This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels. The oxidative stress model was induced in MC3T3-E1 cells by H_2O_2, and 3 mmol·L~(-1) tetraethylammonium(TEA) chloride was used to block the BK channels in MC3T3-E1 cells. MC3T3-E1 cells were divided into a control group, a model group, an EXD group, a TEA group, and a TEA+EXD group. After MC3T3-E1 cells were treated with corresponding drugs for 2 days, 700 μmol·L~(-1) H_2O_2 was added for treatment for another 2 hours. CCK-8 assay was used to detect cell proliferation activity. The alkaline phosphatase(ALP) assay kit was used to detect the ALP activity of cells. Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR) were used to detect protein and mRNA expression, respectively. Alizarin red staining was used to detect the mineralization area of osteoblasts. The results showed that compared with the control group, the model group showed significantly blunted cell proliferation activity and ALP activity, reduced expression of BK channel α subunit(BKα), collagen Ⅰ(COL1), bone morphogenetic protein 2(BMP2), osteoprotegerin(OPG), and phosphorylated Akt, decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2), BMP2, and OPG, and declining area of calcium nodules. EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity, up-regulate the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt, and forkhead box protein O1(FoxO1), promote the mRNA expression of RUNX2, BMP2, and OPG, and enlarge the area of calcium nodules. However, BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt and FoxO1, increasing the mRNA expression of RUNX2, BMP2, and OPG, and enlarging the area of calcium nodules. EXD-containing serum could improve the proliferation activity, osteogenic differentiation, and mineralization ability of MC3T3-E1 cells under oxidative stress, which might be related to the regulation of BK channels and downstream Akt/FoxO1 signaling pathway.
Osteogenesis ; Core Binding Factor Alpha 1 Subunit/pharmacology* ; Large-Conductance Calcium-Activated Potassium Channels/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Calcium/metabolism* ; Cell Differentiation ; RNA, Messenger/metabolism* ; Cell Proliferation ; Osteoblasts

This study aimed to explore the mechanism of n-butanol alcohol extract of Baitouweng Decoction(BAEB) in the treatment of vulvovaginal candidiasis(VVC) in mice based on the negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis. In the experiment, female C57BL/6 mice were divided randomly into the following six groups: a blank control group, a VVC model group, high-, medium-, and low-dose BAEB groups(80, 40, and 20 mg·kg~(-1)), and a fluconazole group(20 mg·kg~(-1)). The VVC model was induced in mice except for those in the blank control group by the estrogen dependence method. After modeling, no treatment was carried out in the blank control group. The mice in the high-, medium-, and low-dose BAEB groups were treated with BAEB at 80, 40, and 20 mg·kg~(-1), respectively, and those in the fluconazole group were treated with fluconazole at 20 mg·kg~(-1). The mice in the VVC model group received the same volume of normal saline. The general state and body weight of mice in each group were observed every day, and the morphological changes of Candida albicans in the vaginal lavage of mice were examined by Gram staining. The fungal load in the vaginal lavage of mice was detected by microdilution assay. After the mice were killed, the degree of neutrophil infiltration in the vaginal lavage was detected by Papanicolaou staining. The content of inflammatory cytokines interleukin(IL)-1β, IL-18, and lactate dehydrogenase(LDH) in the vaginal lavage was tested by enzyme-linked immunosorbent assay(ELISA), and vaginal histopathology was analyzed by hematoxylin-eosin(HE) staining. The expression and distribution of NLRP3, PKCδ, pNLRC4, and IL-1Ra in vaginal tissues were measured by immunohistochemistry(IHC), and the expression and distribution of pNLRC4 and IL-1Ra in vaginal tissues were detected by immunofluorescence(IF). The protein expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by Western blot(WB), and the mRNA expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by qRT-PCR. The results showed that compared with the blank control group, the VVC model group showed redness, edema, and white secretions in the vagina. Compared with the VVC model group, the BAEB groups showed improved general state of VVC mice. As revealed by Gram staining, Papanicolaou staining, microdilution assay, and HE staining, compared with the blank control group, the VVC model group showed a large number of hyphae, neutrophils infiltration, and increased fungal load in the vaginal lavage, destroyed vaginal mucosa, and infiltration of a large number of inflammatory cells. BAEB could reduce the transformation of C. albicans from yeast to hyphae. High-dose BAEB could significantly reduce neutrophil infiltration and fungal load. Low-and medium-dose BAEB could reduce the da-mage to the vaginal tissue, while high-dose BAEB could restore the damaged vaginal tissues to normal levels. ELISA results showed that the content of inflammatory cytokines IL-1β, IL-18, and LDH in the VVC model group significantly increased compared with that in the blank control group, and the content of IL-1β, IL-18 and LDH in the medium-and high-dose BAEB groups was significantly reduced compared with that in the VVC model group. WB and qRT-PCR results showed that compared with the blank control group, the VVC model group showed reduced protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues of mice and increased protein and mRNA expression of NLRP3. Compared with the VVC model group, the medium-and high-dose BAEB groups showed up-regulated protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues and inhibited protein and mRNA expression of NLRP3 in vaginal tissues. This study indicated that the therapeutic effect of BAEB on VVC mice was presumably related to the negative regulation of NLRP3 inflammasome by promoting PKCδ/NLRC4/IL-1Ra axis.
Female ; Animals ; Humans ; Mice ; Candidiasis, Vulvovaginal/drug therapy* ; Inflammasomes/genetics* ; Interleukin-18 ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; 1-Butanol/pharmacology* ; Fluconazole/therapeutic use* ; Interleukin 1 Receptor Antagonist Protein/therapeutic use* ; Mice, Inbred C57BL ; Candida albicans ; Cytokines ; Drugs, Chinese Herbal/pharmacology* ; Ethanol ; RNA, Messenger ; Calcium-Binding Proteins/therapeutic use*

