Effects of T-2 toxin and selenium on expression of aggrecanase in human chondrocyte / 中国地方病学杂志

Objective To determine effects of T-2 toxin and selenium on expression of aggrecanase in human chondrocyte.Methods Human chondrocytes were treated with T-2 toxin(0,1,10,20 μg/L),and/or sodium selenite(final concentration of selenium 0,0.1 mg/L) for 5 days.Aggrecan expression was determined by Western blotting,aggrecanase-1 and aggrecanase-2 mRNA levels were measured by RT-PCR.ResultsSelenium and T-2 toxin had effects on both aggrecan protein levels and its aggrecanases(include aggrecanase-1 and aggrecanase-2 ) mRNA levels(F =0.294,27.71 for aggrecan,F =107.45,362.25 for aggrecanase-l,F =34.68,22.26 for aggrecanase-2,respectively,all P ＜ 0.05),and there was interaction between selenium and T-2 toxin on aggrecan,aggrecanase-1 and aggrecanase-2 expression(F =79.99,230.76,388.33,all P ＜ 0.05).Furthermore,selenium presented significant antagonism to T-2 toxin on aggrecan,aggrecanase-1 and aggrecanase-2 expression.Aggrecan expression levels(0.278 ± 0.015,0.235 ± 0.029,0.195 ± 0.028,0.399 ± 0.028,0.299 ± 0.020,0.263 ±0.019) induced by both 1,10,20 μg/L T-2 toxin and 0,0.1 mg/L selenium were significantly decreased than the levels(0.472 ± 0.0358,0.197 ± 0.018,all P ＜ 0.05) in control group(0 mg/L toxin).Selenium partially blocked the effects induced by 1,10,and 20 μg/L T-2 toxin(all P＜ 0.05).One,10,20 μg/L T-2 toxin and 0,0.1 mg/L selenium increased both aggrecanase-1 mRNA levels(0.535 ± 0.033,1.071 ± 0.043,1.454 ± 0.058,1.057 ±0.048,0.555 ± 0.036,0.902 ± 0.045) and aggrecanase-2 mRNA levels(0.596 ± 0.038,0.656 ± 0.033,0.949 ±0.049,0.600 ± 0.040,0.453 ± 0.031,0.164 ± 0.011),when compared with control(0.481 ± 0.023,0.346 ±0.020 for aggrecanase-1 and 0.387 ± 0.020,1.021 ± 0.046 for aggrecanase-2,respectively,all P ＜ 0.05).Selenium partially blocked 10,20 μg/L T-2 toxins induced upregulation of aggrecanase-1 (all P ＜ 0.05) and aggrecanase-2 (all P ＜ 0.05 ).Conclusions These data suggest a possible molecular mechanism that T-2 toxin could induce cartilage matrix degradation through the upregulation of aggrecanases expression and enzyme activities.Trace element selenium has some protective effect on cartilage proteoglycan degradation induced by T-2 toxins.

Effects of carbon monoxide inhalation on severe limb ischemia/reperfusion injury and its damages lead to shock / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To study the protective effect of carbon monoxide (CO) inhalation on the serious limb ischemia/reperfusion (I/R) injury, and which effects caused to shock in rats.</p><p><b>METHODS</b>36 SD rats were randomly divided into I/R, I/R + CO (RC), sham operation (S) groups. I/R injury models were made by the occlusion of the femoral artery for 8 h and the reperfusion for 12 h, 10 d. Before reperfusion of 2 h, RC group started to breathe medical air containing CO (the volume fraction of CO: 0.075%) continuously, until after reperfusion for 4 h, a total of inhalation 6 h. S, I/R groups exposed to air, breathe freely. Caudal artery pressures (CAP), ten days survival rate, serum lactate dehydrogenase (LDH) and creatine kinase (CK) activity, limb wet - to - dry weight ratio (W/D) and the pathologic changes of limb were observed.</p><p><b>RESULTS</b>Once the reperfusion started, the CAP decreased rapidly in I/R group, and the mean reduced to(5.3259 +/- 0.3832) kPa when reperfusion for 8 h. Compared to I/R group, the CAP decreased slower and smaller in RC group, moreover, its mean reduced to (8.3300 +/- 0.4224) kPa when reperfusion for 8 h. The 10 d survival rate in I/R group was that 8 rats died all between reperfusion for 13 - 20 h. Only 1 rat died in RC group and the other 7 rats were still alive when reperfusion for 10 d. Compared to I/R group, the pathological features of the ischemic limb were significant ly improved, and the figures of W/D, serum LDH and CK value were remarkable lower in RC group (P < 0.05).</p><p><b>CONCLUSION</b>Inhaling exogenous low-dose CO has a reverse regulation in the blood pressure decline caused by serious limb I/R injury in rats. And at the same time, it can effectively prevent the occurrence of shock, reduce physical damage, significantly increase the survival rate of animals.</p>

