[Mechanism of Tougu Xiaotong Capsules in delaying degeneration of osteoarthritis by regulating cholesterol metabolism in chondrocytes through lncRNA MALAT1].

From the perspective of lncRNA MALAT1 regulating cholesterol metabolism in chondrocytes, this paper explores the effect and mechanism of Tougu Xiaotong Capsules(TGXTC) in delaying the degeneration of osteoarthritis. After one week of adaptive feeding, 48(8-week-old) C57BL/6 mice were randomly divided into a blank group(12 mice) and a model group(36 mice) by random number table method. The mice in the model group were anesthetized by inhalation of 5% isoflurane, and the OA model was induced by Hulth method. The experiment randomly divided the mice into a model group(12 mice), a drug-positive group(taururso-deoxycholic acid)(12 mice), and a TGXTC group(12 mice). The drug-positive group was given 500 mg·kg~(-1) taurodeoxycholic acid by intragastric administration. TGXTC group was given TGXTC 368 mg·kg~(-1) by gavage. The blank group and model group were given the same amount of normal saline for four weeks. After the intervention, the mice in each group were killed under anesthesia, and the knee cartilage tissue was separated and collected. The morphologic changes of knee cartilage were observed. The level of lncRNA MALAT1 in the cartilage tissue was detected by real-time PCR. The protein expressions of ABCA1, ApoA1, LXRß, CHOP, and caspase-3 in mouse articular cartilage were detected by Western blot. Lentivirus-coated plasmid was used to transfect mouse chondrocytes with sh-MALAT1. The gene levels of lncRNA MALAT1 in mouse chondrocytes transfected with sh-MALAT1 were detected by real-time PCR. Western blot was used to detect the effect of TGXTC on the protein content of ABCA1, ApoA1, LXRß, CHOP, and caspase-3 in thapsigargin(TG)-induced mouse chondrocytes after lncRNA MALAT1 knockdown. Flow cytometry was used to detect the effect of TGXTC on apoptosis of TG-induced mouse chondrocytes after lncRNA MALAT1 knockdown. The results of HE and saffranine O staining showed that compared with the model group, the structure of the cartilage layer was basically intact; the damage degree of joint structure was significantly improved, and the cartilage matrix was significantly enhanced by saffranine O staining in the TGXTC group and drug-positive group. Compared with the model group, the lncRNA MALAT1 level was significantly decreased in the TGXTC group and drug-positive group. Compared with the model group, the protein content of ABCA1, ApoA1, and LXRß was significantly increased, while that of CHOP and caspase-3 in the TGXTC group and drug-positive group significantly decreased. Compared with the TG group, the lncRNA MALAT1 level in the TG+sh-MALAT1 group was decreased. The lncRNA MALAT1 level in the TG+sh-MA-LAT1+TGXTC group was increased compared with the TG+TGXTC group. Western blot results showed that compared with the model group, protein expressions of ABCA1, ApoA1, LXRß, CHOP, and caspase-3 in the TGXTC group were significantly decreased, after lncRNA MALAT1 knockdown, the regulation and apoptosis of ABCA1, ApoA1, LXRß, CHOP, and caspase-3 in TG-induced mouse chondrocytes were weakened by TGXTC. TGXTC can improve the disorder of cholesterol metabolism in OA chondrocytes and delay OA degeneration, which is closely related to the regulation of lncRNA MALAT1.

Achyranthes bidentata polysaccharides alleviate endoplasmic reticulum stress in osteoarthritis via lncRNA NEAT1/miR-377-3p pathway.

