Network Pharmacology and Experimental Validation to Explore Mechanism of Tetrahydropalmatine on Acute Myocardial Ischemia.

OBJECTIVE: To explore the potential molecular mechanism of tetrahydropalmatine (THP) on acute myocardial ischemia (AMI). METHODS: First, the target genes of THP and AMI were collected from SymMap Database, Traditional Chinese Medicine Database and Analysis Platform, and Swiss Target Prediction, respectively. Then, the overlapping target genes between THP and AMI were evaluated for Grene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction network analysis. The binding affinity between the protein and THP was assessed by molecular docking. Finally, the protective effects of THP on AMI model and oxygen and glucose deprivation (OGD) model of H9C2 cardiomyocyte were explored and the expression levels of target genes were detected by RT-qPCR in vivo and in vitro. RESULTS: MMP9, PPARG, PTGS2, SLC6A4, ESR1, JAK2, GSK3B, NOS2 and AR were recognized as hub genes. The KEGG enrichment analysis results revealed that the potential target genes of THP were involved in the regulation of PPAR and hormone pathways. THP improved the cardiac function, as well as alleviated myocardial cell damage. Furthermore, THP significantly decreased the RNA expression levels of MMP9, PTGS2, SLC6A4, GSK3B and ESR1 (P<0.05, P<0.01) after AMI. In vitro, THP significantly increased H9C2 cardiomyocyte viability (P<0.05, P<0.01) and inhibited the RNA expression levels of PPARG, ESR1 and AR (P<0.05, P<0.01) in OGD model. CONCLUSIONS: THP could improve cardiac function and alleviate myocardial injury in AMI. The underlying mechanism may be inhibition of inflammation, the improvement of energy metabolism and the regulation of hormones.

[Guideline for clinical comprehensive evaluation of Chinese patent medicine (2022 version)].

Currently,the research or publications related to the clinical comprehensive evaluation of Chinese patent medicine are increasing,which attracts the broad attention of all circles. According to the completed clinical evaluation report on Chinese patent medicine,there are still practical problems and technical difficulties such as unclear responsibility of the evaluation organization,unclear evaluation subject,miscellaneous evaluation objects,and incomplete and nonstandard evaluation process. In terms of evaluation standards and specifications,there are different types of specifications or guidelines with different emphases issued by different academic groups or relevant institutions. The professional guideline is required to guide the standardized and efficient clinical comprehensive evaluation of Chinese patent medicine and further improve the authority and quality of evaluation. In combination with the characteristics of Chinese patent medicine and the latest research achievement at home and abroad,the detailed specifications were formulated from six aspects including design,theme selection,content and index,outcome,application and appraisal,and quality control. The guideline was developed based on the guideline development requirements of China Assoication of Chinese medicine. After several rounds of expert consensus and public consultation,the current version of the guideline has been developed.

[Molecular mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion injury in H9c2 cardiomyocytes based on molecular docking].

The aim of this study was to investigate the mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion(OGD/R) injury in H9 c2 cardiomyocytes. First, Discovery Studio 2019 was used for the molecular docking of luteolin with three key enzymes including lipoxygenase 5(ALOX5), lipoxygenase 12(ALOX12), and lipoxygenase 15(ALOX15) in lipoxygenase pathway. The docking results showed that luteolin had high docking score and similar functional groups with the original ligand. From this, H9 c2 cardiomyocytes were cultured in vitro, and then the injury model of H9 c2 cardiomyocytes was induced by deprivation of oxygen-glucose for 8 h, and rehabilitation of oxygen-glucose for 12 h. Cell viability was detected by tetrazolium(MTT) colorimetry. H9 c2 cardiomyocytes were observed with a fluorescence inverted microscope, and colorimetry was used to detect the level of lactate dehydrogenase(LDH) in cell supernatant. The results showed that luteolin could significantly protect the morphology of H9 c2 cells, significantly improve the survival rate of H9 c2 cardiomyocytes in OGD/R injury model, reduce the level of LDH in cell supernatant, inhibit cytotoxicity, and maintain the integrity of cell membrane. The inflammatory cytokines interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with the model group, luteolin can significantly reduce the release of IL-6 and TNF-α. Western blot was employed to detect the protein levels of ALOX5, ALOX12, and ALOX15 in lipoxygenase pathway. After luteolin intervention, the protein levels of ALOX5, ALOX12, and ALOX15 were significantly down-regulated compared with those in model group. These results indicate that luteolin can inhibit the release of IL-6 and TNF-α by restraining the activation of lipoxygenase pathway, thereby playing a protective role in the cardiomyocyte injury model induced by OGD/R.

