Xihai Li

An ultrasensitive electrochemical impedance sensor for a special BRCA1 breast cancer gene sequence based on lambda exonuclease assisted target recycling amplification

A label-free, target recycling electrochemical impedance spectroscopy (EIS) DNA sensor has been developed for detection of a model related to the BRCA1 breast cancer gene with a detection limit of 0.05 nM.

Icariin promotes bone formation via the BMP-2/Smad4 signal transduction pathway in the hFOB 1.19 human osteoblastic cell line.

Icariin, the main active compound of the traditional Chinese medicine, Epimedium, is commonly used for the clinical treatment of osteoporosis. However, the precise molecular mechanism of the therapeutic effect of icariin has not been elucidated. The aim of this study was to examine the effect of icariin on cell viability, alkaline phosphatase (ALP) activity, the amount of calcified nodules, and to delineate the molecular mechanism of icariin-enhanced bone formation by investigating the expression of bone morphogenic protein-2 (BMP-2), Smad4, Cbfa1/Runx2, osteoprotegerin (OPG), receptor activator of nuclear factor κ-B ligand (RANKL) and the OPG/RANKL ratio in the hFOB 1.19 human osteoblastic cell line. We found that icariin significantly increased the cell viability, the activity of ALP and the amount of calcified nodules in the hFOB 1.19 cells. Furthermore, we observed that icariin upregulated the expression of BMP-2, Smad4, Cbfa1/Runx2, OPG, RANKL and the OPG/RANKL ratio. Our results indicate that icariin can modulate the process of bone formation via the BMP-2/Smad4 signal transduction pathway in hFOB 1.19 cells.

Quercetin-mediated apoptosis via activation of the mitochondrial-dependent pathway in MG-63 osteosarcoma cells

Quercetin (a natural polyphenolic compound) is a polyphenolic flavonoid compound found in a variety of plants. It has been demonstrated to exert cytostatic activity against a variety of human cancer cell lines, including the human osteosarcoma cell line, MG-63. However, its effects on osteosarcoma cell apoptosis are still undefined. The present study was undertaken to examine the effect of quercetin on cell viability, apoptosis and mitochondrial membrane potential, and to determine the molecular mechanism of quercetin-induced apoptosis by investigating the expression of Bcl-2 family proteins (Bcl-2, Bax), cytochrome C, caspase-9 and caspase-3 in MG-63 cells. We found that quercetin suppressed the viability of MG-63 cells in a dose- and time- dependent manner. Furthermore, we observed that quercetin induced the loss of mitochondrial membrane potential, upregulated the expression of the proapoptotic proteins, Bax and cytochrome C, and activated caspase-9 and caspase-3, and downregulated the expression of antiapoptotic protein, Bcl-2. These data suggest that quercetin may induce apoptosis via the mitochondrial-dependent pathway in MG-63 cells.

Gallic acid induces apoptosis and inhibits cell migration by upregulating miR-518b in SW1353 human chondrosarcoma cells.

Gallic acid (GA), a natural agent, is widely distri-buted in plants with a range of biological effects and has been of potential interest as anticancer agent. However, its effects on chondrosarcoma cell apoptosis are still undefined. In the present study, the possible mechanisms of GA-induced apoptosis were explored in SW1353 cells, a human chondrosarcoma cell line. Our results showed that GA inhibited cell viability dose- and time-dependently. Morphological examination of GA-treated cells exhibited the typical features of cell death, such as rounding up of the cells and cell shrinkage. Wound-healing assay indicated that GA inhibited the migratory abilities of SW1353 cells. Hoechst 33258 staining assay and Annexin V/PI staining assay exhibited apoptosis induction by GA. To determine the molecular mechanism of GA-induced apoptosis, the expression levels Bcl-2, Bax, caspase-3 and caspase-9 were determined in SW1353 cells treated with GA. We found that GA downregulated the expression of the anti-apoptotic protein Bcl-2, and upregulated the expression of the pro-apoptotic protein Bax, and the activation of caspase-3 and caspase-9. To identify the possible mechanisms, the changes of microRNA expression were tested using the miRCURY™ LNA expression array. It was observed that the miR-518b gene was upregulated in treated cells. Taken together, these data show that GA induces apoptosis and inhibits cell migration by upregulating miR-518b in SW1353 cells.