Carbon monoxide inhalation protects against limb ischemia/reperfusion injury / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To study the protective effect of carbon monoxide(CO) inhalation in the limb ischemia/reperfusion (I/R) injury of rats.</p><p><b>METHODS</b>Forty-four Sprague-Dawley rats were randomly divided into three groups: S, I/R and RC groups. I/R injury model was made by the occlusion of the femoral artery for four hours and the reperfusion for forty-eight hours. RC group was exposed to medical air mixed CO (the volume fraction of CO: 0.05%) during limb reperfusion in rats. The other two groups were exposed to the routine air. Gross morphology of the ischemic limb, wet-to-dry weight ratio (W/D), and skeletal muscle histopathology were observed. The apoptosis index and expression levels of Bax and Bcl-2 in the muscle were assessed with Flow Cytometry. The activities of serum lactate dehydrogenase (LDH) and creatine kinase (CK) were tested by Automatic Biochemical Analyzer.</p><p><b>RESULTS</b>Compared to I/R group, W/D, serum LDH and CK activities, the apoptosis index and Bax expression level in the muscle were all significantly decreased, the Bcl-2 expression level was significantly increased, gross morphology of the ischemic limb and muscle histopathology were obviously improved in RC group.</p><p><b>CONCLUSION</b>Inhaling exogenous CO can attenuate limb I/R injury.</p>

Exogenous carbon monoxide protects against liver injury induced by ischemia/reperfusion of hind limbs in rats / 中国应用生理学杂志

<p><b>AIM</b>To investigate the protective effect of exogenous carbon monoxide (CO) on the liver injury induced by ischemia/reperfusion (I/R) of hind limbs in rats.</p><p><b>METHODS</b>100 SD rats were divided randomly into sham operated group (S), S+ CO group (SC), I/R group (I/R), I/ R+ CO group (RC). A rat model of ischemia in hind limbs and the reperfusion liver injury was established with the occlusion of the femoral arteries for 4 h and re-opening for 6 - 72 h, 10 d. The rats in SC and RC groups were exposed to air containing CO (the volume traction of CO: 0.05%) for 2 h before and after reperfusion or the corresponding control time point, while the other two groups were exposed to the routine air. The pathologic changes of liver tissue were morphologically observed by HE stain. Serum GPT activity was tested by Automatic Biochemical Analyzer. The percentage of apoptosis, expression levels of bax and bcl-2 protein in liver tissue were detected by Flow Cytometry.</p><p><b>RESULTS</b>There was no difference between S and SC groups. Compared with SC group: (1) Pathological changes in liver tissue were significant in I/R and RC groups. (2) The serum GPT activity of I/R and RC groups was obviously increased. (3) In IR and RC groups, the percentage of apoptosis in liver tissue was all significantly increased. (4) The bax expression level was significantly increased. Compared RC group with I/R group: (1) Pathological change was slight. (2) The serum GPT activity was depressed. (3) The percentage of apoptosis and expression level of bax protein in liver tissue were depressed. (4) The expression level of bcl-2 protein in liver tissue was increased.</p><p><b>CONCLUSION</b>Exogenous CO could attenuate liver tissue injury induced by limbs I/R in rats.</p>

Promotion of the articular cartilage proteoglycan degradation by T-2 toxin and selenium protective effect / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Butenolide induces apoptosis of cultured chondrocytes: study of its mechanism / 南方医科大学学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Protective effect of selenium against T-2 toxin-induced inhibition of chondrocyte aggrecan and collagen II synthesis / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the inhibitory effect of T-2 toxin on the expression of aggrecan and collagen II in chondrocytes and the protection of selenium against this effect.</p><p><b>METHODS</b>Human chondrocytes cultured in vitro were treated with T-2 toxin at different concentrations for varied time periods (1-5 days), and the cell viability was measured by MTT assay. Aggrecan expression was detected by toluidine blue staining and collagen II expression by immunostaining using monoclonal antibody of collagen. Aggrecan and collagen II mRNA expressions were measured by semiquantitative RT-PCR.</p><p><b>RESULTS</b>T-2 toxin dose- and time-dependently affected chondrocyte viability within the concentration range of 0.001-2 mg/L, the prolonged treatment time further enhanced the dose dependence of the inhibitory effect. T-2 toxin lowered aggrecan and collagen II synthesis in the chondrocytes and reduced their mRNA expressions. Selenium could partly attenuate the inhibitory effects of T-2 toxin on aggrecan mRNA expression, but showed no such effect against T-2-induced collagen II expression.</p><p><b>CONCLUSION</b>T-2 toxin can obviously inhibit aggrecan and collagen II synthesis in human chondrocytes, and selenium can partly antagonize the inhibitory effects of T-2 toxin on aggrecan.</p>