Landscape of Hepatobiliary Adverse Drug Reactions Related to Preparations Containing Psoraleae Fructus and Its Application in Pharmacovigilance.

OBJECTIVE: To analyze clinical feature and information of medication to explore the risk signals of preparations containing Psoraleae Fructus (BGZP) related with hepatobiliary adverse drug reactions (ADR), in order to reinforce pharmacovigilance. METHODS: A retrospective study was conducted based on hepatobiliary ADR related with BGZP from the China Adverse Drug Reaction Monitoring System in years from January 2012 to December 2016. Serious and general ADRs were analyzed and assessed. RESULTS: There were 355 cases of hepatobiliary ADR related to BGZP. Both the amount of cases and the proportion of serious ADR showed an increasing growth by years (P<0.05). It was found that 10.43% of 355 cases may be involved with irrational drug use, including overdose, repeated medication, and combination of multiple drugs. There were 190 cases which used BGZP (non-combination), and they were mainly for common in diseases caused by abnormal immune activation (accounting for 40.53% of the total cases). Especially at the age group with the most cases with age of 41-50 years, the cases associated with immunological diseases of female were obviously more than that of male (P<0.05). The latency of hepatobiliary ADR related to BGZP ranged from 1 to 386 days, and the median latency was 27.5 days, along with the range of cumulative dose (0.45-520.02 g) as well as the daily dose (0.09-2.64 g/d) after the conversion. CONCLUSIONS: Cases of hepatobiliary ADR related to BGZP showed significant individual differences, and there was no correlation between drug usage duration and dosage and the occurrence of hepatobiliary ADR. It may be similar with idiosyncratic drug-induced liver injury, and recommended that BGZP should be used with more caution under monitoring liver function, especially in female patients with immunological diseases.

[Expert consensus on clinical application of Chinese herbal medicine decoction pieces (First Edition)].

Chinese herbal medicine decoction pieces(CHMDP), one of the main forms of traditional Chinese medicine(TCM) in clinic, have been widely used. However, the irrational use is increasingly serious due to the lack of the indicators for judging the rational use of CHMDP in medical institutions and the codes and standards for the clinical use of CHMDP. In order to regulate the rational clinical use of CHMDP, improve the clinical efficacy and ensure the drug safety for the patients, clinical pharmaceutical experts and clinical medical experts from 40 third-grade class-A hospitals nationwide were organized to give the "expert consensus on clinical application of CHMDP" in terms of prescription writing, combined use of drugs, use of special drugs, and drug use for special population. Detailed analysis and argumentation were conducted in accordance with the laws and regulations, Chinese Pharmacopoeia 2015 edition, Chinese Pharmacopoeia Code Notice for Clinical Use of Medicine, Administrative Regulations for Prescriptions, Administrative Specifications for Hospital Prescription Review(interim), and Chinese Traditional Medicine Prescription Format and Writing Specifications, as well as relevant project findings.

[Analysis of epidemiological characteristics of drug induced liver injury associated with Baixianpi Preparations].

