| Objective1 Pilose Antler belongs to the liver,kidney meridian,and can be used for the treatment of early osteoarthritis(OA),but the mechanism is still not clear,and there is no experimental research report on its meridian tropism.So this study intends to observe the effects of Pilose Antler on the levels of cAMP/cGMP and TGF-? in viscus of rats from the perspective of signal transduction system and receptor theory,and to explore the correlation between this effects and its meridian tropism.To verify the therapeutic effect of Pilose Antler on knee osteoarthritis of rats and to study the effect of Pilose Antler on the expression of TGF-?/Smad signaling pathway downstream downstream molecules Smad2/3/6/7 gene and protein in chondrocytes through intervening the OA rat by Pilose Antler.To identify the differentially expressed proteins in the articular cartilage of the OA rats after the intervention of pilose antler based on the proteomics technology,in order to explore the possible targets and mechanisms of Pilose Antler on regulating the articular cartilage formation of rats.2 The pathogenesis of osteoarthritis is complex,and its genomics research results are varied and different.So this study intends to integrate and analyze the OA related high-throughput microarray data in the NCBI GEO database,in order to identify the common differentially expressed genes(DEGs)by the bioinformatics analysis technology,and to explore the key gene of human OA pathogenesis which combined with the results of proteomics in animal experiment.The PCR technique was used to verify the expression in the clinical tissue samples of key genes.Methods1 Animal experimentForty 3-month old female healthy SD ratswere recruited and randomly divided into 4 groups,i.e.the low dose PA group,the medium dose PA group,high dose PA group,the normal control group,10 in each group.The model was prepared using classic Hulth method except the normal control group.After 4-week modeling,the model rats were fed with low,medium and high dose for 2 weeks according to different groups.The normal control group was fed with the same amount of normal saline.The levels of cAMP/cGMP and TGF-? receptor in visceral tissues were detected by enzyme-linked immunosorbent assay and immunohistochemistry.Trend of effect of PA on visceral tissues was analyzed by comparing the changes of the relative expression of cAMP/cGMP and TGF-? receptor in visceral tissues.Fifty 3-month old female healthy SD ratswere recruited and randomly divided into 5 groups,i.e.the blank control group,the model control group,the PA 2 weeks group,the PA 4 weeks group,the PA 6 weeks group,10 in each group.The classic Hulth method was used to prepare the disease model of osteoarthritis except the blank control group.After 4 weeks modeling,the PA feeding group rats were fed with Pilose Antler for 2,4,6 weeks according to different time points,and the blank control group and the model control group were given the same amount of saline for 6 weeks.The bilateral knee joint cartilage was taken from rats in each group after the last irrigation of 2h,pathological changes of articular cartilage were detected by histopathological HE staining,and the expression of Smad2,3,6,7 mRNA and protein were detected by fluorescence quantitative-PCR and Western blot hybridization.Twenty 3-month old female healthy SD ratswere recruited and the classic Hulth method was used to prepare the disease model of osteoarthritis.After 4 weeks modeling,the SD rats were randomly divided into the model group and the PA feeding group,10 in each group.The PA feeding group rats were fed with 0.042 g Pilose Antler per 100 g body mass,the model control group were given the same amount of saline for 4 weeks,1 times a day.The bilateral knee joint cartilage was taken from rats in each group after the last irrigation of 2h,two dimensional electrophoresis and silver nitrate staining were used to screen the differences of protein points in the knee cartilage of two groups of rats.Identification of protein by mass spectrometry and Coomassie Blue Staining.Database retrieval matches protein information,and the protein coding genes function enrichment by the GO analysis.2 Clinical trialScreening the OA related high-throughput microarray data in the NCBI GEO database using the key words as “osteoarthritis” and ”OA”.Identifying