Achyranthes bidentata polysaccharides activate the Wnt/β-catenin signaling pathway to promote chondrocyte proliferation

Achyranthes bidentata polysaccharides (ABPS) are the active components of Radix Achyranthis Bidentatae (AB), which has been extensively used in Traditional Chinese medicine (TCM) in the treatment of osteoarthritis (OA). Our previous study provided evidence that ABPS regulated the G1/S transition to promote chondrocyte proliferation. However, the precise mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of ABPS on the Wnt/β‑catenin signaling pathway in chondrocytes. Chondrocytes, obtained from the knee cartilage of Sprague-Dawley rats, were identified by type II collagen immunohistochemistry. ABPS upregulated the expression of Wnt-4, Frizzled-2, β-catenin and cyclin D1, and downregulated the expression of glycogen synthase kinase 3β (GSK-3β), as shown by reverse transcription PCR (RT-PCR) and western blot analysis. Using immunofluorescence, we also found that ABPS induced β-catenin nuclear translocation. Importantly, the expression of β-catenin and cyclin D1 was partly inhibited by Dickkopf-1 (DKK-1), an inhibitor of the Wnt/β-catenin signaling pathway. In addition, we found that ABPS increased the expression of type II collagen in chondrocytes. These results suggest that ABPS promote chondrocyte proliferation by activating the Wnt/β-catenin signaling pathway.

Achyranthes bidentata polysaccharides induce chondrocyte proliferation via the promotion of the G1/S cell cycle transition

Achyranthes bidentata polysaccharides (ABPS) are the major bioactive constituents of Radix Achyranthes bidentata (AB), which has been widely used in traditional Chinese medicine for the treatment of osteoarthritis. However, the molecular mechanisms behind the therapeutic effect of ABPS remain unclear. In the present study, chondrocytes were isolated from Sprague-Dawley rats. The effects of ABPS on the G1/S cell cycle transition in primary chondrocytes were investigated. The chondrocytes treated with and without ABPS were analyzed and it was observed that ABPS treatment was able to enhance chondrocyte proliferation in a dose- and time-dependent manner and promote the progression of chondrocyte cell cycle proliferation via the promotion of the G1 to S phase transition. Furthermore, using RT-PCR and western blot analysis, ABPS were observed as significantly upregulating the expression of cyclin D1 and the cyclin-dependent kinases (CDKs) CDK4 and CDK6. These results suggest that ABPS are able to promote chondrocyte proliferation via the promotion of the G1/S cell cycle transition.

Clinical study on the treatment of knee osteoarthritis of Shen (肾)-Sui (髓) insufficiency syndrome type by electroacupuncture

ObjectiveTo study the clinical effificacy of electroacupuncture (EA) on treating knee osteoarthritis (KOA) of Shen (肾)-Sui (髓) insuffificiency (SSI) syndrome type.MethodsA total of 245 patients (279 knees) of KOA-SSI were randomly assigned to two groups by lottery: 141 knees in the treatment group and 138 knees in the control group. The treatment group was managed with EA at the dominant points of Neixiyan (Ex-LE4) and Waixiyan (Ex-LE5) as well as the conjugate points of Xuanzhong (GB39) and Taixi (KI3) for 30 min, once a day, with 15 days as one course; 2 courses were applied with a 5-day interval in between. The control group was treated with intra-articular injection of 2 mL hyaluronic acid into the affected joint every 7 days for 5 times in total. The clinical effects on the patients in different stages were observed, and their symptom scores of knee and contents of cytokines, including interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), prostaglandin E2α (PGE2α) and matrix metalloproteinases-3 (MMP-3), in the knee joint fluid were measured before and after treatment.ResultsThe study was completed in 235 patients (263 knees); four patients (7 knees) in the treatment group and six patients (9 knees) in the control group dropped out. Comparison of therapeutic effects (excellent and effective rates) between the two groups showed insignificant differences (P>0.05). Symptom scores of knee and contents of cytokines in the knee flfluid after treatment were lowered signifificantly in the patients of stage I–III in both groups (P<0.05 or P<0.01). However, the lowering of the total symptom score of knee in the patients of stage III in the treatment group was more signifificant (P<0.05).ConclusionsEA could effectively alleviate the clinical symptoms in KOA patients of stage III, showing an effect superior to that of hyaluronic acid. EA also shows action in suppressing the secretion of IL-1, IL-6, TNF-α, PGE2α and MMP-3 in the knee flfluid.

Experimental study on the suppression of sodium nitroprussiate-induced chondrocyte apoptosis by Tougu Xiaotong Capsule (透骨消痛胶囊)-containing serum