Endoplasmic reticulum stress (ERS) has been identified to be an important factor leading to chondrocyte apoptosis in osteoarthritis (OA). Previous studies have confirmed that Achyranthes bidentata polysaccharides (ABPS) can inhibit chondrocyte apoptosis; however, the mechanism of action of ABPS on chondrocyte ERS remains unclear. Thus in this study, we aim to investigate whether ABPS could inhibit OA-associated chondrocyte apoptosis by regulating ERS, especially by observing the relationship between the lncRNA NEAT1 and miR-377-3p, to explore further the protective mechanism of ABPS in OA. In vitro and in vivo experiments showed that ABPS inhibited chondrocyte ERS by regulating the expression of lncRNA NEAT1 and miR-377-3p. Moreover, both lncRNA NEAT1 silencing and miR-377-3p inhibition could attenuate the therapeutic effect of ABPS on ERS. Dual-luciferase results indicated that miR-377-3p targets the lncRNA NEAT1 gene in mouse chondrocytes. Therefore, we concluded that ABPS could inhibit thapsigargin (TG)-induced chondrocyte ERS through the lncRNA NEAT1/miR-377-3p axis.

Bioinformatics analysis to identify key genes and pathways influencing synovial inflammation in osteoarthritis.

Osteoarthritis (OA) is a chronic arthropathy that occurs in the middle­aged and elderly population. The present study aimed to identify gene signature differences between synovial cells from OA synovial membrane with and without inflammation, and to explain the potential mechanisms involved. The differentially expressed genes (DEGs) between 12 synovial membrane with inflammation and 12 synovial membrane without inflammation from the dataset GSE46750 were identified using the Gene Expression Omnibus 2R. The DEGs were subjected to enrichment analysis, protein­protein interaction (PPI) analysis and module analysis. The analysis results were compared with text­mining results. A total of 174 DEGs were identified. Gene Ontology enrichment results demonstrated that functional molecules encoded by the DEGs primarily had extracellular location, molecular functions predominantly involving 'chemokine activity' and 'cytokine activity', and were associated with biological processes, including 'inflammatory response' and 'immune response'. The Kyoto Encyclopedia of Genes and Genomes results demonstrated that DEGS may function through pathways associated with 'rheumatoid arthritis', 'chemokine signaling pathway', 'complement and coagulation cascades', 'TNF signaling pathway', 'intestinal immune networks for IgA production', 'cytokine­cytokine receptor interaction', 'allograft rejection', 'Toll­like receptor signaling pathway' and 'antigen processing and presentation'. The top 10 hub genes [interleukin (IL)6, IL8, matrix metallopeptidase (MMP)9, colony stimulating factor 1 receptor, FOS proto­oncogene, AP1 transcription factor subunit, insulin­like growth factor 1, TYRO protein tyrosine kinase binding protein, MMP3, cluster of differentiation (CD)14 and CD163] and four gene modules were identified from the PPI network using Cytoscape. In addition, text­mining was used to identify the commonly used drugs and their targets for the treatment of OA. It was initially verified whether the results of the present study were useful for the study of OA treatment targets and pathways. The present study provided insight for the molecular mechanisms of OA synovitis. The hub genes and associated pathways derived from analysis may be targets for OA treatment. IL8 and MMP9, which were validated by text­mining, may be used as molecular targets for the OA treatment, while other hub genes require further validation.

Ultrastructural change of the subchondral bone increases the severity of cartilage damage in osteoporotic osteoarthritis of the knee in rabbits.

Osteoporotic osteoarthritis is a phenotype of osteoarthritis (OA) manifested as fragile and osteoporotic subchondral bone. However, the ultrastructural features of subchondral bone in osteoporosis OA have not been determined. The study was aimed to investigate the ultrastructural dynamic changes of subchondral bone in osteoporotic OA model and how the ultrastructural damage in the subchondral bone caused by osteoporosis deteriorated the cartilage damage in OA. Eighteen rabbits were equally randomized to three groups, including the control, the OA and the osteoporotic OA groups. The structural changes of cartilage were evaluated by HE and safranin-O fast green staining, the Mankin's grading system was used to assess the stage of OA progression. And microstructural or ultrastructural changes in subchondral bone were assessed by micro-computed tomography or by scanning electron microscopy. According to the changes of cartilage histopathology, the OA group was in the early pathological stage of OA while the osteoporotic OA group was in the middle stage of OA based on Mankin's grading system. In addition, the damage of cartilage surface, reduction in the number of chondrocytes and the matrix staining were more increased in the osteoporotic OA group compared to the OA group. Compared to the OA group, the subchondral bone in the microstructure and ultrastructure in the osteoporotic OA group showed more microfracture changes in trabecular bone with more destructions of the tree-like mesh. Moreover, the collagen fibers were random rough with a fewer amount of bone lacunae in subchondral cortical plate in the osteoporotic OA group compared to the OA group. These findings indicated that the subchondral bone ultrastructure in the osteoporotic OA model was characterized by the destruction of the network structure and collagen fibers. The subchondral bone ultrastructural damage caused by osteoporosis may change mechanical properties of the upper cartilage and aggravate OA cartilage. Therefore, early diagnosis and treatment of osteoporosis is of great significance to prevent early OA from further developing osteoporotic OA.

