Mingxia Wu

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.

Duhuo Jisheng Decoction promotes chondrocyte proliferation through accelerated G1/S transition in osteoarthritis

Duhuo Jisheng Decoction (DHJSD), a well known traditional Chinese folk medicine, is used for eliminating stagnation, removing blood stasis, promoting blood circulation and alleviating pain; it is commonly used for the treatment of various diseases, including osteoarthritis (OA). However, the molecular mechanisms behind the therapeutic effects of OA remain unclear. In the present study, the effects of DHJSD on the morphology of articular cartilage and the G1/S cell cycle progression in chondrocytes, as well as the underlying mechanisms, were investigated. A total of 27 two‑month‑old male Sprague Dawley rats were randomly divided into 3 groups: the control group (no papain-induced OA; received an equivalent amount of saline only), the model group (papain-induced OA; received an equivalent amount of saline only) and the DHJSD group [papain-induced OA; received a clinical oral dose of DHJSD (9.3 g/kg/day)]. After 8 consecutive weeks of treatment, the morphological changes in articular cartilage were observed under an optical microscope and by transmission electron microscopy (TEM) and the mRNA and protein expression levels of cyclin D1, CDK4, CDK6, retinoblastoma protein (Rb) and p16 were measured by RT‑PCR and immunohistochemistry, respectively. Treatment with DHJSD significantly improved the arrangement of collagen fibers in the articular cartilage, as well as its structure and reduced cell degeneration compared with the model group. The mRNA and protein expression levels of cyclin D1, CDK4, CDK6 and Rb in the DHJSD‑treated group were significantly increased compared with those in the model group, whereas p16 expression was significantly downregulated. Taken together, these results indicate that DHJSD treatment promotes chondrocyte proliferation by promoting the G1/S checkpoint transition in the cell cycle and by upregulating the expression of cyclin D1, CDK4, CDK6 and Rb and downregulating the expression of p16 and this may, in part, explain its clinical efficacy in the treatment of osteoarthritis.

Millimeter wave treatment promotes chondrocyte proliferation via G1/S cell cycle transition

Millimeter waves, high-frequency electromagnetic waves, can effectively alleviate the clinical symptoms in osteoarthritis patients, as a non-pharmaceutical and non-invasive physical therapy regimen. However, the molecular mechanisms of the therapeutic effects of millimeter wave treatment are not well understood. In the present study, the effect of millimeter waves on the G1/S cell cycle progression in chondrocytes and the underlying mechanism was investigated. Chondrocytes isolated from the knee of SD rats were cultured and identified using toluidine blue staining. The second generation chondrocytes were collected and stimulated with or without millimeter waves for 48 h. Chondrocyte viability was analyzed using the MTT assay. The cell cycle distribution of chondrocytes was analyzed by flow cytometry. mRNA and protein expression levels of cyclin D1, cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) and p21 were detected using real-time PCR and western blotting, respectively. Millimeter wave stimulation was found to significantly enhance chondrocyte viability. Moreover, the percentage of chondrocytes in the G0/G1 phase was significantly decreased, whereas that in the S phase was significantly increased. In addition, following millimeter wave treatment, cyclin D1, CDK4 and CDK6 expression was significantly upregulated, whereas p21 expression was significantly downregulated. The results indicate that millimeter wave treatment promotes chondrocyte proliferation via cell cycle progression.

Potential Synergistic and Multitarget Effect of Herbal Pair Chuanxiong Rhizome-Paeonia Albifora Pall on Osteoarthritis Disease: A Computational Pharmacology Approach

ObjectiveTo study the polypharmacological mechanism of herbal pair Chuanxiong Rhizome-Paeonia Albifora Pall (HP CXR-PAP) on the treatment for osteoarthritis (OA).MethodsChemical space was used to discuss the similarities and differences between the molecule sets of HP CXR-PAP and drugs. Docking protocol was used to study the interaction between HP CXR-PAP and OA target enzymes. The similarities and differences of HP CXR-PAP and drugs in target spaces were elucidated by network features.ResultsThe plots between the molecule sets of HP CXR-PAP and drugs in chemical space had the majority in the same region, and compounds from HP CXR-PAP covered a much larger additional region of space than drug molecules, which denoted the diverse structural properties in the molecule set of HP CXR-PAP. The molecules in HP CXR-PAP had the properties of promiscuous drugs and combination drug, and both HP CXR-PAP ligand-target interaction network and drug ligand-target interaction network were similar in the interaction profiles and network features, which revealed the effects of multicomponent and multitarget.ConclusionThe clue of potential synergism was obtained in curing OA disease by Chinese medicine, which revealed the advantages of Chinese medicine for targeting osteoarthritis disease.

Experimental study of low-frequency electroacupuncture-induced differentiation of bone marrow mesenchymal stem cells into chondrocytes.

In the present study, we investigated the effect of low-frequency electroacupuncture (EA) on the differentiation of bone mesenchymal stem cells (BMSCs) into chondrocytes and the molecular mechanism involved. We isolated BMSCs from Sprague-Dawley (SD) rat bone marrow. Third-generation SD rat BMSCs (P3 BMSCs) were harvested and characterized by flow cytometry with FITC staining. Data indicated that the positive rates of CD90 and CD45 were 98.22 and 1.91%, respectively, indicating the high purity of the BMSCs. The P3 BMSCs were treated with EA for 15 or 30 min daily for 7 or 14 days. Using optical microscopy and transmission electron microscopy, we found that EA induced morphological changes in the BMSCs, displaying typical morphology of early chondrocytes. In addition, we found that the cytoplasm and extracellular matrices were metachromatically stained by toluidine blue in the treated cells in a dose-dependent manner, indicating that EA treatment resulted in the expression of glycosaminoglycan. Furthermore, upon immunohistocytochemical staining and Western blotting, we found that EA treatment significantly and dose-dependently induced expression of chondrocyte-specific matrix protein type II collagen, which may have been mediated by the transcription factor Sox9, as the mRNA expression of Sox9 was found to be significantly increased after EA treatment. Taken together, these results suggest that EA can be employed as a novel non-drug-inducing method for the differentiation of BMSCs into chondrocytes.