the common differentially expressed genes(DEGs)by the online tool GEO2 R.And the Funrichnew software is used to extract the common differentially genes in each microarray.The DAVID tool is used to annotate the GO function and the enrichment of KEGG pathway of the DEGs.Combining the DEGs identified from microarray data and the protein encoding gene of the pilose antler action target screened by proteomics in animal experiments.Uploading those genes to the online analysis tool String for protein interaction network analysis,and applying the software Cytoscape MCODE toolkit to analyze the protein interaction results and carry out protein-protein interaction network subset analysis,then identifying the key genes of human OA.Selecting the Second Affiliated Hospital of Guangzhou University of Chinese Medicine Othopdeic inpatients,recruiting research patients in accordance with the established inclusion / exclusion criteria,Informing and signing the informed consent to the patient.Includeding 6 OA patients who were intended for knee replacement into the OA group,and Includeding 6 femoral neck fracture patients who were intended for hip replacement into the control group.Clinical samples were collected during the operation after the patients were registered and preserved.The polymerase chain reaction(PCR)was used to detect and compare the expression of the key genes of human OA in the two groups of clinical samples,and to verify the key position of the selected genes in the laboratory.Result1 Animal experiment1.1 The enzyme-linked immunosorbent:The levels of cAMP/cGMP in spleen of the low dose PA group increased significantly(P<0.05);The levels of cAMP/cGMP in kidney and heart of the medium dose PA group increased significantly(P<0.05);While the levels of cAMP/cGMP in liver and ovary of the high dose PA group increased significantly(P<0.05);And the rank of the relative expression of cAMP/cGMP from large to small order,the Pilose Antler low-dose group: kidney,liver,ovary,heart,spleen,brain,stomach,lung and intestine,the Pilose Antler medium-dose group:kidney,liver,brain,spleen,heart,ovary,lung,stomach and intestine,and the Pilose Antler high-dose group:kidney,liver,ovary,spleen,heart,intestine,brain,stomach and lung.1.2 The immunohistochemistry:The levels of TGF-? receptor in kidney and liver of the low and medium dose PA group increased significantly(P<0.05);The levels of TGF-? receptor in spleen and stomach of the low and medium dose PA group,in spleen of the medium dose PA group,and in intestine and stomach of the high dose PA group decreased significantly(P<0.05);The rank of the relative expression of TGF-? receptor from large to small order,the Pilose Antler low-dose group: kidney,liver,ovary,intestine,brain,heart,lung,stomach and spleen,the Pilose Antler medium-dose group: kidney,liver,heart,brain,ovary,stomach,intestine,lung and spleen,and the Pilose Antler high-dose group:ovary,kidney,liver,spleen,heart,lung,intestines,stomach and brain.1.3 The histopathological HE staining:Pathological result showed that the articular cartilage of the rats in the model control group was disordered,the cartilage surface was ulcerated,the number of cells increased in a diffuse,cluster and disorder distribution.The pathological condition of articular cartilage in pilose antler feeding groups were between those in the blank control group and the model control group,and the damage of cartilage structure improved gradually according to the feeding time.1.4 The fluorescence quantitative-PCR:Comparison between the model group,normal group and antler fed groups: Smad2 mRNA expression in group model group decreased compared with the normal group(P < 0.01),and increased when antler fed group compared to normal control group(P < 0.05);Smad3 mRNA expression in group model group increased slightly compared with the normal group(P> 0.05),while increased significantly when antler fed group compared to normal control group(P < 0.01);Smad6,7 mRNA expression in model group both increased than those in the normal group(P < 0.01),and decreased slightly when antler fed group compared to normal control group(P > 0.05).Comparison between 2,4,6weeks in the antler fed group:Smad2 mRNA expression at 4 weeks decreased comparing to that at 2 weeks(P < 0.01),and increased at 6 weeks than 4 weeks(P < 0.05);Smad3 mRNA expression at 