A retrospective study was performed in drug-induced liver injury(DILI) cases associated with Dictamni Cortex(Baixianpi,BXP) Preparations,which were treated at grade Ⅲ class A liver disease hospitals from 2008 to 2016 and spontaneously reported for adverse reactions between 2012 and 2016 at HILI Cloud(hilicloud.net). The results showed 25 DLII cases associated with BXP Preparations treated at grade Ⅲ class A liver disease hospitals during the 9 years,including only 14 cases in line with the clinical diagnostic criteria of Guidelines for the Diagnosis and Treatment of Herb-Induced Liver Injury. And 74 DILI cases associated with BXP Preparations spontaneously reports adverse reactions,and 18. 92% of them had unreasonable medication,including polypharmacy(21. 43%),overdose(28. 57%) and repeated dosage(50%). And 47 DILI cases used BXP Preparations to treat psoriasis and vitiligo(a total of59. 57%). The time range of taking BXP Preparations until liver injury occurred was 1-366 d,with the median of 18 d. The dose of BXP Preparations was estimated to be 0. 09-12 g·d-1. And the cumulative dosage of taking drugs until liver injury occurred was 1. 1-336 g. Obvious associations with time-toxicity as well as quantity-toxicity could not be found based on the wide range of time-toxicity relations and quantity-toxicity relations. On the basis of the study,we found that DILI cases associated with BXP Preparations commonly occurred in patients with immune diseases,such as psoriasis and vitiligo,indicating specific individual differences. The results suggested that DILI cases associated with BXP Preparations would be correlated with the property of idiosyncratic drug-induced liver injury. In conclusion,the risk of liver injury clinically caused by BXP Preparations should be paid more attention,and the studies on the mechanism of idiosyncratic drug-induced liver injury must be enhanced,and those on risk factors,like irrational drug use,should be strengthened. Moreover,the evaluation of the risk-to-benefit ratio is supposed to be performed for the sake of improving the risk prevention and control standards for BXP preparations,and ensuring safe and rational clinical application of BXP Preparations.

[Standardization of names in prescriptions of traditional Chinese medicines].

Chinese medicine prescriptions are a type of medical documents written by doctors after they understand the patients' conditions for syndrome differentiation. Chinese medicine prescriptions are also the basis for pharmacy personnel to dispense medicines and guide patients to use drugs. It has the legal, technical and economic significances. Chinese medicine prescriptions contain such information of names, quantity and usage. Whether the names of drugs in Chinese medicine prescriptions are standardized or not is directly related to the safety and efficacy of the drugs. At present, nonstandard clinical prescriptions are frequently seen. With "Chinese medicine prescription", "names of drug in Chinese medicine prescription" and "standards of Chinese medicine prescription" as key words, the author searched CNKI, Wanfang and other databases, and consulted nearly 100 literatures, so as to summarize current names of drugs in traditional Chinese medicine prescription, analyze the reasons, and give suggestions, in the expectation of standardizing the names of drugs used in traditional Chinese medicine prescriptions.

Urine metabolomics study on the liver injury in rats induced by raw and processed Polygonum multiflorum integrated with pattern recognition and pathways analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonum multiflorum L. is a famous traditional Chinese medicine that has always been perceived to be safe. Recently, the increasing case reports on hepatotoxicity induced by Raw P. multiflorum (RP) have attracted particular attention. However, the diagnosis and identification of RP-induced hepatotoxicity are still very difficult for its unknown mechanism and the lack of specific biomarkers. AIM OF THE STUDY: To further explore the toxicity and metabolic mechanisms involved in the hepatotoxicity induced by RP. MATERIALS AND METHODS: The hepatotoxicity induced by RP and its processed products (PP) (dosed at 20g/kg for 4 weeks) on rats were investigated using conventional approaches including the biochemical analysis and histopathological observations. Further, a urinary metabolomic approach was developed to study the metabolic disturbances caused by RP and PP, followed by the pattern recognition approach and pathways analysis. RESULTS: RP showed obvious hepatotoxity whereas PP did not. 16 potential biomarkers (pyridoxamine, 4-pyridoxic acid, citrate et al.) differentially expressed in RP group were identified compared with the control and PP-treated groups. The pathways analysis showed that vitamin B6 metabolism, tryptophan metabolism and citrate cycle might be the major enriched pathways involved in the hepatotoxicity of the herb. CONCLUSION: 16 differentially expressed metabolites were identified to be involved in the RP-induced hepatotoxicity. Vitamin B6 metabolism might be mostly related to the hepatotoxicity induced by RP. This finding may provide a potential therapeutic target or option to treat hepatotoxicity induced by RP.

Assessment on the Efficacy and Safety of Aidi Injection Combined with Vinorelbine and Cisplatin for Treatment of Advanced Nonsmall Cell Lung Cancer.