Xiao Jin Wan, a traditional Chinese herbal formula, inhibits proliferation via arresting cell cycle progression at the G2/M phase and promoting apoptosis via activating the mitochondrial‑dependent pathway in U-2OS human osteosarcoma cells

Xiao Jin Wan (XJW) is a well-known traditional Chinese folk-medicine, which is commonly used for the treatment of various types of diseases including cancers. However, the mechanism of the anticancer activity of XJW against U-2OS human osteosarcoma cells, have not yet been reported. In the present study, we investigated the cellular effects of the XJW on the U-2OS human osteosarcoma cell line. Our results showed that XJW induced cell morphological changes, reduced cell viability in a dose- and time-dependent manner and arrested in the G2/M phase of the cell cycle suggesting that XJW inhibited the proliferation of U-2OS cells. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. In addition, XJW treatment caused loss of plasma membrane asymmetry, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, the results indicate that the U-2OS cell growth inhibitory activity of XJW was due to cell cycle arrested and mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of Xiao Jin Wan.

Electroacupuncture serum inhibits TNF‑α‑mediated chondrocyte inflammation via the Ras‑Raf‑MEK1/2‑ERK1/2 signaling pathway

The Ras-Raf-mitogen-activated protein kinase kinase (MEK)1/2-extracellular signal-regulated kinase (ERK)1/2 signaling pathway contributes to the release of chondral matrix-degrading enzymes and accelerates the degradation of articular cartilage. Electroacupuncture (EA) treatment has been widely used for the treatment of osteoarthritis (OA); however, the mechanism underlying the effects of EA on OA remains unclear. Therefore, the present study evaluated the anti-inflammatory effects and potential underlying mechanisms of EA serum (EAS) on tumor necrosis factor (TNF)-α-mediated chondrocyte inflammation. A total of 30 Sprague Dawley rats were randomly divided into three groups: The blank group; experimental group I, which received 15 min of EA treatment; and experimental group II, which received 30 min of EA treatment. Subsequently, serum samples were obtained. Chondrocytes were isolated from the knee cartilage of Sprague Dawley rats, and were identified using collagen type II immunohistochemistry. TNF-α-treated chondrocytes were used as a cell model, and subsequently the cells were treated with EAS from each group for various durations. The results demonstrated that EAS treatment significantly promoted the viability and inhibited the apoptosis of TNF-α-treated chondrocytes. In addition, interleukin (IL)-1β concentration was significantly increased in the model group compared with in the control group, whereas EAS significantly reduced IL-1β concentration in TNF-α-treated chondrocytes. Furthermore, the protein expression levels of Ras, Raf and MEK1/2 were reduced in the EAS groups compared with in the model group. EAS also significantly inhibited the phosphorylation of ERK1/2, and the expression of downstream regulators matrix metalloproteinase (MMP)-3 and MMP-13. In conclusion, these results indicated that EAS may inhibit TNF-α-mediated chondrocyte inflammation via the Ras-Raf-MEK1/2-ERK1/2 signaling pathway in vitro, thus suggesting that EAS may be considered a potential therapeutic strategy for the treatment of OA.

Electroacupuncture inhibits sodium nitroprusside‑mediated chondrocyte apoptosis through the mitochondrial pathway

In China, electroacupuncture (EA) is a therapeutic method that is extensively applied in the clinical treatment of osteoarthritis (OA); however, the underlying molecular mechanism remains unclear. Chondrocyte apoptosis may be observed in cartilage tissue in OA, and is often considered a key target for the treatment of this condition. Therefore, the present study aimed to determine the effects of EA on sodium nitroprusside (SNP)-induced chondrocyte apoptosis. Chondrocytes were obtained from the knee joints of Sprague Dawley rats by type II collagenase digestion. Following microscopic observation and authentication with type II collagen immunohistochemistry, articular cartilage cells were used in subsequent experiments. Using inverted phase contrast microscopy, DAPI staining and flow cytometry, it was revealed that chondrocytes treated with SNP became apoptotic, whereas EA inhibited SNP-induced chondrocyte apoptosis. Subsequently, JC-1 single staining, reverse transcription-quantitative polymerase chain reaction analysis, western blotting, colorimetric assays and immunofluorescence staining were performed for further investigation. The results demonstrated that, when compared with normal chondrocytes, the mitochondrial membrane potential of SNP-treated chondrocytes was markedly lowered, B-cell lymphoma 2 (Bcl-2) expression was reduced, and the expression levels of Bcl-2-associated X protein (Bax), cytochrome c, caspase-9 and caspase-3 were increased. Compared with in SNP-treated chondrocytes, the decrease in the mitochondrial membrane potential of chondrocytes treated with SNP and EA was smaller, Bcl-2 expression was increased, and the expression levels of Bax, cytochrome c, caspase-9 and caspase-3 were decreased following EA intervention. In conclusion, the present study demonstrated that EA modulated the mitochondrial pathway to suppress SNP-mediated chondrocyte apoptosis. Therefore, EA may be of value in the treatment of OA.