ObjectiveTo study the mechanism of action of Tougu Xiaotong Capsule (透骨消痛胶囊, TGXTC) ex vivo in suppressing chondrocyte (CD) apoptosis induced by sodium nitroprussiate (SNP).MethodsThirty New Zealand rabbits, 2 months old, were randomized by lottery into five groups, six in each: the blank group treated with saline, the positive control group treated with Zhuanggu Guanjie Pill (壮骨关节丸, 70 mg/kg), and the three experimental groups, EGA, EGB, and EGC, treated with low dose (35 mg/kg), moderate dose (70 mg/kg), and high dose (140 mg/kg) of TGXTC, respectively. All treatments were administered via gastrogavage twice a day for 3 days. Arterial blood was collected from the abdominal aorta and drug or drug metabolites-containing serum was prepared. CDs obtained from knee joints of 16 four-week-old New Zealand rabbits were cultured to the third passage and confirmed by toluidine blue staining. SNP of various final concentrations (0, 0.5, 1.0, and 2.0 mmol/L) was used to induce CD apoptosis, and the dosage-effect relationship of SNP in inducing CD apoptosis was determined. Serum samples from the blank, control, and three dosages of TGXTC-treated rabbits were tested in the CD culture in the presence of SNP. Cell apoptosis was determined by Hoechst 33342 staining, viability of CDs was quantified by MTT, CD apoptosis rate was determined by annexin V-FITC/PI staining, levels of p53 and Bcl-2 mRNA expression in CDs were determined with RT-PCR, and contents of caspase-3 and caspase-9 proteins were determined by colorimetry.ResultsCD apoptosis was induced by SNP at all concentrations tested and in a dose-dependent manner. The SNP concentration of 1 mmol/L and treatment duration of 24 h appeared to be optimal and were selected for the study. Serum samples from the positive control rabbits and from the two higher doses of TGXTC-treated rabbits showed reduction of SNP-induced CD apoptosis, decrease in p53 mRNA expression, inhibition of catalytic activities of caspase-3 and caspase-9, and increase in Bcl-2 mRNA expression when compared with the serum from the blank group (P<0.05).ConclusionTGXTC-containing sera antagonized SNP-induced CD apoptosis and the molecular basis for the action was associated with up-regulation of Bcl-2, down-regulation of p53 expression, and inhibition of caspase-3 and caspase-9 catalytic activities.

In vitro study of inhibitory millimeter wave treatment effects on the TNF-α-induced NF-κB signal transduction pathway

Abnormal activation of the nuclear factor-κB (NF-κB) in chondrocytes initiates the transcription of inflammatory mediators, promotes their generation and release, and amplifies initial inflammatory signals. This results in the release of chondral matrix-degrading enzymes and accelerates the degeneration of articular cartilage. As a non-pharmaceutical and non-invasive physical therapy regimen, millimeter wave treatment has been successfully used for the treatment of osteoarthritis. In this study, chondrocytes were derived from the cartilages of knee joints of 4-week-old male Sprague-Dawley rats and were mechanically digested by collagenase type II treatment for further culture in vitro. The third-passage chondrocytes were stained with toluidine blue and treated with a gradient of tumor necrosis factor-α (TNF-α) for various times. Chondrocytic activity was measured by MTT assay, and the apoptotic rate of the chondrocytes was determined with Hocehst 33342 staining to identify effective treatment concentrations and durations and to establish an apoptosis model for the chondrocytes in response to TNF-α. Using this model, the chondrocytes were randomly divided to receive millimeter wave treatment for various times. The apoptotic rate of the chondrocytes was measured by Annexin V-FITC staining and the protein expression levels of RIP, TAK1, IκB kinase (IKK)-ß, IκB-α and NF-κB, were determined by Western blotting. Chondrocytic structure was examined by transmission electronic microscopy. The apoptotic rates were significantly lower at 4 and 8 h of treatment than at 0 and 2 h. The expression levels of RIP, TAK1, IKK-ß and NF-κB were also significantly lower at 4 and 8 h than at 0 and 2 h, whereas that of IκB-α was significantly higher at 4 and 8 h than at 0 and 2 h. Therefore, we can conclude that millimeter wave treatment can inhibit the activation of the TNF-α-mediated NF-κB signal transduction pathway through the down-regulation of RIP, TAK1, IKK-ß and NF-κB, and the up-regulation of IkB-α, in chondrocytes.

Overexpression of Shox2 Leads to Congenital Dysplasia of the Temporomandibular Joint in Mice

Our previous study reported that inactivation of Shox2 led to dysplasia and ankylosis of the temporomandibular joint (TMJ), and that replacing Shox2 with human Shox partially rescued the phenotype with a prematurely worn out articular disc. However, the mechanisms of Shox2 activity in TMJ development remain to be elucidated. In this study, we investigated the molecular and cellular basis for the congenital dysplasia of TMJ in Wnt1-Cre; pMes-stop Shox2 mice. We found that condyle and glenoid fossa dysplasia occurs primarily in the second week after the birth. The dysplastic TMJ of Wnt1-Cre; pMes-stop Shox2 mice exhibits a loss of Collagen type I, Collagen type II, Ihh and Gli2. In situ zymography and immunohistochemistry further demonstrate an up-regulation of matrix metalloproteinases (MMPs), MMP9 and MMP13, accompanied by a significantly increased cell apoptosis. In addition, the cell proliferation and expressions of Sox9, Runx2 and Ihh are no different in the embryonic TMJ between the wild type and mutant mice. Our results show that overexpression of Shox2 leads to the loss of extracellular matrix and the increase of cell apoptosis in TMJ dysplasia by up-regulating MMPs and down-regulating the Ihh signaling pathway.