Objectives： To investigate the mechanism of Liuwei Dihuang Pill （六味地黄丸, LDP） in treating postmenopausal osteoporosis （PMOP） with Shen （Kidney） yin deficiency. Methods： In this study, 205 cases of PMOP wer...Objectives： To investigate the mechanism of Liuwei Dihuang Pill （六味地黄丸, LDP） in treating postmenopausal osteoporosis （PMOP） with Shen （Kidney） yin deficiency. Methods： In this study, 205 cases of PMOP were divided into the PMOP Shen-yin deficiency group （Group A）, PMOP Shen-yang deficiency group （Group B）, PMOP without Shen deficiency group （Group C）, and control group （Group N）. Real-time polymerase chain reaction （RT-PCR） and Western blot techniques were used to observe the effects of LDP treatment on the cardiotrophin-like cytokine factor 1 （CLCF1）, ankyrin repeat and SOCS box containing 1 （ASB1）, and proldneticin 2 （PROK2） genes and the Janus kinase/signal transducer and activator of transcription （JAK/STAT） signaling pathway. Results： The mRNA （P〈0.05） and protein （P〈0.01） expression levels of the CLCF1 gone in Group A were significantly lower than the corresponding levels in Group N. After LDP treatment for 3 months, the mRNA expression levels of the CLCF1 gone were obviously up-regulated （P〈0.01）. After 6-month treatment, the expression levels of CLCF1 mRNA and protein were significantly up-regulated （both P〈0.01）, and the average bone density of the top femur had significantly increased （P〈0.05）. In vitro, CLCF1 overexpression resulted in a significant increase in the total protein and phosphorylated protein levels of JAK2 and STAT3. Conclusions： The CLCF1 gone is an important gone associated with PMOP Shen-yin deficiency and the therapeutic effects of LDP may be mediated by up-regulation of CLCF1 gone expression and activation of the JAK/STAT signaling pathway.展开更多
目的观察心脏营养素-1(CT-1)是否能促进经诱导分化剂5-氮杂胞苷(5-aza)诱导的骨髓间充质干细胞(BMMSCs)分化为心肌样细胞,并研究其相关机制。方法自成年大鼠骨髓中分离BMMSCs,分别以普通培养基(A组)、加入含CT-1的培养基(B组)培养、经5-...目的观察心脏营养素-1(CT-1)是否能促进经诱导分化剂5-氮杂胞苷(5-aza)诱导的骨髓间充质干细胞(BMMSCs)分化为心肌样细胞,并研究其相关机制。方法自成年大鼠骨髓中分离BMMSCs,分别以普通培养基(A组)、加入含CT-1的培养基(B组)培养、经5-aza诱导后加入普通培养基(C组)及5-aza加入含CT-1的培养基(D组)培养。观察细胞形态的改变,并通过免疫组化分析分化后细胞表达心脏特异性肌钙蛋白T(cTnT)的情况。电镜观察分化后细胞的超微结构及实时荧光定量检测α-actin、β-myosin heavy chain(β-MHC)、Nkx2.5、GATA4基因表达。结果C、D组的BMMSCs在培养4周后均形成心肌样细胞形态,并且均表达cTnT;D组BMMSCs分化的心肌样细胞形成了肌管样结构;D组α-actin、β-MHC、Nkx2.5、GATA4基因表达明显高于C组。结论CT-1可能通过对GATA4、Nkx2.5基因表达的调控而促进5-aza诱导的BMMSCs分化为心肌样细胞。展开更多
AIM:To elucidate the role of cytokine receptor-like factor 1(CRLF1) in hepatic stellate cells and liver fibrosis.METHODS:Rat hepatic stellate cells(HSCs) were isolated by Nykodenz gradient centrifugation and activated...AIM:To elucidate the role of cytokine receptor-like factor 1(CRLF1) in hepatic stellate cells and liver fibrosis.METHODS:Rat hepatic stellate cells(HSCs) were isolated by Nykodenz gradient centrifugation and activated by culturing in vitro.Differentially expressed genes in quiescent and culture activated HSCs were identified using microarrays.Injections of carbon tetrachloride(CCl 4) for 4 wk were employed to induce liver fibrosis.The degree of fibrosis was assessed by Sirius red staining.Adenovirus expressing CRLF1 was injected through tail vein into mice to achieve overexpression of CRLF1 in the liver.The same adenovirus was used to overexpress CRLF1 in quiescent HSCs cultured in vitro.Expression of CRLF1,CLCF1 and ciliary neurotrophic factor receptor(CNTFR) in hepatic stellate cells and fibrotic livers was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and Western blotting.Expression of profibrotic cytokines and