目的探讨不同病理类型宫颈组织中TNF-α和TNFR1的表达及其与宫颈鳞癌之间的关系。方法应用实时定量RT-PCR法检测正常宫颈(正常组,48例)、宫颈上皮内瘤样病变(CIN组,47例)和宫颈鳞癌(SCC组,39例)组织中TNF-α和TNFR1、TNFR2 m RNA表达水...目的探讨不同病理类型宫颈组织中TNF-α和TNFR1的表达及其与宫颈鳞癌之间的关系。方法应用实时定量RT-PCR法检测正常宫颈(正常组,48例)、宫颈上皮内瘤样病变(CIN组,47例)和宫颈鳞癌(SCC组,39例)组织中TNF-α和TNFR1、TNFR2 m RNA表达水平,采用免疫组织化学SP法检测TNF-α蛋白表达,Western blot法检测TNFR1、TNFR2蛋白表达,将TNF-α和TNFR1 m RNA表达结果与临床病理结果进行相关性分析。结果 TNF-α和TNFR1的m RNA表达水平随宫颈癌变程度的增加而升高,组间两两比较差异均有统计学意义(P<0.05),而TNFR2在各组中的表达差异无统计学意义(P>0.05);TNF-α在宫颈组织中的表达随病理程度的增加有显著升高,正常组为6.25%、CIN组为58.82%、SCC组为71.79%,组间比较差异有统计学意义(P<0.01);TNFR1蛋白表达水平随宫颈癌变程度的增加而升高,差异均有统计学意义(P<0.05),TNFR2在各组中的表达变化不明显,均与其m RNA在各组间的表达结果一致;SCC组中的TNF-α和TNFR1 m RNA的表达量与肿瘤大小、临床分期、浸润深度以及淋巴结转移情况呈正相关性,差异均有统计学意义(P<0.05),与患者年龄以及细胞分化程度无关。结论 TNF-α和TNFR1的激活与宫颈鳞癌的发生、发展相关,参与肿瘤微环境的变化,两者将有望成为治疗宫颈鳞癌的靶标。展开更多
Quinolones (QNs) are widely used for their broad antibacterial spectrum and good antibacterial activities,desirable pharmacokinetic characteristics and few cross reactions with other therapeutic agents. However,QNs ha...Quinolones (QNs) are widely used for their broad antibacterial spectrum and good antibacterial activities,desirable pharmacokinetic characteristics and few cross reactions with other therapeutic agents. However,QNs have adverse effects including chondrotoxicity in juvenile animals,so the use of QNs is contraindicated in children and adolescents. In regarding to the chondrotoxicity of QNs,numerous studies have been done. The current hypothesis suggests that QNs compete with the β1 integrin receptors residing on chondrocyte surface for extracellular Mg ions,which leads to alternation in β1 integrin expression,or function and eventually results in chondrocyte death. Stupack et al (2001)demonstrated that caspase-8 could be recruited to unligated integrins in adherent cells and further initiate apoptosis in a death receptor-independent manner. Sheng et al (2008) found that ofloxacin induced rabbit's chondrocyte apoptosis by causing disturbance of β1 integrin functions and subsequently through caspase-8-dependent mitochondrial pathway.Apoptosis could be initiated through the stimulation of death receptors and through an intrinsic pathway from mitochondria. Santangelo and Bertone (2011) and Wu et al (2011) found that TNFα could be expressed in human primary condrocytes inducing by interleukin-1beta (IL-1) or lipopolysaccharide (LPS). However,to date there have not been sufficient results to support that signaling from the death receptors was involved in QNs-induced chondrocyte apoptosis.In addition,dilated cisternae of rough endoplasmic reticulum (ER) have been noticed in QNs-induced arthropathy.ER can participate in the initiation of apoptosis. Varieties of harmful cellular stimuli could lead to ERs (ER stress). Elevated ERs results in cellular apoptosis. But whether ERs mediates apoptosis in QNs-treated chondrocytes is not clear yet.In this study,we chose two QNs agents and chondrocytes were treated with ofloxacin and marbofloxacin at final concentrations of 20 μg / mL,50 μg / mL and 100 μg / mL respectively in vitro for 2 h,8 h and 24 h. Cell survival rate,cell apoptosis rate and death receptor pathway factors TNFα (intracellular tumor necrosis factor-alpha),TNFR1 (TNF receptor-1),TRADD (TNF receptor 1 associated via death domain),FADD (Fas-associated protein with death domain),caspase-8 and ERs mediated apoptosis factors caspase-12,GADD153 (CHOP or DDIT3),GRP78 (Bip),calpain,and anti-apoptosis factors Bcl-2 (B-cell lymphoma 2),NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) gene expression levels were measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) analysis to determine the dose-response relationship. We further silenced the expression of TNFR1 successfully by transferring TNFR1-siRNA to chondrocytes to confirm whether TNFα / TNFR1 signaling pathways are involved ofloxacin and marbofloxacin-induced apoptosis. Furthermore,expression of death receptor pathway representative proteins TNFα / TNFR1 and endocytoplasmic reticulum (ER) pathway representative protein caspase-12 were confirmed using Western Blot.We have found that ofloxacin and marbofloxacin could induce apoptosis of chondrocytes in a time-and dose-dependent fashion within 24 h. mRNA of TNFα,TNFR1,TRADD,FADD and caspase-8 (caspase-8 of ofloxacin treated group were at 24 h) were highly expressed at 8 h,and GADD153,GRP78,calpain and caspase-12 at 8 h or 24 h,and antiapoptosis factors NF-κB and Bcl-2 were also raised after 2 h,all in a dose-dependent fashion. Expression of caspase-8was downregulated after silenced TNFR1. TNFα and TNFR1 proteins were expressed at 8 h and caspase-12 proteins were expressed at 24 h. In addition,ofloxacin showed a higher toxicity.Our results indicate that death receptor pathway TNF / TNFR1 and ERs mediated apoptosis factors are involved in ofloxacin and marbofloxacin-induced apoptosis of in vitro cultured juvenile dog joint chondrocytes within 24 h.展开更多