Sympathetic cues via the adrenergic signaling critically regulate bone homeostasis and contribute to neurostress-induced bone loss, but the mechanisms and therapeutics remain incompletely elucidated. Here, we reveal an osteoclastogenesis-centered functionally important osteopenic pathogenesis under sympatho-adrenergic activation with characterized microRNA response and efficient therapeutics. We discovered that osteoclastic miR-21 was tightly regulated by sympatho-adrenergic cues downstream the β2-adrenergic receptor (β₂AR) signaling, critically modulated osteoclastogenesis in vivo by inhibiting programmed cell death 4 (Pdcd4), and mediated detrimental effects of both isoproterenol (ISO) and chronic variable stress (CVS) on bone. Intriguingly, without affecting osteoblastic bone formation, bone protection against ISO and CVS was sufficiently achieved by a (D-Asp₈)-lipid nanoparticle-mediated targeted inhibition of osteoclastic miR-21 or by clinically relevant drugs to suppress osteoclastogenesis. Collectively, these results unravel a previously underdetermined molecular and functional paradigm that osteoclastogenesis crucially contributes to sympatho-adrenergic regulation of bone and establish multiple targeted therapeutic strategies to counteract osteopenias under stresses.
Adrenergic Agents/pharmacology* ; Apoptosis Regulatory Proteins/pharmacology* ; Bone Diseases, Metabolic/metabolism* ; Humans ; Liposomes ; MicroRNAs/genetics* ; Nanoparticles ; Osteoclasts ; Osteogenesis/physiology* ; RNA-Binding Proteins/pharmacology*