collagens was analyzed by the same method.RESULTS:CRLF1 is a secreted cytokine with unknown function.Human mutations suggested a role in development of autonomous nervous system and a role of CRLF1 in immune response was implied by its similarity to interleukin(IL)-6.Here we show that expression of CRLF1 was undetectable in quiescent HSCs and was highly upregulated in activated HSCs.Likewise,expression of CRLF1 was very low in normal livers,but was highly upregulated in fibrotic livers,where its expression correlated with the degree of fibrosis.A cofactor of CLRF1,cardiotrophin-like cytokine factor 1(CLCF1),and the receptor which binds CRLF1/CLCF1 dimer,the CNTFR,were expressed to similar levels in quiescent and activated HSCs and in normal and fibrotic livers,indicating a constitutive expression.Overexpression of CLRF1 alone in the normal liver did not stimulate expression of profibrotic cytokines,suggesting that the factor itself is not pro-inflammatory.Ectopic expression in quiescent HSCs,however,retarded their activation into myofibroblasts and specifically decreased expression of type Ⅲ collagen.Inhibition of type Ⅲ collagen expression by CRLF1 was also seen in the whole liver.Our results suggest that CLRF1 is the only component of the CRLF1/CLCF1/CNTFR signaling system that is inducible by a profibrotic stimulus and that activation of this system by CLRF1 may regulate expression of type Ⅲ collagen in fibrosis.CONCLUSION:By regulating activation of HSCs and expression of type Ⅲ collagen,CRLF1 may have an ability to change the composition of extracellular matrix in fibrosis.展开更多
基金Supported by National Natural Science Foundation of China(Nos.81173280,81302995,81403420)Fujian Medical Innovation project(No.2011-CX-30)+1 种基金Science and Technology Department of Fujian Province autonomous non-profit research institutes topics project(No.2011R1038-5)Fujian Academy of Traditional Chinese autonomous topics Project(No.2012fjzyyk-5)
文摘Objectives： To investigate the mechanism of Liuwei Dihuang Pill （六味地黄丸, LDP） in treating postmenopausal osteoporosis （PMOP） with Shen （Kidney） yin deficiency. Methods： In this study, 205 cases of PMOP were divided into the PMOP Shen-yin deficiency group （Group A）, PMOP Shen-yang deficiency group （Group B）, PMOP without Shen deficiency group （Group C）, and control group （Group N）. Real-time polymerase chain reaction （RT-PCR） and Western blot techniques were used to observe the effects of LDP treatment on the cardiotrophin-like cytokine factor 1 （CLCF1）, ankyrin repeat and SOCS box containing 1 （ASB1）, and proldneticin 2 （PROK2） genes and the Janus kinase/signal transducer and activator of transcription （JAK/STAT） signaling pathway. Results： The mRNA （P〈0.05） and protein （P〈0.01） expression levels of the CLCF1 gone in Group A were significantly lower than the corresponding levels in Group N. After LDP treatment for 3 months, the mRNA expression levels of the CLCF1 gone were obviously up-regulated （P〈0.01）. After 6-month treatment, the expression levels of CLCF1 mRNA and protein were significantly up-regulated （both P〈0.01）, and the average bone density of the top femur had significantly increased （P〈0.05）. In vitro, CLCF1 overexpression resulted in a significant increase in the total protein and phosphorylated protein levels of JAK2 and STAT3. Conclusions： The CLCF1 gone is an important gone associated with PMOP Shen-yin deficiency and the therapeutic effects of LDP may be mediated by up-regulation of CLCF1 gone expression and activation of the JAK/STAT signaling pathway.