BACKGROUND: The aim of this study was to assess the efficacy and safety of vinorelbine and cisplatin (NP chemotherapy) alone or in combination with Aidi injection for the treatment of advanced nonsmall cell lung cancer (NSCLC). METHODS: Pertinent publications were identified in PubMed, EMBASE, Cochrane Library, CNKI, CQVIP, and Wanfang databases, up to December 8, 2015. After quality assessment of all included randomized controlled trials evaluating Aidi injection combined with NP chemotherapy for the treatment of advanced NSCLC, a meta-analysis was performed by Review Manager 5.2 and STATA 12.0 for statistical analyses. RESULTS: Twelve studies including 509 and 503 cases in the experimental and control groups, respectively, were finally analyzed. The meta-analysis revealed that when cisplatin dose ranging from 20 to 40 mg/m 2 , combination of Aidi injection and NP chemotherapy was statistically different compared with NP chemotherapy alone in enhancing efficiency (relative risk [RR] = 1.24, 95% confidence interval [CI] [1.05-1.47], P = 0.010) and reducing the incidence of Grade II or above nausea and vomiting (RR = 0.49, 95% CI [0.30-0.80], P = 0.005). Meanwhile, with cisplatin ranging from 80 to 120 mg/m 2 , no significant differences in efficiency (RR = 1.11, 95% CI [0.87-1.42], P = 0.390) and Grade II or above nausea and vomiting (RR = 0.88, 95% CI [0.71-1.10], P = 0.260) were obtained. In addition, Aidi injection combined with NP chemotherapy was superior to NP chemotherapy alone in improving the quality of life, alleviating Grade II or above leukopenia and thrombocytopenia. CONCLUSIONS: Aidi injection combined with NP chemotherapy can enhance efficiency, improve the quality of life, and decrease adverse effects in patients with advanced NSCLC.

[Brief introduction of research methods of commodity specification and grade of Chinese medicinal materials].

The commodity specification and grade of Chinese medicinal materials is a measure of the quality of traditional Chinese medicines (TCMs), which directly impacts on the safety and effectiveness of clinical medicines. It is an urgent problem to establish a set of standards which can both interpret the scientific connotation of the commodity specification and grade of Chinese medicinal materials and play a significant role on clinical medicines as well as markets. This paper reviews the research methods of the commodity specification and grade of Chinese medicinal materials such as sensory evaluation, chemical assessment, biological evaluation, and cited the applications of various methods for the classification of TCMs. It provides technical support for establishing standards of the commodity specification and grade of Chinese medicinal materials, and also constructs scientific basis for clinical rational drug use.

[Effect of Nivalenol and selenium on IL-1beta and TNF-alpha secretion in cultured chondrocytes].

AIM: To investigate the effect of Nivalenol(NIV) and Selenium(Se) on the levels of IL-1beta and TNF-alpha in the cultured chondrocytes. METHODS: Human chondrocytes cultured in vitro were treated with or without NIV and Se. The morphology of chondrocytes was observed by optic microscope. The DNA content was determined by UV Spectrophotometry. The levels of IL-1beta and TNF-alpha in cultured medium were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Hematoxylin & eosin staining indicated there was cell necrosis in the cartilage reconstructed in vitro from both NIV group and NIV+Se group. Compared with the group of NIV toxin, the damage of chondrocytes was less severe when Se was added. NIV could inhibit chondrocyte DNA synthesis. The content of DNA with NIV was lowest than that in other groups. The levels of IL-1beta and TNF-alpha with NIV were significantly higher than control group (P<0.05). After Se was added, the levels did not change significantly compared with the groups without Se. CONCLUSION: NIV toxin could superinduce IL-1beta and TNF-alpha secretion in chondrocytes, which may be the key mechanism of chondrocyte injury by NIV. Se can partially alleviate the effects of NIV on chondrocytes cultured in vitro.

Effects of moniliformin and selenium on human articular cartilage metabolism and their potential relationships to the pathogenesis of Kashin-Beck disease.

OBJECTIVE: To investigate the effects of mycotoxin moniliformin (MON) on the metabolism of aggrecan and type II collagen in human chondrocytes in vitro and the relationship between MON and Kashin-Beck disease (KBD). METHODS: Human chondrocytes were isolated and cultured on bone matrix gelatin to form an artificial cartilage model in vitro with or without MON toxin. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of aggrecan and type II collagen in the cartilage was determined using immunocytochemical staining. RESULTS: MON toxin inhibited chondrocyte viability in dose-dependent and time-dependent manners. MON reduced aggrecan and type II collagen syntheses in the tissue-engineered cartilage. MON also increased the expression of matrix metalloproteinase-1 (MMP-1), MMP-13, BC4 epitopes, and CD44 in cartilages. However, the expression of 3B3(-) epitopes in cartilages was inhibited by MON. Selenium partially alleviated the damage of aggrecan induced by MON toxin. CONCLUSION: MON toxin promoted the catabolism of aggrecan and type II collagen in human chondrocytes.

