wallichii,dangshen,twotooth achyranthes root,radixglycyrrhizae,radix paeoniae alba,radix rehmanniae,rhizomaarisaematis,clematis root,radix scutellariae,papaya.The analysis of thecondition of current has not yet been detected asarum,cinnamon,radixgentianae macrophyllae,angelica four flavor ingredient.(3)After the separation of New Zealand rabbits knee articularcartilage tissue,primary culture and identification,third generationcartilage cells and bone marrow mesenchymal stem cell growth,cellviability was the strongest,the best stage of the experiment.The resultsshowed that the toluidine blue staining nuclei stained blue,cytoplasmand interstitial cells visible red staining results of.II collagenimmunofluorescence showed that the cartilage cell cytoplasm wasstained brown nuclei..MTT method was used to detect differentconcentrations of Modified Duhuo Jisheng mixture intervention after 24 hours after cell activity,Modified Duhuojisheng mixture concentration,the OD value of the cells,and the cell activity is maximum;With theModified Duhuojisheng mixture group cell proliferation compared withthe control group,was statistically significant(P < 0.05).(4)Flow cytometry was used to detect the normal cartilage cellsshowed that each intervention after 48,24 h,12h,Wnt signal pathwayblocker group and blank serum group compared to the number of G1 cells increased significantly and decreased in G2 phase,S phasedecreased.Wnt signal pathway blocker group compared with drug group,G1 increased significantly,G2 reduced,S phase decrease,PI lower thanthe drug group(P < 0.05).12 h,24h,48 h,drug group and blank serumgroup G1,no significant G2 phase and S phase difference(P > 0.05),butfrom the trend with the extension of time of different drug groups.Theapoptosis of the cells was slower than that of the blank serum group.(5)The results of Western Blot showed that: low dose group,middle dose group,high dose group,Wnt pathway blocker Wnt5 acompared with the blank serum group,expression of beta-cateninprotein significantly increased,the difference was statisticallysignificant(P < 0.05).The cells were SOX9 protein expression,differentdose of Modified Duhuo parasitic mixture containing serum group andblocker group and were higher than the normal group,the differencewas statistically significant(P <0.05);Modified Duhuojisheng Decoctiongroup,the expression of SOX9 protein concentration dependentlyincreased.Aggrecan and CollThe trend of agese,type and II proteinexpression was consistent with that of SOX9 protein.The results ofRT-PCR and immunohistochemical results of Western and Blot wasconsistent with the test results.(6)The results of Tunel method to detect apoptosis showed thatthe apoptosis of cells in the model group;low,high ModifiedDuhuojisheng mixture containing serum and Wnt blocker group andmodel group were significantly reduced compared to the difference wasstatistically significant(P< 0.05),which contained serum group ofapoptosis decreased with the increase of drug concentration.(7)Flow cytometry results showed that: the model groupcompared with normal group,Wnt inhibitor group in G1 cells and thecell number in S phase was decreased,the difference was statisticallysignificant(P < 0.05).Compared with the normal group,high dose group,the number of G1 cells increased,the number of cells in S phase reduce,the difference was statistically significant(P < 0.05).(8)The results of Western Blot showed that: the interleukin-1stimulation,model group of Modified Duhuo Jisheng mixture containingserum significantly increased on Wnt5 a and beta-catenin proteindegeneration of chondrocytes.The expression of low,high dose groupcompared with model group significantly decreased,there wasstatistical significant differences(P < 0.05).The degree of decrease wasin a concentration dependent manner,high dose group,middle dosegroup > low dose group.Model group than the normal group of SOX9,Aggrecan,type II collagen expression was significantly increased(P<0.05);Modified AngelicaThe intervention group(mixture of parasiticdegenerative cartilage cells)and compared to the model group SOX9,Aggrecan,the expression of type II collagen concentration dependentlyincreased,the difference was statistically significant(P< 0.05);Wntblocker group compared with model group,SOX9,Aggrecan,type IIcollagen expression was significantly increased(P < 0.05).(9)The MSC into chondrocytes showed by flow cytometry: eachintervention groups after 24 h cell cycle,Wnt blocker group and blankserum group compared to the number of G1 cells increased significantlyand decreased in G2 phase,S phase decreased compared to.Wnt blockergroup and drug group,G1 increased significantly,G2 reduced,S phasedecrease,PI lower than the drug group(P < 0.05).(10)RT-PCR test results show: low dose group,middle dose group,high dose group,Wnt pathway blocker Wnt5 a compared with the blankserum group,the expression of beta-catenin mRNA increasedsignificantly,and there was statistically significant difference(P < 0.05),consistent with trends and changes in the expression of Wnt5 a beta-catenin expression in each group.SOX9 mRNA cells,different dose ofModified Duhuo Jisheng mixture containing serum group and Wntblocker group were higher than the normal group,the difference wassignificant(P< 0.05);Modified Duhuojisheng mixture group,SOX9 MTheexpression of RNA concentration dependently increased the expressionof.Aggrecan and Collagese type II mRNA and the change trend of SOX9 protein.Casp3 cells were Casp8,Bcl2,expression of BAX mRNA weredifferent dose of Modified Duhuo Jisheng mixture containing serumgroup compared with the control group,the differences werestatistically significant(P< 0.05)group with the Wnt block,thedifference was statistically significant(P<0.05).Immunohistochemistrywas used to detect MSC chondrogenic cells with the results of RT-PCRassay results.Conclusion:(1)Modified Duhuojisheng mixture in direct material might havebeen made in the serum of 33 drug-induced constituents,combined withserum pharmacology further studies will help to clarify the materialbasis for the efficacy of its preparation.(2)Modified Duhuojisheng mixture containing serum can promotenormal rabbit cartilage cells and bone marrow mesenchymal stem cellsinto cartilage induced cell proliferation,and its mechanism may beaffected by the Wnt signal pathway in Wnt5 a,beta-catenin,SOX9,Aggrecan,Collagese,II,Casp3/8,Bcl2,BAX gene expression andregulation of Wnt signaling pathway then,adjust the G1 phase of the cellcycle,promote normal cartilage cell proliferation.(3)Modified Duhuojisheng mixture containing serum can promotethe apoptosis of cartilage degeneration in rabbits,and its mechanismmay be affected by the Wnt signal pathway in Wnt5 a,beta-catenin,SOX9,Aggrecan,Collagese II,Casp3/8,Bcl2,BAX gene expression andregulation of Wnt signaling pathway,thereby promoting apoptosis ofcartilage degeneration,Modified Duhuojisheng mixture treatment ofKOA patients,and the mechanisms of TCM Blood Stasis to the newtheory of conformity. |