To study the mechanism and action of Cinnamomi Ramulus in ameliorating intrahepatic cholestasis induced by α-isothiocyanate( ANIT) in rats by regulating FXR pathway. Forty SD rats were randomly divided into normal group,model group,positive control( ursodeoxycholic acid) group( 60 mg·kg~(-1)),Cinnamomi Ramulus treatment( 60 mg·kg~(-1)·d~(-1)) group,and Cinnamomi Ramulus treatment( 20 mg·kg~(-1)·d~(-1)) group,with 8 rats in each group. Except for the normal control group,the other groups were intragastrically administered with the corresponding concentrations of continuous aqueous solution( 0. 005 m L·g~(-1)),once a day,for 7 days.Except for the normal group,the other groups were treated with ANIT( 100 mg·kg~(-1)),once a day,for 3 days. Blood was taken from the abdominal aorta 24 hours after the last administration,and serum alanine aminotransferase( ALT),aspartate aminotransferase( AST),total bilirubin( TBi L),and total bile acid( TBA) were measured. 1. 5-2 cm of rat liver tissue was taken. After fixation with10% formaldehyde,paraffin-embedded sections were taken,HE staining was performed,and immunohistochemistry( IHC) was used to analyze the expression of FXR. RNA and protein were extracted from rat liver tissue to detect FXR mRNA expression,as well as bile acid synthesis and detoxification,transport related SHP,UGT2 B4,BSEP protein expressions at downstream of FXR. Compared with the normal group,serum ALT,AST,TBi L,and TBA levels were elevated in the model group( P<0. 01),liver damage was severe,FXR protein's optical density decreased,FXR mRNA expression decreased,and SHP,UGT2 B4,BSEP protein expressions were decreased( P<0. 05,P<0. 01). Compared with the model group,the drug group could reduce serum ALT,AST,TB,TBA levels to different degrees( P<0. 05,P<0. 01),alleviate liver tissue damage,increase the optical density of FXR protein,and promote the expressions of FXR mRNA and FXR,SHP,BSEP and UGT2 B4 proteins( P<0. 05,P<0. 01). Cinnamomi Ramulus can alleviate ANIT-induced intrahepatic cholestasis,and reduce hepatocyte injury and serum ALT,AST,TBi L and TBA levels. The mechanism may be through FXR-SHP,FXR-UGT2 B4,FXR-BSEP signaling pathways. Therefore,in the pathogenesis of intrahepatic cholestasis,we can try to further explore in alleviating intrahepatic cholestasis with Cinnamomi Ramulus,so as to provide effective drugs for clinical treatment of intrahepatic cholestasis.
Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Bile Acids and Salts ; blood ; Bilirubin ; blood ; Cholestasis, Intrahepatic ; chemically induced ; drug therapy ; Cinnamomum ; chemistry ; Isothiocyanates ; Liver ; Plant Extracts ; pharmacology ; RNA-Binding Proteins ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley

To explore the mechanism for the role of autophagy in endometriosis, and to provide a theoretical basis for prevention and treatment of endometriosis.
 Methods: The endometrial CRL-7566 cells were treated with ATG5 siRNA, autophagic activator rapamycin and autophagic inhibitor 3-MA, respectively. The cell proliferation and invasion were detected by clonal formation, cell growth curve and MTT assay. The clinical specimens of endometriosis were collected from 20 cases. The expression of autophagy marker LC3II and autophagy substrate protein P62 were detected.
 Results: Rapamycin inhibited the proliferation and clonal formation of CRL-7566 cells, while autophagy inhibitor 3-MA and ATG5 siRNA showed opposite effect. Moreover, rapamycin inhibited filopodia growth in endometriosis, whereas overexpression of filopodia-relevant protein fascin-1 inhibited the decrease in invasiveness caused by rapamycin. In clinical samples, we also found a significant decrease of LC3II while an increase in P62 compared with the control group.
 Conclusion: Autophagy inhibition may contribute to an increase in endometrial cell proliferation and invasiveness. Autophagy activation could be a potential strategy for endometriosis therapy.
Autophagy ; drug effects ; genetics ; Carrier Proteins ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; drug effects ; Endometriosis ; physiopathology ; Endometrium ; cytology ; Female ; Gene Expression Regulation ; Humans ; Microfilament Proteins ; genetics ; metabolism ; Microtubule-Associated Proteins ; genetics ; RNA-Binding Proteins ; genetics ; Sirolimus ; pharmacology

OBJECTIVETo predict and identify the target gene of miR-145, and to explore the underlying mechanism of the inhibition of miR-145 on drug resistance to Oxaliplatin (L-OHP) in human colorectal cancer cells.