[Construction of tissue-engineered cartilage by seeding chondrocytes on allogeneic cancellous bone matrix gelatin].

OBJECTIVE: To evaluate the use of cancellous bone matrix gelatin (BMG) combined with chondrocytes in constructing tissue-engineered cartilage by observing the growth, proliferation and differentiation of chondrocytes on allogeneic cancellous BMG. METHODS: The articular chondrocytes isolated from a 1-month-old rabbit were multiplied to a monolayer and seeded onto cancellous BMG to construct tissue-engineered cartilage in vitro during a period of 6 weeks. Samples were taken from the construct after 1, 2, 4, and 6 weeks of culture and evaluated by histology, immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The chondrocytes excreted matrix proteoglycan and collagen on cancellous BMG. With the prolongation of the culture time, the cells proliferated in the construct and the cells in the lacunae increased. Numerous chondrocytes were present the central region of the cancellous BMG and surrounded by extracellular matrix. By 6 weeks of culture, the BMG was covered with 15-20 layers of chondrocytes and cartilaginous tissue occurred in the pores throughout the cancellous BMG. Immunohistochemical staining showed rich and evenly distributed type II collagen around the chondrocytes, and TEM revealed an ultrastructure of the chondrocyte similar to that of native chondroctyes, with abundant extracellular matrix produced around the cells. CONCLUSION: Tissue-engineered cartilage can be constructed in vitro using allogeneic cancellous BMG combined with chondrocytes. Allogeneic cancellous BMG serves as a good scaffold material for tissue-engineered cartilage to promote the growth and proliferation of the seeded chondrocytes and allows maintenance of the differentiation phenotype of the cells.

[Establishment of bioassay method for antivirus potency of radix isatidis based on chemical fluorometric determination].

In the present study, the in vitro inhibitory activity of extracts from radix isatidis on neuraminidase (NA) was investigated by the chemical fluoremetic determination to establish the quality control method for antivirus action of radix isatidis. The initial study indicated that radix isatidis had obvious in vitro inhibitory activity on NA with IC50 = (0.90 +/- 0.20) mg (herb) x mL(-1). The correlation between logarithmic dose and reaction rate showed a "S" shape and a linear curve (linear equation, y = 8.7259 + 1.2169 x log(D), R = 0.9992) when the reaction rate was converted to probit-quite similar to Tamiflu's reaction curve, which hinted that radix isatidis had the same inhibitory function on NA as Tamiflu. According to the reaction type and the regularity of "parallel lines of qualitative effect", the experimental condition was optimized and a statistic method was confirmed based on the principle of bioassay statistic. Then the bioassay method for antivirus potency of radix isatidis based on fluorometric determination was established. The results of bio-potency assay showed the qualitative differences of radix isatidis samples sensitively and quantitatively. Meanwhile, this method has good reproducibility with RSD = 5.78% and reliability. The quality bioassay control method based on chemical fluorometric determination can reflect the pharmaco-dynamic features of Chinese medicine herb.

T-2 toxin-induced apoptosis involving Fas, p53, Bcl-xL, Bcl-2, Bax and caspase-3 signaling pathways in human chondrocytes.

OBJECTIVE: To investigate the effects of T-2 toxin on expressions of Fas, p53, Bcl-xL, Bcl-2, Bax and caspase-3 on human chondrocytes. METHODS: Human chondrocytes were treated with T-2 toxin (1-20 ng/ml) for 5 d. Fas, p53 and other apoptosis-related proteins such as Bax, Bcl-2, Bcl-xL, caspase-3 were determined by Western blot analysis and their mRNA expressions were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Increases in Fas, p53 and the pro-apoptotic factor Bax protein and mRNA expressions and a decrease of the anti-apoptotic factor Bcl-xL were observed in a dose-dependent manner after exposures to 1-20 ng/ml T-2 toxin, while the expression of the anti-apoptotic factor Bcl-2 was unchanged. Meanwhile, T-2 toxin could also up-regulate the expressions of both pro-caspase-3 and caspase-3 in a dose-dependent manner. CONCLUSION: These data suggest a possible underlying molecular mechanism for T-2 toxin to induce the apoptosis signaling pathway in human chondrocytes by regulation of apoptosis-related proteins.

