| Objective1.Bioinformatics,pathological staining and molecular analysis were used to predict the possible molecular mechanism of Bushen Jianpi Huoxue recipe mediating CDH19 to regulate JAK1/STAT1 in the treatment of osteoporosis.2.Establishing the cell model of osteoporosis,validation of spleen qi and party mediated CDH19 zhichuan at a cellular level adjustment JAK1/STAT1 molecular mechanism for the treatment of osteoporosis.3.To establish a rat model of osteoporosis,in the animal level validation of spleen qi and party mediated CDH19 zhichuan regulating JAK1/STAT1 molecular mechanism for the treatment of osteoporosis.Methods1.Bioinformatics analysis of the molecular mechanism of Bushen Jianpi Huoxue recipe in preventing and treating osteoporosis(1)application TCMSP database and pick out the kidney and spleen and invigorate the circulation of the active ingredients,using Drugbank database to predict the target genes of these active ingredients,from the predicted target genes and GEO pathological state of different genes in the database,Then Bisogenet and CytoNCA technology were used to construct PPI(protein interaction)network so as to identify the core regulatory genes of OP disease,and GO and KEGG enrichment analysis of these key genes were performed.(2)Four samples of hip bone tissues from non-osteoporosis patients and four samples of hip bone tissues from osteoporosis patients were selected.The mRNA expression levels of hip bone tissues from osteoporosis patients were measured by the second generation high-throughput sequencing technology,and the two-end sequencing technology of illuminaNovaseq6000 sequencing platform was used.In different measure the expression level of STAR in osteoporosis patients.Then,using DESeq2 software to compare the level of gene expression in the sample,and use the rMATS software to analyze the variability of shear.Finally,GO analyzer was used to detect gene expression levels,and KEGG enrichment analysis was used to determine the changes in gene expression levels and screen out differentially expressed genes.(3)patients with osteoporosis and the genetic variation analysis of patients with osteoporosisA total of 20 patients with non-osteoporosis and postmenopausal osteoporosis were selected,10 in each Group.The patients with osteoporosis were selected as the Observation Group,and the patients with non-osteoporosis were selected as the Control Group.The hip bone tissues were selected,and the sequencing results were further verified by WB,QPCR,and IHC combined with the results of high-throughput sequencing.2.Cytological study of Bushen Jianpi Huoxue decoction mediating CDH19 to regulate JAK1/STAT1 pathway in the treatment of osteoporosis(1)The effect of Bushen Huoxue Jianpi recipe on MC3TC cellsMC3T3 and rat osteoclasts were cultured according to culture conditions.After stable growth,CDH19JAK/STAT pathway indicators,and osteogenic indicators of the two groups of cells were detected.Osteoblast MC3T3 cells were treated with different concentrations of Bushen Jianpi Huoxue recipe to determine the best concentration to promote the growth of osteoblasts.Osteoblast MC3T3 cells were treated with high,medium and low concentrations of Bushen Jianpi Huoxue Decoction,and the growth curve of MC3T3 cells in the concentration of Bushen Jianpi decoction was established.With WB,QPCR,IHC and alizarin red staining detection CDH19,JAK1/STAT1 pathway,gene and protein expression in osteogenesis related indicators.(2)CDH19 expression of osteoblast MC3TC indicators of developmentCHD19 and si-CDH19 were transfected into MC3TC,and the expression of CDH19 protein and gene was detected by WB and PCR.Stable MC3T3 cell lines with CDH19 overexpression and knockdown were established.In contrast,express group,interfere with the group,detection of osteogenesis related index expression.(3)The effect of CDH19 on JAK1/STAT1 signaling pathway in MC3TC cellsThrough transfection CHD19 and si-CDH19 to MC3TC,with WB and PCR detection CDH19 protein and gene expression,establish MC3T3 CDH19 expression and the interference of stable cell lines.The expression of JAK1/STAT1 pathway related indicators was detected in control group,overexpression group and interference group.Through the above research,the CDH19 in kidney and spleen and invigorate the circulation of different concentrations on the activity of osteoblasts,the biggest effect when the 20 times diluted.This experiment USES the traditional Chinese medicine(TCM)when the 20 times diluted concentration,with O-CDH19 after transfection,reoccupy qRT PCR and Western blot test this CDH19 expression in osteoblast.Then,the recipe of Bushen Jianpi Huoxue was used for recovery verification.BSJPHXF(1/20),BSJPHXF(1/20)+NC and BSJPHXF(1/20)+si-CDH19 were used to treat osteoblasts,and WB was used to detect the expression of CDH19.Jak1/Stat1 and osteoblast-related proteins in osteoblasts.The overexpression and silencing of CDH19 mRNA in osteoblasts were detected by PCR.