OBJECTIVE: To examine whether electroacupuncture(EA) treatment inhibited cell apoptosis of intervertebral annulus fibrosis(AF) via tumor necrosis factor-α(TNF-α)-tumor necrosis factor receptor 1(TNFR1)-caspase-8 and...OBJECTIVE: To examine whether electroacupuncture(EA) treatment inhibited cell apoptosis of intervertebral annulus fibrosis(AF) via tumor necrosis factor-α(TNF-α)-tumor necrosis factor receptor 1(TNFR1)-caspase-8 and integrin β1/Akt signaling pathways in a rat model of cervical intervertebral disc degeneration caused by unbalanced dynamic and static forces.METHODS: Thirty-two Sprague-Dawley rats were included in this study, of which 24 rats underwent surgery to induce cervical intervertebral disc degeneration, while eight rats received EA treatment at Dazhui(GV 14). Immunohistochemical staining was used to detect TNF-α, TNFR1, and caspase-8Apoptosis of AF cells was examined with terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL) staining. The m RNA and protein expression levels of integrin β1 andAkt were evaluated with real-time polymerase chain reaction and western blot analysis, respectively.RESULTS: Treatment with EA decreased TUNEL-positive AF cells and lowered TNF-α, TNFR1 and caspase-8 positive cells compared with control groups. EA treatment also increased integrin β1and Akt m RNA and protein levels compared with controls.CONCLUSION: Treatment with EA inhibits AF cell apoptosis through suppression of the TNF-α-TNFR1-caspase-8 signal pathway and increases the expression of integrin β1 and Akt. EA may be a good alternative therapy for treating cervical spondylosis.展开更多
Aim： To evaluate the immunohistopathological changes in the contralateral testis of rats after an experimental spermatic cord torsion. Methods： Male Sprague-Dawley rats of 45-50 days old were subjected to a 720°...Aim： To evaluate the immunohistopathological changes in the contralateral testis of rats after an experimental spermatic cord torsion. Methods： Male Sprague-Dawley rats of 45-50 days old were subjected to a 720° unilateral spermatic cord torsion for 10, 30 and 80 days （experimental group, E）, respectively or sham operation （control group, C）. Histopathology of the contralateral testis as well as germ cell apoptosis were studied using the Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling （TUNEL） technique. The number of testicular lymphocytes, mast cells and macrophages, and the expression of tumor necrosis factor-α （TNF-α） and its receptor （TNFR1） in testicular cells of the contralateral testis were quantified by histochemistry and immunohistochemistry. TNF-α concentration in testicular fluid was determined by ELISA. Results： In the contralateral testis of rats from the E group, the maximal degree of damage of the germinal epithelium was seen 30 days after torsion. At this time we observed in the E group vs. the C group increases： （i） the number of testicular T-lymphocytes; （ii） the number of testicular mast cells and macrophages; （iii） the percentage of macrophages expressing TNF-α; （iv） TNF-α concentration in testicular fluid; （v） the number of apoptotic germ cells; and （vi） the number of TNFR1^＋ germ cells. Conclusion： Experimental spermatic cord torsion induces, in the contralateral testis, a focal damage of seminiferous tubules characterized by apoptosis and sloughing of germ cells. Results suggest humoral and cellular immune mediated testicular cell damage in which macrophages and mast cells seem to be involved in the induction of germ cell apoptosis through the TNF-α/TNFR1 system and in the modulation of the inflammatory process.展开更多