METHODSL-OHP-resistant human colorectal cancer cell line (HCT116/L-OHP) was established in vitro by exposing to increased concentrations of L-OHP in cell culture medium. MiR-145-mimics and its negative control (NC-miRNA) were transfected into HCT116/L-OHP cells using liposome to establish HCT116/L-OHPover-expressing miR-145 and HCT116/L-OHP. The target genes of miR-145 were predicted by bioinformatic analysis, and validated by dual luciferase activity assay. After determination of G protein coupled receptor 98(GPR98) as target gene, corresponding plasmids were constructed and transfected to establish HCT116/L-OHPover-expressing GPR98 and HCT116/L-OHP. HCT116/L-OHP cells over-expressing both GPR98 and miR-145 (HCT116/L-OHP) were acquired through modification of the binding sites of GPR98 cDNA with miR-145. CCK-8 assay was used to assess the proliferation (A value) and sensitivity to L-OHP (the lower the IC50, the stronger the sensitivity) in HCT116/L-OHP cells. Real-time quantitative PCR was used to measure the mRNA expression of miR-145 and GPR98. Western blot was used to examine the protein expression of GPR98 and drug-resistant associated protein, such as P-glycoprotein (gp), multiple drug-resistance protein 1(MRP1), cancer-inhibition gene PTEN.

RESULTSHCT116/L-OHP cell line was successfully established with ICof (42.34±1.05) mg/L and miR-145 mRNA expression of 0.27±0.04, which was higher than (9.81±0.95) mg/L (t=39.784, P=0.000) and lower than 1.00±0.09 (t=13.021, P=0.000) in HCT116 cells. Based on HCT116/L-OHP cells, HCT116/L-OHPcells were established successfully, with relative miR-145 expression of 10.01±1.05, which was higher than 1.06±0.14 in HCT116/L-OHPand 1.00±0.16 in HCT116/L-OHP (F=161.797, P=0.000). GPR98 was identified to be the target gene of miR-145. The relative mRNA and protein expressions of GPR98 in HCT116/L-OHPcells were 8.48±0.46 and 1.71±0.09, respectively, which were higher than those in HCT116/L-OHP(mRNA: 3.65±0.40, protein: 1.21±0.10) and HCT116/L-OHP (mRNA: 3.49±0.35, protein: 1.22±0.08; all P<0.05). The A value was 1.31±0.10, and the relative protein expressions of P-gp and MRP1 were 1.53±0.18 and 1.49±0.20 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHP (A value: 0.82±0.08, relative protein expression: 1.00±0.06 and 1.21±0.13, all P<0.05). The A value was 0.89±0.08, and the relative protein expressions of P-gp and MRP were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHP(A value: 0.20±0.05, relative protein expression: 0.20±0.07, 0.55±0.10, all P<0.05). The relative protein expression of PTEN in HCT116/L-OHPcells was 0.12±0.03, which was lower than 1.25±0.14 in HCT116/L-OHP cells(P<0.05). In addition, relative protein expressions of P-gp and MRP1 were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHPcells (0.20±0.07 and 0.55±0.10), while PTEN expression in HCT116/L-OHPcells was lower as compared to HCT116/L-OHPcells (1.41±0.16 vs. 1.98±0.13, P<0.05).

CONCLUSIONMiR-145 inhibits drug resistance to L-OHP of HCT116 cells through suppressing the expression of target gene GPR98.

ATP Binding Cassette Transporter, Sub-Family B ; drug effects ; ATP-Binding Cassette, Sub-Family B, Member 1 ; drug effects ; Cell Line, Tumor ; drug effects ; physiology ; Colorectal Neoplasms ; physiopathology ; Down-Regulation ; drug effects ; genetics ; Drug Resistance, Neoplasm ; drug effects ; genetics ; physiology ; HCT116 Cells ; drug effects ; physiology ; Humans ; In Vitro Techniques ; MicroRNAs ; genetics ; pharmacology ; Multidrug Resistance-Associated Proteins ; drug effects ; Organoplatinum Compounds ; pharmacology ; PTEN Phosphohydrolase ; drug effects ; RNA, Messenger ; Receptors, G-Protein-Coupled ; drug effects ; genetics