Cibotium barometz polysaccharides stimulate chondrocyte proliferation in vitro by promoting G1/S cell cycle transition.

Cibotium barometz polysaccharides (CBPS) are one of the most important bioactive components extracted from the Cibotium barometz plant, which belongs to the Dicksoniaceae family. It has been widely used for the treatment of orthopedic diseases in traditional Chinese medicine. However, the molecular mechanisms behind the therapeutic effects of CBPS remain to be clarified. In the present study, the concentration of CBPS was detected by phenol-vitriol colorimetry. Furthermore, the effects stimulated by CBPS on the viability and G1/S cell cycle transition in primary chondrocytes from Sprague-Dawley rats were investigated. A cell viability assay demonstrated that chondrocyte proliferation may be enhanced by CBPS in a dose­ and time­dependent manner. The mechanism underlying the promotion of chondrocyte cell cycle was suggested to involve the stimulation of G1 to S phase transition. To further confirm the results, reverse transcription­quantitative polymerase chain reaction and western blot analyses were used to detect the expression of mRNA and protein levels of cyclin D1, cyclin­dependent kinase 4 and retinoblastoma protein. The results suggested that CBPS may stimulate chondrocyte proliferation via promoting G1/S cell cycle transition. Since osteoarthritis is characterized by deficient proliferation in chondrocytes, the present study indicates that CBPS may potentially serve as a novel method for the treatment of osteoarthritis.

Ethanolic extract of Tulipa edulis Bak induces apoptosis in SGC-7901 human gastric carcinoma cells via the mitochondrial signaling pathway.

Tulipa edulis Bak (TEB) is an active ingredient in various traditional Chinese medicine compounds and is commonly used to treat swelling and redness, remove toxicity and eliminate stagnation, as well as to prevent and treat certain cancer types. However, the underlying molecular mechanism of the anticancer activity of TEB remains unclear. The aim of the current study was to investigate the effect and underlying mechanism of the ethanolic extract of TEB (EETEB) on SGC-7901 human gastric carcinoma cells. An MTT assay was performed to analyze cell viability. In addition, transmission electron microscopy, an Annexin V/fluorescein isothiocyanate assay, a JC-1 assay and laser scanning confocal microscopy with DAPI staining were used to determine the rate of apoptosis. Furthermore, reverse transcription-polymerase chain reaction and western blot analysis were used to detect the expression levels of the apoptosis gene and protein. EETEB was identified to inhibit the growth of SGC-7901 cells in a dose-dependent manner and induce changes in cell morphology. At the molecular level, EETEB induced SGC-7901 cell DNA fragmentation, loss of plasma membrane and asymmetrical collapse of the mitochondrial membrane potential, while it increased the expression of pro-apoptotic B-cell lymphoma-2 (Bcl-2)-associated X protein and reduced expression of anti-apoptotic Bcl-2. Thus, the results of the current study revealed that the application of EETEB may inhibit the growth of the SGC-7901 cells due to mitochondria-mediated apoptosis.

Protective effects of Tougu Xiaotong capsule on tumor necrosis factor-α-injured UMR-106 cells.