文摘目的探讨不同病理类型宫颈组织中TNF-α和TNFR1的表达及其与宫颈鳞癌之间的关系。方法应用实时定量RT-PCR法检测正常宫颈(正常组,48例)、宫颈上皮内瘤样病变(CIN组,47例)和宫颈鳞癌(SCC组,39例)组织中TNF-α和TNFR1、TNFR2 m RNA表达水平,采用免疫组织化学SP法检测TNF-α蛋白表达,Western blot法检测TNFR1、TNFR2蛋白表达,将TNF-α和TNFR1 m RNA表达结果与临床病理结果进行相关性分析。结果 TNF-α和TNFR1的m RNA表达水平随宫颈癌变程度的增加而升高,组间两两比较差异均有统计学意义(P<0.05),而TNFR2在各组中的表达差异无统计学意义(P>0.05);TNF-α在宫颈组织中的表达随病理程度的增加有显著升高,正常组为6.25%、CIN组为58.82%、SCC组为71.79%,组间比较差异有统计学意义(P<0.01);TNFR1蛋白表达水平随宫颈癌变程度的增加而升高,差异均有统计学意义(P<0.05),TNFR2在各组中的表达变化不明显,均与其m RNA在各组间的表达结果一致;SCC组中的TNF-α和TNFR1 m RNA的表达量与肿瘤大小、临床分期、浸润深度以及淋巴结转移情况呈正相关性,差异均有统计学意义(P<0.05),与患者年龄以及细胞分化程度无关。结论 TNF-α和TNFR1的激活与宫颈鳞癌的发生、发展相关,参与肿瘤微环境的变化,两者将有望成为治疗宫颈鳞癌的靶标。
文摘Quinolones (QNs) are widely used for their broad antibacterial spectrum and good antibacterial activities,desirable pharmacokinetic characteristics and few cross reactions with other therapeutic agents. However,QNs have adverse effects including chondrotoxicity in juvenile animals,so the use of QNs is contraindicated in children and adolescents. In regarding to the chondrotoxicity of QNs,numerous studies have been done. The current hypothesis suggests that QNs compete with the β1 integrin receptors residing on chondrocyte surface for extracellular Mg ions,which leads to alternation in β1 integrin expression,or function and eventually results in chondrocyte death. Stupack et al (2001)demonstrated that caspase-8 could be recruited to unligated integrins in adherent cells and further initiate apoptosis in a death receptor-independent manner. Sheng et al (2008) found that ofloxacin induced rabbit's chondrocyte apoptosis by causing disturbance of β1 integrin functions and subsequently through caspase-8-dependent mitochondrial pathway.Apoptosis could be initiated through the stimulation of death receptors and through an intrinsic pathway from mitochondria. Santangelo and Bertone (2011) and Wu et al (2011) found that TNFα could be expressed in human primary condrocytes inducing by interleukin-1beta (IL-1) or lipopolysaccharide (LPS). However,to date there have not been sufficient results to support that signaling from the death receptors was involved in QNs-induced chondrocyte apoptosis.In addition,dilated cisternae of rough endoplasmic reticulum (ER) have been noticed in QNs-induced arthropathy.ER can participate in the initiation of apoptosis. Varieties of harmful cellular stimuli could lead to ERs (ER stress). Elevated ERs results in cellular apoptosis. But whether ERs mediates apoptosis in QNs-treated chondrocytes is not clear yet.In this study,we chose two QNs agents and chondrocytes were treated with ofloxacin and marbofloxacin at final concentrations of 20 μg / mL,50 μg / mL and 100 μg / mL respectively in vitro for 2 h,8 h and 24 h. Cell survival rate,cell apoptosis rate and death receptor pathway factors TNFα (intracellular tumor necrosis factor-alpha),TNFR1 (TNF receptor-1),TRADD (TNF receptor 1 associated via death domain),FADD (Fas-associated protein with death domain),caspase-8 and ERs mediated apoptosis factors caspase-12,GADD153 (CHOP or DDIT3),GRP78 (Bip),calpain,and anti-apoptosis factors Bcl-2 (B-cell lymphoma 2),NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) gene expression levels were measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) analysis to determine the dose-response relationship. We further silenced the expression of TNFR1 successfully by transferring TNFR1-siRNA to chondrocytes to confirm whether TNFα / TNFR1 signaling pathways are involved ofloxacin and marbofloxacin-induced apoptosis. Furthermore,expression of death receptor pathway representative proteins TNFα / TNFR1 and endocytoplasmic reticulum (ER) pathway representative protein caspase-12 were confirmed using Western Blot.We have found that ofloxacin and marbofloxacin could induce apoptosis of chondrocytes in a time-and dose-dependent fashion within 24 h. mRNA of TNFα,TNFR1,TRADD,FADD and caspase-8 (caspase-8 of ofloxacin treated group were at 24 h) were highly expressed at 8 h,and GADD153,GRP78,calpain and caspase-12 at 8 h or 24 h,and antiapoptosis factors NF-κB and Bcl-2 were also raised after 2 h,all in a dose-dependent fashion. Expression of caspase-8was downregulated after silenced TNFR1. TNFα and TNFR1 proteins were expressed at 8 h and caspase-12 proteins were expressed at 24 h. In addition,ofloxacin showed a higher toxicity.Our results indicate that death receptor pathway TNF / TNFR1 and ERs mediated apoptosis factors are involved in ofloxacin and marbofloxacin-induced apoptosis of in vitro cultured juvenile dog joint chondrocytes within 24 h.