4 weeks decreased slightly compared to 2 weeks(P > 0.05),and 6 weeks increased slightly than 4 weeks(P > 0.05);Smad6 mRNA expression at 4 weeks decreased slightly comparing to that at 2 weeks(P > 0.05),6 weeks increased slightly than 4 weeks(P > 0.05);Smad7 mRNA expression at 4 weeks decreased comparing to that at 2 weeks(P < 0.01),6 weeks increased than 4 weeks(P < 0.01).1.5 The Western blot hybridization:Comparison between the model group,normal group and antler fed groups: Smad2 protein expression in group model group decreased compared with the normal group(P < 0.01),and increased when antler fed group compared to normal control group(P < 0.01);Smad3 protein expression in group model group increased significantly compared with the normal group(P < 0.01),and increased significantly when antler fed group compared to normal control group(P < 0.01);Smad6,7 protein expression in model group both increased than those in the normal group(P < 0.01),and decreased slightly when antler fed group compared to normal control group(P < 0.01).Comparison between 2,4,6weeks in the antler fed group:Smad2/3 protein expression at 4 weeks decreased comparing to that at 2 weeks(P < 0.01),and increased at 6 weeks than 4 weeks(P < 0.01);Smad6 protein expression at 4 weeks increased comparing to that at 2 weeks(P < 0.01),6 weeks decreased than 4 weeks(P < 0.01);Smad7 protein expression at 4 weeks decreased comparing to that at 2 weeks(P < 0.01),6 weeks decreased slightly than 4 weeks(P > 0.05).1.6 The two-dimensional electrophoresis results: silver nitrate staining combined with two-dimensional electrophoresis isolated 4027 and 4573 protein spots from the model control group and antler fed group,35 differentially varied protein spots,of which 18 were up-regulated and 17 were down regulated.the original Kaumas Coomassie blue staining screened 16 differential protein spots,of which 9 were up-regulated and 7 were down regulated,the difference ratio of different proteins were more than 1.3 times.1.7 Mass spectrometry results :All the Coomassie brilliant blue stained proteins identificatied by the matrix assisted laser desorption ionization time-of-flight mass spectrometry,14 target proteins were obtained by matching the database,of which 9 were up-regulated by the deer antler irrigation group.including two hydrogen pyrimidine related protein 2(Dpysl2),serum transferrin(Tf),serine protease inhibitor(Serpina3n),lactate dehydrogenase B subunit protein(Ldhb)and bone inducing factor(Ogn),adiponectin(Adipoq),nitrilase 1(Nit1),apoptosis inducing factor family protein PEF1(PEF1),small heat shock proteins(Hspb1),down regulate the expression of 7 protein spots in three protein spots by mass spectrometry analysis,for the same protein,we identified 5 proteins down regulated expression,including C type lectin domain family protein(Clec3b),calreticulin(Calr),Endoplasmic reticulum calcium binding protein 3(Rcn3),endoplasmic reticulum calcium binding protein 1(Rcn1)and nuclear heterogeneous ribonucleoprotein(Hnrnpu).All the protein points matching significantly with the Mascot scores over 58(P < 0.05).1.8 GO analysis results:The differential protein identified mainly enrichment biological processes including response to drug(P=4.89E-02),negative regulation of smooth muscle cell proliferation(P=2.70E-02),cellular response to organic substance(P=2.77E-02),cellular response to cAMP(P=1.22E-03)and retina homeostasis(P= 2.37E-02);cell component enrichment analysis showed that these proteins mainly located in cell exosomes(P=3.58E-06)and extracellular space(P=8.10E-05)and extracellular matrix(P=5.12E-04)and membrane structure(P=9.09E-03);molecular function enrichment analysis showed that the calcium ion binding(P=1.60E-03)and the hydrolase activity,acting on carbon-nitrogen(but not peptide)bonds(P=8.56E-03)is the main molecular function of those proteins.2 Clinical trial2.1 Screening the DEGs from the gene microarrays:Searching the human OA related gene microarray data from the NCBI GEO database and selecting 3 microarray data.Identifying 39 common DEGs of human cartilage and subchondral bone tissue by the online tool GEO2 R and the Funrichnew software,including 17 up-regulated genes and 22 down-regulated genes.All the common DEGs are significantly different with the absolute values of fold change greater than 1.5(P < 0.05).2.2 GO