Tumor necrosis factor-α (TNF-α) plays an important role in the abnormal metabolism of osteoblasts (OBs), which leads to subchondral bone (SB) alterations in osteoarthritis. In the present study, Tougu Xiaotong capsule (TXC), a traditional Chinese medicine, was used to treat TNF-α-injured OB-like cells. The cellular viability, mortality and ultramicroscopic morphology were evaluated. Thereafter, the activity of alkaline phosphatase (ALP), secretion of osteocalcin (OCN) and mineralization of nodules were analyzed. The results showed that TXC treatment significantly promoted cell proliferation, reduced cellular mortality and improved cellular ultrastructure, particularly that of the endoplasmic reticulum and nucleus. These data indicate that TXC is able to promote cell growth, as well as prevent inflammation in OB-like cells. Furthermore, the activity of ALP, secretion of OCN and mineralization of nodules were accelerated, and the calcium content of the TNF-α-injured OB-like cells was promoted by TXC treatment. These results indicate that TXC protected the OB-like cells from TNF-α-induced injuries. This may be a potential mechanism through which TXC regulates SB remodeling in the clinical treatment of osteoarthritis.

Deciphering the underlying mechanisms of Diesun Miaofang in traumatic injury from a systems pharmacology perspective.

Diesun Miaofang (DSMF) is a traditional herbal formula, which has been reported to activate blood, remove stasis, promote qi circulation and relieve pain. DSMF holds a great promise for the treatment of traumatic injury in an integrative and holistic manner. However, its underlying mechanisms remain to be elucidated. In the present study, a systems pharmacology model, which integrated cluster ligands, human intestinal absorption and aqueous solution prediction, chemical space mapping, molecular docking and network pharmacology techniques were used. The compounds from DSMF were diverse in the clusters and chemical space. The majority of the compounds exhibited drug-like properties. A total of 59 compounds were identified to interact with 16 potential targets. In the herb-compound-target network, the majority of compounds acted on only one target; however, a small number of compounds acted on a large number of targets, up to a maximum of 12. The comparison of key topological properties in compound-target networks associated with the above efficacy intuitively demonstrated that potential active compounds possessed diverse functions. These results successfully explained the polypharmacological mechanism underlying the efficiency of DSMF for the treatment of traumatic injury as well as provided insight into potential novel therapeutic strategies for traumatic injury from herbal medicine.

Millimeter Wave Treatment Inhibits Apoptosis of Chondrocytes via Regulation Dynamic Equilibrium of Intracellular Free Ca (2+).

The molecular mechanisms of TNF-α-induced apoptosis of chondrocyte and the role of Ca(2+) mediating the effects of MW on TNF-α-induced apoptosis of chondrocytes remained unclear. In this study, we investigated the molecular mechanism underlying inhibiting TNF-α-induced chondrocytes apoptosis of MW. MTT assay, DAPI, and flow cytometry demonstrated that MW significantly increased cell activity and inhibited chromatin condensation accompanying the loss of plasma membrane asymmetry and the collapse of mitochondrial membrane potential. Our results also indicated that MW reduced the elevation of [Ca(2+)] i in chondrocytes by LSCM. Moreover, MW suppressed the protein levels of calpain, Bax, cytochrome c, and caspase-3, while the expressions of Bcl-2, collagen II, and aggrecan were increased. Our evidences indicated that MW treatment inhibited the apoptosis of chondrocytes through depression of [Ca(2+)] i . It also inhibited calpain activation, which mediated Bax cleavage and cytochrome c release and initiated the apoptotic execution phase. In addition, MW treatment increased the expression of collagen II and aggrecan of chondrocytes.

Duhuo Jisheng Decoction­containing serum promotes proliferation of interleukin­1ß­induced chondrocytes through the p16­cyclin D1/CDK4­Rb pathway.