文摘目的观察心脏营养素-1(CT-1)是否能促进经诱导分化剂5-氮杂胞苷(5-aza)诱导的骨髓间充质干细胞(BMMSCs)分化为心肌样细胞,并研究其相关机制。方法自成年大鼠骨髓中分离BMMSCs,分别以普通培养基(A组)、加入含CT-1的培养基(B组)培养、经5-aza诱导后加入普通培养基(C组)及5-aza加入含CT-1的培养基(D组)培养。观察细胞形态的改变,并通过免疫组化分析分化后细胞表达心脏特异性肌钙蛋白T(cTnT)的情况。电镜观察分化后细胞的超微结构及实时荧光定量检测α-actin、β-myosin heavy chain(β-MHC)、Nkx2.5、GATA4基因表达。结果C、D组的BMMSCs在培养4周后均形成心肌样细胞形态,并且均表达cTnT;D组BMMSCs分化的心肌样细胞形成了肌管样结构;D组α-actin、β-MHC、Nkx2.5、GATA4基因表达明显高于C组。结论CT-1可能通过对GATA4、Nkx2.5基因表达的调控而促进5-aza诱导的BMMSCs分化为心肌样细胞。
基金Supported by Scleroderma Research Foundation and NIH grants,to Stefanovic B
文摘AIM:To elucidate the role of cytokine receptor-like factor 1(CRLF1) in hepatic stellate cells and liver fibrosis.METHODS:Rat hepatic stellate cells(HSCs) were isolated by Nykodenz gradient centrifugation and activated by culturing in vitro.Differentially expressed genes in quiescent and culture activated HSCs were identified using microarrays.Injections of carbon tetrachloride(CCl 4) for 4 wk were employed to induce liver fibrosis.The degree of fibrosis was assessed by Sirius red staining.Adenovirus expressing CRLF1 was injected through tail vein into mice to achieve overexpression of CRLF1 in the liver.The same adenovirus was used to overexpress CRLF1 in quiescent HSCs cultured in vitro.Expression of CRLF1,CLCF1 and ciliary neurotrophic factor receptor(CNTFR) in hepatic stellate cells and fibrotic livers was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and Western blotting.Expression of profibrotic cytokines and collagens was analyzed by the same method.RESULTS:CRLF1 is a secreted cytokine with unknown function.Human mutations suggested a role in development of autonomous nervous system and a role of CRLF1 in immune response was implied by its similarity to interleukin(IL)-6.Here we show that expression of CRLF1 was undetectable in quiescent HSCs and was highly upregulated in activated HSCs.Likewise,expression of CRLF1 was very low in normal livers,but was highly upregulated in fibrotic livers,where its expression correlated with the degree of fibrosis.A cofactor of CLRF1,cardiotrophin-like cytokine factor 1(CLCF1),and the receptor which binds CRLF1/CLCF1 dimer,the CNTFR,were expressed to similar levels in quiescent and activated HSCs and in normal and fibrotic livers,indicating a constitutive expression.Overexpression of CLRF1 alone in the normal liver did not stimulate expression of profibrotic cytokines,suggesting that the factor itself is not pro-inflammatory.Ectopic expression in quiescent HSCs,however,retarded their activation into myofibroblasts and specifically decreased expression of type Ⅲ collagen.Inhibition of type Ⅲ collagen expression by CRLF1 was also seen in the whole liver.Our results suggest that CLRF1 is the only component of the CRLF1/CLCF1/CNTFR signaling system that is inducible by a profibrotic stimulus and that activation of this system by CLRF1 may regulate expression of type Ⅲ collagen in fibrosis.CONCLUSION:By regulating activation of HSCs and expression of type Ⅲ collagen,CRLF1 may have an ability to change the composition of extracellular matrix in fibrosis.