[Effects of 3 genosides on bio-thermodynamic expression of splenic lymphocyte in mice].

OBJECTIVE: To establish a new method for screening active ingredients of Chinese herbs by determining different bio-thermodynamic effects of 3 genosides on splenic lymphocyte of mice. METHODS: Using a thermal bioactivity monitoring system, the maximum heat output (mHO), average metabolic heat (MH) and constant of decrease rate (DR) of lymphocyte were determined based on the growth metabolic power-time curve, and the outcomes were verified by MIT. RESULTS: The mHO and MH increased and the DR decreased after lymphocytes being exposed to the 3 genosides in different concentrations, arranged upon their potency as genoside Rg3 > genoside Rg2 > genoside Rg1 (merely insignificant effect). MTT showed the same results. CONCLUSION: Heat activity monitoring system could precisely display the different bio-thermal dynamic effects of 3 genosides on splenic lymphocyte.

Promotion of the articular cartilage proteoglycan degradation by T-2 toxin and selenium protective effect.

OBJECTIVE: To identify the relationship between T-2 toxin and Kashin-Beck disease (KBD), the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in vitro. METHODS: Chondrocytes were isolated from human articular cartilage and cultured in vitro. Hyaluronic acid (HA), soluble CD44 (sCD44), IL-1beta and TNF-alpha levels in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). CD44 content in chondrocyte membrane was determined by flow cytometry (FCM). CD44, hyaluronic acid synthetase-2 (HAS-2) and aggrecanases mRNA levels in chondrocytes were determined using reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemical method was used to investigate expressions of BC-13, 3-B-3(-) and 2-B-6 epitopes in the cartilage reconstructed in vitro. RESULTS: T-2 toxin inhibited CD44, HAS-2, and aggrecan mRNA expressions, but promoted aggrecanase-2 mRNA expression. Meanwhile, CD44 expression was found to be the lowest in the chondrocytes cultured with T-2 toxin and the highest in control plus selenium group. In addition, ELISA results indicated that there were higher sCD44, IL-1beta and TNF-alpha levels in T-2 toxin group. Similarly, higher HA levels were also observed in T-2 toxin group using radioimmunoprecipitation assay (RIPA). Furthermore, using monoclonal antibodies BC-13, 3-B-3 and 2-B-6, strong positive immunostaining was found in the reconstructed cartilage cultured with T-2 toxin, whereas no positive staining or very weak staining was observed in the cartilage cultured without T-2 toxin. Selenium could partly inhibit the effects of T-2 toxin above. CONCLUSION: T-2 toxin could inhibit aggrecan synthesis, promote aggrecanases and pro-inflammatory cytokines production, and consequently induce aggrecan degradation in chondrocytes. These will perturb metabolism balance between aggrecan synthesis and degradation in cartilage, inducing aggrecan loss in the end, which may be the initiation of the cartilage degradation.

[Butenolide induces apoptosis of cultured chondrocytes: study of its mechanism].

OBJECTIVE: To observe cell apoptosis and Bcl-2 and Bax expression changes of chondrocytes induced by butenolide (BUT) and the inhibitory effect of selenium against BUT-induced chondrcyte apoptosis, to gain insights into the mechanism by which BUT induces chondrcyte apoptosis. METHODS: Cartilage tissue reestablished from human fetal articular chondrocytes in vitro were treated with BUT at the concentrations of 0.1, 1.0 and 5.0 microg/ml and with the protective factor selenium. TUNEL method was used to detect chondrocyte apoptosis, which was quantified by flow cytometry. Immunohitochemistry was performed to analyze the expression of Bcl-2 and Bax in the reestablished cartilage tissue. RESULTS: BUT exposure induced chondrocyte apoptosis, and the apoptosis rate increased with the concentration increment of BUT from 0 to 1.0 mg/ml, resulting also increased positive expression rate of Bcl-2 and Bax(P<0.05). The apoptosis rate of chondrocytes in BUT+ selenium group was significantly lower than that of BUT groups (P<0.05), as was the positivity rate of Bcl-2 and Bax expression (P<0.05). CONCLUSION: BUT induces chondrocyte apoptosis in positive relation with BUT concentration (from 0 to 1.0 mg/ml) and causes increased expressions of Bcl-2 and Bax. Selenium can inhibit the chondrocyte apoptosis induced by BUT.