(4)the kidney invigorate the circulation of spleen party restrain JAK1/STAT1 to improve osteoporosis is implemented by promoting CDH19 expression.Establish a MC3T3 controls,MC3T3+kidney invigorate the circulation of the spleen,MC3T3+kidney invigorate the circulation of spleen+NC,MC3T3++kidney invigorate the circulation of spleen party CHD19 expression grouping,training after 48 hours,the detection and osteogenesis JAK1/STAT1 pathway,alizarin red staining,etc.,To verify the kidney invigorate the circulation of spleen party restrain JAK1/STAT1 to improve osteoporosis is achieved by promoting CDH19 expression.3.The therapeutic effect of Bushen Jianpi Huoxue decoction on osteoporosis by regulating JAK1/STAT1 pathway through CDH19 in animal model(1)Establishment of osteoporosis rat modelSixty SPF healthy 6-month-old female SD rats were randomly divided into five groups according to body weight:normal group(Control),Sham operation group(Sham),OVX group(OVX),OVX+BSJPF group(OVX+BSJPF),OVX+alendronate group(OVX+ALN),with 12 rats in each group.In these experiments,rat ovariectomies were performed in OVX,OVX+BSJP,and OVX+ALN groups.The recovery time of rats in each group was 1 week,and after 1 week,the rats were given OVX+BSJPF and OVX+ALN by gavage for 12 weeks.At 3 months after operation,the following indexes were measured:①whole body bone mineral density(BMD)and lumbar spine BMD;② Bone mineral density of the right femur was detected;(3)in the rat right proximal femoral bone mineral density;④bone mineral density of left tibia in isolated rats;⑤Tibial Micro CT scanning;⑥HE staining of the tibia.(2)the WB test and PCR detectionWestern blotting and PCR were used to detect the expression of CDH19,Jakl/Statl and osteogenesis-related proteins and mrnas in bone tissues.(3)Chinese medicine safety assessment-function of liver and kidney toxicity assessmentThe body weight,white blood cell count and liver and kidney function of rats were detected to evaluate whether Bushen Jianpi Huoxue decoction had good safety in rats.Results1.Bioinformatics analysis of the molecular mechanism of Bushen Jianpi Huoxue recipe in preventing and treating osteoporosis(1)Through bioinformatics analysis,176 highly active compounds in the recipe were screened,254 target genes of highly active compounds were obtained,and 17 intersection target genes were obtained.The Bisogenet and CytoNCA method get seven core gene screening:CDKN1 A,AR,STAT1 and STAT3,NPM1,PCNA and VCAM1,speculated that these genes are kidney and spleen and invigorate the circulation of party is the key to the treatment of osteoporosis.Enrichment of core GO gene functions in multiple biological processes.KEGG pathways were mainly enriched in JAK-STAT signaling pathway,HIF-1 signaling pathway,FoxO signaling pathway,tumor,inflammation,cell cycle,hormone regulation and other signaling pathways.(2)After the second generation high-throughput sequencing and KEGG enrichment analysis,Found above the main enrichment of mRNA pathways involved in the growth of bone cells and death,Jak1/Stat1 pathway,P53 signal transduction pathway,cell factor interactions,phospholipase D signal transduction pathway,cell aging,antigen processing and presentation,chemokine signaling path,the phagosome formation,homologous recombination,adhesion molecule O-chitosan and its biosynthesis And so on.Through the analysis of gene differences between non-osteoporosis and osteoporosis patients,we found that CDH19 was significantly different between the two groups.(3)WB,PCR,IHC and other methods were used to detect the relationship between CDH19,Jak1,Statl and osteoporosis.The results showed that the expression of CDH19 was high in non-osteoporosis bone tissues,but low in osteoporosis bone tissues.The expression of Jak1/Statl is low in osteoporotic bone tissues,but high in osteoporotic bone tissues.OPG,OPN,Osterix high quantity,expressed in the osteoporosis bone tissue in patients with osteoporosis bone tissue expression quantity is low.The predicted results showed that CDH19 could promote the proliferation of osteoblasts and play a role in the treatment of osteoporosis,while Jak1/Statl pathway could inhibit the proliferation of osteoblasts and promote the occurrence and development of osteoporosis.2.Cytological study of Bushen Jianpi Huoxue decoction mediating CDH19 to regulate JAK1/STAT1 pathway in the treatment of osteoporosis(1)The effect of CDH19 on the growth of osteocytes and the expression of JAK1/STAT1 signaling pathway indicatorsCell experiments showed that CDH19 could promote the activity of osteoblasts,inhibit the expression of p-Jak1,and significantly reduce the expression of p-Stat1.The expressions of OPG,OPN,Osterix and RUNX2 were significantly increased.