基金the National Natural Science Foundation of China(No.81273836,Based on Ineegrin/FAK signaling pathways research the effects of electroacupuncture on the contents of apoptosisNo.81001554,Based on Wnt/β-catenin signaling pathways research the effects of electroacupuncture on the annulus fibrosis cells)
文摘OBJECTIVE: To examine whether electroacupuncture(EA) treatment inhibited cell apoptosis of intervertebral annulus fibrosis(AF) via tumor necrosis factor-α(TNF-α)-tumor necrosis factor receptor 1(TNFR1)-caspase-8 and integrin β1/Akt signaling pathways in a rat model of cervical intervertebral disc degeneration caused by unbalanced dynamic and static forces.METHODS: Thirty-two Sprague-Dawley rats were included in this study, of which 24 rats underwent surgery to induce cervical intervertebral disc degeneration, while eight rats received EA treatment at Dazhui(GV 14). Immunohistochemical staining was used to detect TNF-α, TNFR1, and caspase-8Apoptosis of AF cells was examined with terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL) staining. The m RNA and protein expression levels of integrin β1 andAkt were evaluated with real-time polymerase chain reaction and western blot analysis, respectively.RESULTS: Treatment with EA decreased TUNEL-positive AF cells and lowered TNF-α, TNFR1 and caspase-8 positive cells compared with control groups. EA treatment also increased integrin β1and Akt m RNA and protein levels compared with controls.CONCLUSION: Treatment with EA inhibits AF cell apoptosis through suppression of the TNF-α-TNFR1-caspase-8 signal pathway and increases the expression of integrin β1 and Akt. EA may be a good alternative therapy for treating cervical spondylosis.
基金Project supported by the National Natural Science Foundation of China （No. 81171651） and the Science and Technology Breakthrough Project of the Science and Technology Department of Sichuan Province （No. 2010FZ0044）, China
文摘Aim： To evaluate the immunohistopathological changes in the contralateral testis of rats after an experimental spermatic cord torsion. Methods： Male Sprague-Dawley rats of 45-50 days old were subjected to a 720° unilateral spermatic cord torsion for 10, 30 and 80 days （experimental group, E）, respectively or sham operation （control group, C）. Histopathology of the contralateral testis as well as germ cell apoptosis were studied using the Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling （TUNEL） technique. The number of testicular lymphocytes, mast cells and macrophages, and the expression of tumor necrosis factor-α （TNF-α） and its receptor （TNFR1） in testicular cells of the contralateral testis were quantified by histochemistry and immunohistochemistry. TNF-α concentration in testicular fluid was determined by ELISA. Results： In the contralateral testis of rats from the E group, the maximal degree of damage of the germinal epithelium was seen 30 days after torsion. At this time we observed in the E group vs. the C group increases： （i） the number of testicular T-lymphocytes; （ii） the number of testicular mast cells and macrophages; （iii） the percentage of macrophages expressing TNF-α; （iv） TNF-α concentration in testicular fluid; （v） the number of apoptotic germ cells; and （vi） the number of TNFR1^＋ germ cells. Conclusion： Experimental spermatic cord torsion induces, in the contralateral testis, a focal damage of seminiferous tubules characterized by apoptosis and sloughing of germ cells. Results suggest humoral and cellular immune mediated testicular cell damage in which macrophages and mast cells seem to be involved in the induction of germ cell apoptosis through the TNF-α/TNFR1 system and in the modulation of the inflammatory process.