Duhuo Jisheng Decoction (DHJSD) is a traditional Chinese herbal medicine that has multiple uses, including as a treatment for osteoarthritis (OA). However, the molecular mechanisms underlying the therapeutic effects of DHJSD on OA remain unknown. In the present study, a serum pharmacological method was applied to investigate the effects of DHJSD on the proliferation of chondrocytes treated with interleukin­1ß (IL­1ß) in vitro. This is a cell model commonly used to reproduce the mechanisms involved in degenerative arthropathies, including OA. The most effective intervention conditions of DHJSD serum were examined by MTT assay. The degenerative chondrocyte model was established by IL­1ß­culture for 24 h, and was verified by optical microscopy and immunohistochemical analyses. Following the successful establishment of the degenerative chondrocyte model, the chondrocytes were subsequently randomly divided into two groups: The blank serum group and the DHJSD treatment group. Subsequent to treatment with the corresponding serum, cell proliferation was detected by MTT assay and DNA staining followed by FACS analysis, and the mRNA and protein expression levels of cyclin D1, cyclin­dependent kinase 4 (CDK4), retinoblastoma tumor suppressor protein (Rb) and p16 were measured by reverse transcription polymerase chain reaction and western blotting, respectively. The results indicated that the most effective condition for the promotion of chondrocyte proliferation was 10% concentration of DHJSD 2­h serum, and the degenerative chondrocyte model was successfully reproduced by IL­1ß­treatment for 24 h. The mRNA and protein expression levels of cyclin D1, CDK4 and Rb in the DHJSD serum­treated cells were significantly increased compared with those in the blank serum group, whereas p16 expression was significantly downregulated. These results indicate that treatment of cells with DHJSD­containing serum is able to promote IL­1ß­induced chondrocyte proliferation by promoting G1/S phase transition via modulating the expressions of cyclin D1, CDK4, Rb and p16, which contribute to the clinical efficacy of DHJSD in OA.

In silico search for multi-target therapies for osteoarthritis based on 10 common Huoxue Huayu herbs and potential applications to other diseases.

Huoxue Huayu (HXHY) has been widely used in traditional Chinese medicine (TCM) as a key therapeutic principle for osteoarthritis (OA), and related herbs have been widely prescribed to treat OA in the clinic. The aims of the present study were to explore a multi-target therapy for OA using 10 common HXHY herbs and to investigate their potential applications for treatment of other diseases. A novel computational simulation approach that integrates chemical structure, ligand clusters, chemical space and drug­likeness evaluations, as well as docking and network analysis, was used to investigate the properties and effects of the herbs. The compounds contained in the studied HXHY herbs were divided into 10 clusters. Comparison of the chemical properties of these compounds to those of other compounds described in the DrugBank database indicated that the properties of the former are more diverse than those of the latter and that most of the HXHY-derived compounds do not violate the 'Lipinski's rule of five'. Docking analysis allowed for the identification of 39 potential bioactive compounds from HXHY herbs and 11 potential targets for these compounds. The identified targets were closely associated with 49 diseases, including neoplasms, musculoskeletal, nervous system and cardiovascular diseases. Ligand­target (L­T) and ligand­target­disease (L­T­D) networks were constructed in order to further elucidate the pharmacological effects of the herbs. Our findings suggest that a number of compounds from HXHY herbs are promising candidates for mult­target therapeutic application in OA and may exert diverse pharmacological effects, affecting additional diseases besides OA.

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 two­month­old 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 RT­PCR 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 DHJSD­treated 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.

Electroacupuncture promotes chondrocyte proliferation via accelerated G1/S transition in the cell cycle.

The aim of the present study was to investigate the effects of electroacupuncture (EA) on the proliferation of chondrocytes and the molecular mechanism(s) involved. Passage 2 chondrocytes were randomly divided into four groups and treated with EA or nocodazole. After treatment, cell proliferation was determined using an MTT assay and DNA staining followed by FACS. The mRNA expression levels of cyclin D1, cyclin-dependent kinase (CDK)4, CDK6, phosphorylated retinoblastoma (pRb) and P16 were detected by RT-PCR, and the protein levels of cyclin D1, CDK4, CDK6, pRb and P16 were detected by western blotting. EA treatment significantly increased cell viability in a time-dependent manner and decreased the number of G0/G1 and G2/M phase chondrocytes and increased the number of S phase cells. The mRNA and protein levels of cyclin D1, CDK4, CDK6, (p)Rb and P16 consistently demonstrated a reverse trend with the levels in the chondrocytes treated with nocodazole. The expression levels of cyclin D1, CDK4, CDK6 and Rb were higher in chondrocytes receiving EA treatment when compared to levels in the untreated cells while expression of P16 was lower. In conclusion, EA treatment promotes chondrocyte proliferation via promotion of G1/S checkpoint transition in the cell cycle dependent on the activity of the P16-cyclin D1-CDK4/6-pRb pathway and this may, in part, explain its clinical effect in the treatment of osteoarthritis.