analysis and pathway enrichment analysis:The common DEGs identified from human articular cartilage and subchondral bone tissue mainly enrichment biological processes including the development of the skeletal system(P=7.78E-06),cartilage cell differentiation(P=4.49E-05),morphogenesis of the skeletal system(P=8.53E-05),(P=6.32E-04),development of articular cartilage the differentiation of osteoblasts(P=8.01E-04)and ossification process(P=9.86E-04);locate in the extracellular domain(P=4.08E-03)and extracellular matrix(P=5.65E-03),extracellular matrix protein(P= 7.78E-03),extracellular domain(P=9.97E-03)and extracellular matrix(P=3.22E-02)and the top of the cells(P=4.44E-02);The ferrous binding(P=4.59E-02),transmembrane signal receptor activity(P=5.57E-02),molecular transduction activity(P=6.03E-02),receptor activity(P=6.03E-02),Transmembrane receptor activity(P=6.54E-02)function and signal receptor activity(P=7.60E-02)is the main molecular function of those common DEGs.They mainly enriched in the osteoclast differentiation(P=6.44E-02),PPAR(P=1.93E-01)signaling pathway,ECM receptor interaction(P=2.44E-01),cell adhesion molecule(CAMs)pathway(P=3.67E-01),chemokine signaling pathway(P=4.52E-01),focal adhesion pathway(P=4.87E-01)and cytokine receptor interaction(P=5.26E-01),PI3K/Akt(P=6.76E-01)signal pathway.2.3 Protein-protein interaction network analysis:Combining the common DEGs identified from microarray data and the protein encoding gene of the pilose antler action target screened by proteomics in animal experiments.Uploading those genes to the online analysis tool String for protein interaction network analysis,and selecting 52 genes whose node PPI score >0.15 with 129 nodes interaction,the average value is 4.96,P=2.54e-11.Analyzing the protein interaction results by the software Cytoscape MCODE toolkit,we carry out 2 protein-protein interaction network clusters.And the cluster 1 including genes CLAR,CA2,HSPB1,ADIPOQ,MMP1,TF,PPARG,GLI2,CXCR4,the cluster 2 including genes CHAD,OGN,SFRP2,NTN1.2.4 The expression of gene verified in the clinical specimens: Polymerase chain reaction detection of the identified gene(CA2,MMP1,PPARG,GLI2,CXCR4,SFRP2,NTN1,CHAD)mRNA showed that,Compared with the control group,the expression of MMP1,CXCR4 and PPARG mRNA increased significant in group OA(P < 0.05),and the expression of CA2,GLI2,SFRP2,NTN1 and CHAD were down-regulated(P< 0.05).Conclusion1 Animal experiment1.1 The effect of pilose antler on cAMP/cGMP and TGF-? are mostly in kidney and liver,and this view conform to the traditional meridian trpism of pilose antler in traditional Chinese medicine theory,and can be used as the basis of experimental research of its meridian trpism attribution.1.2 It is possible that Pilose Antler promotes the repair of articular cartilage and treats the OA rat by regulating the expression of Smad2,3,6,7 gene and protein in chondrocytes,and there may be other non Smad signaling pathways targets besides the TGF-?/Smad signaling pathway when Pilose Antler egulates the expression of Smads gene and proteins in chondrocytes.1.3 Pilose Antler may exerts its effect through up-regulating the expression of the two hydrogen pyrimidine associated protein 2,serum transferrin,serine protease inhibitors,lactate dehydrogenase B subunit protein,osteoinductive factor,adiponectin,nitrilase 1,apoptosis inducing factor family protein PEF1,small heat shock protein expression,and down-regulation the C type lectin domain family protein,calreticulin,ER calcium binding protein 3,endoplasmic reticulum calcium binding protein 1,heterogeneous nuclear ribonucleoprotein when Pilose Antler intervening the OA rats.And these differential proteins may be the targets of the Pilose Antler on OA.2 Clinical trialThere were 8 common differentially expressed genes(DEGs)obtained through integrating and analyzing the OA related high-throughput microarray data in the NCBI GEO database.In human OA cartilage the expression of MMP1,CXCR4 and PPARG mRNA were up-regulated,the expression of CA2,GLI2,SFRP2,NTN1 and CHAD mRNA were down-regulated by PCR verification.And we fund those genes are important in OA through the GO function analysis,KEGG Pathway enrichment analysis combined with previous literature,so we predicted those 8 gene may be the key genes of human OA. |
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