(2)The effects of different concentrations of Bushen Jianpi Huoxue recipe on the growth of osteoblasts and the expression of related proteinsThe results showed that the activity of osteoblast MC3T3 was enhanced with the increase of drug concentration within a certain concentration range of traditional Chinese medicine,and there was no cytotoxicity.Kidney and spleen and huoxue fang again divided into high concentrations of H(diluted 10 times as much as 1/10),the concentration of M(20 times dilution 1/20)and low concentration L(1/40 100 times dilution)and no dilution of four groups,do 72 hours of growth curve and related MC3TC protein detection.It was found that the medium concentration M(diluted 20 times 1/20)was the best for MC3T3 cell growth.When the concentration of M,OPG,OPN,Osterix,RUNX2 expression quantity,most Jak1/Statl pathway p-Jakl and p-Statl protein expression in the opposite.After alizarin red staining,calcium nodules were the most at medium concentrations.Therefore,concentration of MC3T3 cells grow best in osteogenesis effect is best.With the increase of the concentration of traditional Chinese medicine,the expression of Jak1/Stat1 protein was gradually decreased,while the expression of Osterix,RUNX2,OPG and OPN protein was gradually increased.(3)Bushen Jianpi Huoxue recipe mediated the effect of CDH19 on the activity of osteoblasts through Jak1/Stat1The results show that si-CDH19-1,2 and si si-CDH19-CDH19-3 transfection MC3T3 cells to screen the optimal interference fragment si-CDH19-1.The effect of CDH19 on osteogenesis and JAK1-STAT1 pathway in osteoblasts showed that CDH19 significantly promoted osteogenesis markers OPG,OPN,Osterix,RUNX2,and promoted the formation of calcium nodules.The expression of p-JAK1 and p-STAT1 was significantly inhibited.3.The therapeutic effect of Bushen Jianpi Huoxue decoction on osteoporosis by regulating JAK1/STAT1 pathway through CDH19 in animal model(1)Effect of Bushen Jianpi Huoxue decoction on bone mineral density in postmenopausal osteoporosis ratsThe results showed that Bushen Jianpi Huoxue decoction could significantly improve the systemic bone mineral density of postmenopausal osteoporosis rats,and its effect was stronger than that of Formejia.Invigorating the spleen and activating blood in the aspect of improving(density and blessed with fairly;F in improving the femur and tibia bone mineral density in the us and Canada is kidney and spleen and invigorate the circulation of the weak.Bushen Jianpi Huoxue Decoction can effectively improve the bone microstructure of proximal tibia by increasing BV/TV,BS/TV,Tb.N,Conn.D,and reducing Tb.Pf,Tb.(2)Effects of Bushen Jianpi Huoxue recipe on osteogenic related protein and mRNA in tibia of ratsFound that in WB and PCR detection in the kidney and spleen,invigorate the party group,compared with OVX group,significantly increased the CDH19 protein and mRNA expression of bone tissue within,and p-Jak1,p-Statl protein and RNA expression significantly reduced;Among the osteogenesis-related proteins,OPG,OPN,Osterix and RUNX2 protein expressions in the bone tissue of the BSHP-HXR group were significantly higher than those of the OVX group.The results showed that the expression of CDH19 and related osteogenic proteins and RNA in the osteoporosis group was decreased,while the expression of p-Jak1 and p-Stat1 was increased.When the traditional Chinese medicine Bushen Jianpi-Huoxue decoction was added,the expression of CDH19 and related osteogenic proteins and mRNA in the bone tissue was increased.Promote Jak1/Stat1 expression,thus improve the osteogenesis related protein and mRNA expression of the quantity,have the effect of improving osteoporosis.(3)the toxicity of Bushen Jianpi Huoxue recipe on liver and kidney function in ratsThe body weight,white blood cell count and liver and kidney function of the rats were detected.It was found that the Bushen Jianpi Huoxue recipe had no significant effect on white blood cell count and liver and kidney function,and the Bushen Jianpi Huoxue recipe was safe.Conclusion1.The significantly different gene between non-osteoporosis and osteoporosis patients was CDH19,which was lowly expressed in bone tissues of osteoporosis patients.The main pathways enriched in core genes were Jak/Stat,p-JAK1 and p-STAT1,which were highly expressed in bone tissues of osteoporosis patients.2.The kidney and spleen and activating blood can be mediated CDH19,inhibit Jak1/Statl signal pathway,so as to promote the growth of osteoblasts and osteogenesis index expression.3.The kidney and spleen and promoting blood party after adjust CDH19 and Jak1/Stat1 expression,can effectively improve ovarian rats bone mineral density,finally achieve the goal of ease or eliminate the osteoporosis. |
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