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 mitochondrial­dependent 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.

Millimeter wave treatment induces apoptosis via activation of the mitochondrial-dependent pathway in human osteosarcoma cells.

Millimeter wave (MW) is an electromagnetic wave with a wavelength between 1 and 10 mm and a frequency of 30-300 GHz that causes multiple biological effects and has been used as a major component in physiotherapies for the clinical treatment of various types of diseases including cancers. However, the precise molecular mechanism of the anticancer activity of millimeter wave remains to be elucidated. In the present study, we investigated the cellular effects of the MW in the U-2OS human osteosarcoma cell line. Our results showed that MW induced cell morphological changes and reduced cell viability in a dose- and time-dependent manner suggesting that MW inhibited the growth of U-2OS cells as demonstrated. 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, MW treatment caused loss of plasma membrane asymmetry, release of cytochrome c, collapse of mitochondrial membrane potential, activation of caspase-9 and -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 MW was due to mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of millimeter wave treatment.

Millimeter wave radiation induces apoptosis via affecting the ratio of Bax/Bcl-2 in SW1353 human chondrosarcoma cells.

The efficacy and safety of millimeter wave radiation has been proven for various types of malignant tumors. However, the mechanisms underlying effects of millimeter wave radiation on apoptosis are still unclear. The present study was undertaken to examine the effects of millimeter wave radiation on cell apoptosis and mitochondrial membrane potential, and to determine the molecular mechanism of millimeter wave radiation-induced apoptosis by investigating the expression of Bcl-2 family proteins (Bcl-2, Bax), caspase-9 and caspase-3 in SW1353 cells. We found that millimeter wave radiation suppressed the viability of SW1353 cells, demonstrating that millimeter wave radiation induced cell apoptosis and reduced cell viability in a time-dependent manner. Furthermore, we observed that treatment of cells with millimeter wave radiation significantly induced loss of mitochondrial membrane potential, upregulated proapoptotic Bax, caspase-9 and caspase-3, but did not significantly change levels of antiapoptotic Bcl-2. These data suggested that millimeter wave radiation may induce apoptosis via affecting the ratio of Bax/Bcl-2 in SW1353 cells.

[Determination of protopine in Corydalis racemose by HPLC].

OBJECTIVE: To develop a HPLC method for determining the content of protopine in Corydalis racemose. METHOD: Analysis was performed on a Gemini C18 column (4.6 mm x 250 mm, 5 microm) eluted with acetonitrile-water containing 0.8% triethylamine and 3% acetic acid acetum (20:80) as the mobile phase. The flow rate was 1.0 mL x min(-1). The detection wavelength was 289 nm. RESULT: The average content of protopine in Herb of Racemose Corydalis was 0.905%. The calibration curve of protopine was linear between 0.124-1.36 microg (r = 0.9999). The average recovery was 98.49% with RSD 1.9%. CONCLUSION: This method is simple, reproducible and can be used to determine the content of protopine in C. racemose.

[Effect of prim-o-glucosylcimifugin and 4'-O-beta-D-glucosyl-5-O-methylvisamminol con on proliferation of smooth muscle cell stimulated by TNF-alpha].