Repairing articular cartilage defects with tissue-engineering cartilage in rabbits.

OBJECTIVE: To investigate the effect of cancellous bone matrix gelatin (BMG) engineered with allogeneic chondrocytes in repairing articular cartilage defects in rabbits. METHODS: Chondrocytes were seeded onto three-dimensional cancellous BMG and cultured in vitro for 12 days to prepare BMG-chondrocyte complexes. Under anesthesia with 2.5% pentobarbital sodium (1 ml/kg body weight), articular cartilage defects were made on the right knee joints of 38 healthy New Zealand white rabbits (regardless of sex, aged 4-5 months and weighing 2.5-3 kg) and the defects were then treated with 2.5% trypsin. Then BMG-chondrocyte complex (Group A, n=18), BMG (Group B, n=10), and nothing (Group C, n=10) were implanted into the cartilage defects, respectively. The repairing effects were assessed by macroscopic, histologic, transmission electron microscopic (TEM) observation, immunohistochemical examination and in situ hybridization detection, respectively, at 2, 4, 8, 12 and 24 weeks after operation. RESULTS: Cancellous BMG was degraded within 8 weeks after operation. In Group A, lymphocyte infiltration was observed around the graft. At 24 weeks after operation, the cartilage defects were repaired by cartilage tissues and the articular cartilage and subchondral bone were soundly healed. Proteoglycan and type II collagen were detected in the matrix of the repaired tissues by Safranin-O staining and immunohistochemical staining, respectively. In situ hybridization proved gene expression of type II collagen in the cytoplasm of chondrocytes in the repaired tissues. TEM observation showed that chondrocytes and cartilage matrix in repaired tissues were almost same as those in the normal articular cartilage. In Group B, the defects were repaired by cartilage-fibrous tissues. In Group C, the defects were repaired only by fibrous tissues. CONCLUSIONS: Cancellous BMG can be regarded as the natural cell scaffolds for cartilage tissue engineering. Articular cartilage defects can be repaired by cancellous BMG engineered with allogeneic chondrocytes. The nature of repaired tissues is closest to the normal cartilage. Local administration of trypsin can promote the adherence of repaired tissues to host tissues. Transplantation of allogeneic chondrocytes has immunogenicity, but the immune reaction is weak.

[Toxic effect of butenolide on chondrocyte differentiation and the protective effect of selenium].

OBJECTIVE: To study the effect of butenolide (BUT) on cultured chondrocytes differentiation and the possible protective effects of selenium (Se). METHODS: Ex-vivo cultured chondrocytes were divided into six groups: (1) Control group (without BUT and Se); (2) Se 0.1 microg/ml control group; (3) BUT 0.1 microg/ml group; (4) BUT 1.0 microg/ml group; (5) BUT 5.0 microg/ml group; and (6) BUT 1.0 microg/ml + Se 0.1 microg/ml group. The expression of collagen II (Col II), collagen X (ColX), basic fibroblast growth factor (bFGF), and parathyroid hormone-related peptide (PTHrP) in (or around) chondrocytes in all groups were analyzed by immunohistochemistry. RESULTS: The expressions of Col II in 1.0 microg/ml BUT group and 5.0 microg/ml BUT group were significantly lower than those in the control group (P < 0.05). The expression of Col II in 1.0 microg/ml BUT + Se group were significantly higher than those in the 1.0 microg/ml BUT group and 5.0 microg/ml BUT group (P < 0.05). The expressions of bFGF and PTHrP of BUT groups were significantly higher than those in the Se and control groups (P < 0.05). No expression of ColX was observed in all groups. CONCLUSION: BUT can affect the collagen II synthesis of the chondrocytes. Selenium supplementation may play a protective role.