Objective: To explore the effect of the AXL expression on the chemosensitivity of prostate cancer PC-3 and DU145 cells to docetaxel and possible mechanisms. METHODS: Using Western blot, we examined the expressions of the AXL protein, p-AXL and Gas6 in the docetaxel-resistant PC-3 (PC-3-DR) and DU145 (DU145-DR) cells stimulated with gradually increased concentrations of docetaxel. We transfected the PC-3 and DU145 cells with negative NC ShRNA and AXL-ShRNA, respectively, which were confirmed to be effective, detected the proliferation, apoptosis and cycle distribution of the cells by CCK8, MTT and flow cytometry after treated with the AXL-inhibitor MP470 and/or docetaxel, and determined the expression of the ABCB1 protein in the PC-3-DR and DU145-DR cells after intervention with the AXL-inhibitor R428 and/or docetaxel. RESULTS: The expression of the AXL protein in the PC-3 and DU145 cells was significantly increased after docetaxel treatment (P <0.05). The expressions AXL and p-AXL were remarkably higher (P <0.05) while that of Gas6 markedly lower (P <0.05) in the PC-3 and DU145 than in the PC-3-DR and DU145-DR cells. The inhibitory effect of docetaxel on the proliferation and its enhancing effect on the apoptosis of the PC-3 and DU145 cells were significantly decreased at 48 hours after AXL transfection (P <0.05). MP470 obviously suppressed the growth and promoted the apoptosis of the PC-3-DR and DU145-DR cells, with a higher percentage of the cells in the G2/M phase when combined with docetaxel than used alone (P <0.05). R428 markedly reduced the expression of ABCB1 in the PC-3-DR and DU145-DR cells, even more significantly in combination with docetaxel than used alone (P <0.05). CONCLUSIONS The elevated expression of AXL enhances the docetaxel-resistance of PC-3 and DU145 prostate cancer cells and AXL intervention improves their chemosensitivity to docetaxel, which may be associated with the increased cell apoptosis in the G2/M phase and decreased expression of ABCB1.
ATP Binding Cassette Transporter, Subfamily B, Member 1 ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Count ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Docetaxel ; Drug Resistance, Neoplasm ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Male ; Prostatic Neoplasms ; drug therapy ; metabolism ; pathology ; Proto-Oncogene Proteins ; drug effects ; genetics ; metabolism ; Pyrimidines ; pharmacology ; RNA, Small Interfering ; Receptor Protein-Tyrosine Kinases ; drug effects ; genetics ; metabolism ; Taxoids ; pharmacology

OBJECTIVETo explore the effect of MSH3 knock-down on sensitivity of tongue cancer cells to cisplatin.

METHODSThree small interfering RNA (siRNA) fragments targeting MSH3 CDS region were synthesized and transfected into CAL27 cells via Lipofectamine. Real-time PCR and Western blotting were used to assess the efficiency of MSH3 silencing. MTS, apoptosis staining and cell immunofluorescence assay were used to examine the cisplatin sensitivity, apoptosis and DNA repair of transfected CAL27 cells.

RESULTSs One of the 3 siRNAs was found to significantly reduce the expression of MSH3 protein in CAL27 cells (P<0.05). MTS assay showed that MSH3 silencing resulted in an significant reduction of IC50 of cisplatin from 21.32 to 13.95 µmol/L (P<0.05) and increased the apoptotic index of the exposed cells from 4.23∓1.27 to 11.32∓1.82 (P<0.05). Immunofluorescence assay demonstrated that silencing MSH3 markedly reduced the number of γ-H2AX foci.

CONCLUSIONSilencing MSH3 can significantly increase cisplatin sensitivity of tongue cancer cells, the mechanism of which involves mainly attenuation of repair of DNA double-strand damage in the cells.

Apoptosis ; Cell Line, Tumor ; Cisplatin ; pharmacology ; DNA-Binding Proteins ; genetics ; metabolism ; Gene Silencing ; Humans ; MutS Homolog 3 Protein ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Tongue Neoplasms ; drug therapy ; genetics ; Transfection

OBJECTIVEThis study observed attenuating effect of hydroxysafflor yellow A (HSYA), an effective ingredient of aqueous extract of Carthamus tinctorius L, on lipopolysaccharide (LPS)-induced endothelium inflammatory injury.