OBJECTIVE: To investigate the effect of prim-O-glucosylcimifugin and 4'-O-p-D-glucosyl-5-O-methylvisa-mminol con on the proliferation of smooth muscle cell stimulated by TNF-alpha. METHOD: The primary cell culture method of smooth muscle cell (SMC) was established by attachment-block. The SMC was identificated by immunochemistry method, and the growth curve was drawn by cytometry. The third generation of SMC was adopted in the experiment. The effect of prim-O-glucosylcimif-ugin and 4'-O-beta-D-glucosyl-5-O-methylvisamminol con on the proliferation and cell cycle of SMC was investigated by MTT and flow cytometry respectively. RESULT: TNF-alpha of 5 micro g x L(-1) can stimulate the proliferation of SMC and increase the proportion of G2 phase and S phase in cell cycle which has great significant difference (P < 0.01) compared with control. The three dose groups of prim-O-glucosylcimifugin and 4'-O-beta-D-glucosyl-5-O-methylvisammin-ol con can inhibit the proliferation of SMC and increase the proportion of G0/G1 phase, which has great significant difference (P < 0.01) compared with model group. CONCLUSION: Prim-O-glucosylcimifugin and 4'-O-beta-D-glucosyl-5-O-methylvisamminol con can inhibit the proliferation of SMC stimulated by TNF-alpha.

[Protection and its mechanism of catechin morphon on hypoxia-reoxygenation [corrected] induced injury in myocardial cells].

OBJECTIVE: To observe the protective effects of catechin morphon (GCG and EGCG) on hypoxia-reoxygenation induced injury in myocardial cells and to explore the mechanisms. METHOD: In cultured neonatal rat cardiomyocytes, we investigated the preconditioning protection by GCG and EGCG on the spontaneous beating, the survival rate, the release of LDH, MDA, SOD, GSH-Px and the ATP enzyme activity of cardiomyocyte cellular membrane in cultured rat cardiomyocytes treated during the reoxygenation 1h following hypoxia 3 h. The blocking agent of protien kinase C staurosporine (10 nmol x L(-1)) or the deactivator of Gi/o protein pertussis toxin (PTX, 200 microg x mL(-1)) were added before the catechin treatment. RESULTS: Preconditioning by GCG and EGCG increased the spontaneous beating and the survival rate, and decreased the release of LDH and MDA with the rise of SOD and ATP enzyme activity. Inhibition of PKC by staurosporine and Gi/o protein by PTX abolished the protection by catechin with the reduction of the beating, survival rate and activity of SOD, and the increase of the release of LDH and MDA. The results indicated that the activation of signal transduction pathway from PKC and Gi/o protein seemed to be involved in the cardioprotection of preconditioning by GCG and EGCG. CONCLUSION: The protection by GCG and EGCG on hypoxia-reoxygenation injury in cultured neonatal rat cardiomyocytes is found, which is related with scavenging of free radicals, and PKC Gi/o signal transduction pathway.

[ESR study and protection of water extract of Carthamus tinctorius on ox-LDL induced injury in rat cardiac microvascular endothelial cell].

OBJECTIVE: To observe the antioxidant effects of water extract of Carthamus tinctorius on ox-LDL induced injury in rat cardiac microvascular endothelial cell and detecting oxygen derived free radicals (OFR) to explore the antioxidant mechanisms. METHOD: By using the third generation of rat cardiac microvascular endothelial cells (rCMEC), the protection of water extract of C. tinctorius was investigated after ox-LDL (100 mg x L(-1) induced damage. The supernatant was collected for detecting lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), xanthine oxidase (XOD), glutathione peroxidase (GSH-Px), nitric oxide (NO), nitric oxide synthase (NOS) activity, and cell suspension was collected for detecting reactive oxygen species (ROS) by electron spin resonance (ESR). RESULT: Water extract of C. tinctorius increased the rCMEC survival rate, reduced LDH, MDA and XOD levels, and improved SOD, GSH-Px and NOS activity, while in the cell suspension ROS signal decreased significantly. CONCLUSION: Water extract of C. tinctorius has antioxidation. The mechanisms are likely related with scavenging of free radicals, enhancing its clearance, enhancing endogenous antioxidant activity.