Toxic effect of butenolide on chondrocyte differentiation and the protective effect of selenium / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the effect of butenolide (BUT) on cultured chondrocytes differentiation and the possible protective effects of selenium (Se).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>BUT can affect the collagen II synthesis of the chondrocytes. Selenium supplementation may play a protective role.</p>

Changes of iNOS genes expression in liver following ischemia and reperfusion of limbs and their significance in rats / 中国应用生理学杂志

<p><b>AIM</b>To detect the changes of inducible nitric oxide synthase (iNOS) expression in liver following ischemia/reperfusion (I/R) of hindlimbs and to elucidate their significance.</p><p><b>METHODS</b>I/R was established using the occlusion of the femoral arteries for 4 h and reopening for 2-24 h in rats. The expression of iNOS mRNA, and iNOS protein and the nitrotyrosine (NT), a marker of peroxynitrite (ONOO-), in liver tissue were detected with reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical technique, respectively. The liver superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents were spectrophotometrically measured. The observation of pathologic changes of liver was made following the inhibition of iNOS by aminoguanidine (AG).</p><p><b>RESULTS</b>Compared with control groups, the relative expression level of iNOS mRNA significantly increased in I/R group. There were more iNOS positive hepatocytes and more NT positive hepatocytes in I/R group than control groups. The contents of MDA markedly increased, while the activity of SOD significantly decreased in I/R group, compared with those in the control groups. The pathologic changes of rat liver became milder in I/R group following the inhibition of iNOS by AG.</p><p><b>CONCLUSION</b>The expressions of iNOS mRNA and protein in liver are significantly upregulated, excess induction of iNOS-NO is contributed to the liver injury during the I/R of hindlimbs.</p>

Changes of HO-1 genes expression in liver following ischemia/reperfusion of limbs and their significance in rats / 中国应用生理学杂志

<p><b>AIM</b>To detect the changes of inducible heme oxygenase (HO-1) expression in liver following ischemia/reperfusion (I/R) of hindlimbs and to elucidate their significance.</p><p><b>METHODS</b>I/R was established using the occlusion of the femoral arteries for 4h and reopening for 2-24 h in rats. The expression of HO-1 mRNA and HO-1 protein in liver tissue were detected with reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical technique, respectively. The observation of pathologic changes of liver was made following the inhibition of HO-1 by zinc protoporphyrin (ZnPP).</p><p><b>RESULTS</b>Compared with control groups, the relative expression level of HO-1 mRNA significantly increased in I/R group. There were more HO-1 positive hepatocytes in I/R group than control groups. The pathologic changes of liver tissue became more severe in I/R + ZnPP group.</p><p><b>CONCLUSION</b>The expressions of HO-1 mRNA and protein in liver tissue are significantly upregulated, induction of HO-1 is involved in protection for hepatocytes during the I/R of hindlimbs.</p>

Endogenous carbon monoxide attenuates lung injury following ischemia-reperfusion in the hind limbs of rats / 生理学报

To investigate the role of endogenous heme oxygenase (HO)/carbon monoxide (CO) system in the lung injury as assessed by lung histology, polymorphonuclear count, malondialdehyde content and wet-to-dry weight ratio following ischemia-reperfusion (I/R) of hind limbs, zinc protoporphyrin (ZnPP), an inhibitor of HO activity, was used, and the lung HO activity and blood carboxyhemoglobin (COHb) level were measured. The results showed that HO activity and COHb level were increased significantly and lung injury occurred after limb I/R. After administration of ZnPP, the lung injury was further aggravated while the HO activity and COHb level were significantly decreased. These findings suggest that upregulation of HO activity followed by subsequent CO production attenuates the lung injury induced by limb I/R in rats.