METHODSEahy926 human endothelium cell (EC) line was used; thiazolyl blue tetrazolium bromide (MTT) test was assayed to observe the viability of EC; Luciferase reporter gene assay was applied to measure nuclear factor-κB (NF-κB) p65 subunit nuclear binding activity in EC; Western blot technology was used to monitor mitogen activated protein kinase (MAPKs) and NF-κB activation. Reverse transcription polymerase chain reaction (RT-PCR) method was applied to observe intercellular cell adhesion molecule-1 (ICAM-1) and E-selectin mRNA level; EC surface ICAM-1 expression was measured with flow cytometry and leukocyte adhesion to EC was assayed with Rose Bengal spectrophotometry technology.

RESULTSHSYA protected EC viability against LPS-induced injury (P <0.05). LPS-induced NF-κB p65 subunit DNA binding (P <0.01) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) phosphorylation was inhibited by HSYA. HSYA attenuated LPS triggered ICAM-1 and E-selectin mRNA levels elevation and phosphorylation of p38 MAPK or c-Jun N-terminal kinase MAPK. HSYA also inhibited LPS-induced cell surface ICAM-1 protein expression P <0.01) and leukocyte adhesion to EC (P <0.05).

CONCLUSIONHSYA is effective to protect LPS-induced high expression of endothelium adhesive molecule and inflammatory signal transduction.

Cell Adhesion ; drug effects ; Cell Nucleus ; drug effects ; metabolism ; Cell Survival ; drug effects ; Chalcone ; analogs & derivatives ; chemistry ; pharmacology ; therapeutic use ; E-Selectin ; genetics ; metabolism ; Endothelium, Vascular ; drug effects ; pathology ; Gene Expression Regulation ; drug effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; pathology ; Humans ; I-kappa B Proteins ; metabolism ; Inflammation ; drug therapy ; pathology ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Leukocytes ; cytology ; drug effects ; Lipopolysaccharides ; MAP Kinase Signaling System ; drug effects ; NF-KappaB Inhibitor alpha ; Phosphorylation ; drug effects ; Protective Agents ; pharmacology ; Protein Binding ; drug effects ; Quinones ; chemistry ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo explore the mechanism of Bushen Qiangji Granule (, BSQJ) in restraining the osteogenic differentiation of ankylosing spondylitis (AS) fifibroblasts.

METHODSHip joint capsules were obtained from AS patients (n=10) receiving total hip replacement and healthy hip joint capsules from patients with hip fracture (n=10) receiving surgery as a control. Finite fifibroblast lines were established from these tissue samples to observe the effect of BSQJ on suppressing osteogenic differentiation of fifibroblasts. The expression of osteogenic marker gene corebinding factor a1 (Cbfa1) and Smad family proteins were examined by Western blot and real-time quantitative polymerase chain reaction (qPCR).

RESULTSThe mRNA expression level of Cbfa1 was significantly higher in AS fibroblasts than that in normal fibroblasts and the expression of pSmad1, pSmad5, Smad4 and Cbfa1 in AS fibroblasts was also higher, demonstrating the activation of the BMP/Smads signal pathway in AS fifibroblasts. BSQJ-medicated serum not only restrained the mRNA and protein expression levels of Cbfa1 and inhibited protein expression level of Smad4 but also decreased the expression quantities of pSmad1 and pSmad5.

CONCLUSIONSBSQJ can inhibit osteogenic differentiation of AS fifibroblasts in vitro by suppressing the activation of the BMP/Smads signal pathway. This may be the important molecular mechanism of BSQJ in regulating AS ossifification.

Adult ; Bone Morphogenetic Proteins ; metabolism ; Cell Differentiation ; drug effects ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; pathology ; Humans ; Middle Aged ; Osteogenesis ; drug effects ; genetics ; Phosphorylation ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Serum ; metabolism ; Signal Transduction ; drug effects ; Smad Proteins ; metabolism ; Spondylitis, Ankylosing ; genetics ; pathology ; Young Adult