AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10<sup>-4</sup>mol/L-10<sup>-7</s...AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10<sup>-4</sup>mol/L-10<sup>-7</sup>mol/L SA-A for 22 h.The cell viability wasassayed by[<sup>3</sup>H]proline incorporation,cellproliferation by[<sup>3</sup>H]TdR incorporation,cellcollagen synthetic rate was measured with[<sup>3</sup>H]proline impulse and collagenase digestionmethod.The total RNA was prepared from thecontrol cells and the drug treated cellsrespectively,and α（1）I pro-collagen mRNAexpression was semi-quantitatively analyzedwith RT-PCR.RESULTS 10<sup>-4</sup>mol/L SA-A decreased cellviability and exerted some cytotoxiciy,while10<sup>-5</sup>mol/L-10<sup>-7</sup>mol/L SA-A did not affect cellviability,but inhibited cell proliferationsignificantly,and 10<sup>-6</sup>mol/L SA-A had the besteffect on cell viability among theseconcentrations of drugs.10<sup>-5</sup>mol/L-10<sup>-6</sup>mol/LSA-A inhibited intracellular collagen syntheticrate,but no significant influence on extracellularcollagen secretion.Both 10<sup>-5</sup>mol/L and10<sup>-6</sup>mol/L SA-A could decrease α（1）I pro-collagen mRNA expression remarkably.CONCLUSION SA-A had potent action againstliver fibrosis.It inhibited NIH/3T3 fibroblastproliferation,intracellular collagen syntheticrate and type I pro-collagen gene expression,which may be one of the main mechanisms of thedrug.展开更多
Background The CREG is an important lysosomal protein involved in a variety of cellular functions including promoting cell differentiation,sustaining mature homeostasis, and antagonizing apoptosis.Deficiency of CREG i...Background The CREG is an important lysosomal protein involved in a variety of cellular functions including promoting cell differentiation,sustaining mature homeostasis, and antagonizing apoptosis.Deficiency of CREG in cell and tissue results in a pathologic apoptosis.The present study aimed to elucidate the mechanism of CREG regulation apoptosis.Methods We firstly generated stable NIH3T3 fibroblasts by transfection of pDS_shCREGs vectors.Furthermore, PI-Annexin V and TUNEL staining were used to identify that CREG knockdown promoted the cell apoptosis in NIH3T3 fibroblasts.Western blotting and immunofluorescence staining was used to identify the expression and localization of M6P/ IGF2R and cathepsin L in cytoplasm.Results pDS_shCREGs vector transfection produced an approximately 80%decrease in CREG levels both in the lysate and in the media.The expression and localization of M6P/IGF2R and cathepsin L in cytoplasma changed obviously associated with down-regulated of CREG.In addition,the retention and secretion of cathepsin L enhanced significantly.Using the specific inhibitor or siRNA to block cathepsin L activation attenuated the apoptosis mediated by CREG downregulation.Conclusions Our findings indicated that inhibition of CREG expression in NIH3T3 fibroblasts leads to impaired cathepsin L sorting function mediated by M6P/IGF2R and subsequently promotes pathological cell apoptosis.展开更多
Aim: To observe the apoptotic changes following exposure to EMP and to explore the possible injury mechanism in NIH - 3T3 fibroblasts. Methods: Following NIH - 3T3 cells were exposed to EMP,the proliferation and viabi...Aim: To observe the apoptotic changes following exposure to EMP and to explore the possible injury mechanism in NIH - 3T3 fibroblasts. Methods: Following NIH - 3T3 cells were exposed to EMP,the proliferation and viability of NIH - 3T3 fibroblasts were estimated by hemacytometer and MTT Measurements. Apoptotic rate was measured by flow cytometer. The imnmohistochemical SP method was used to determine bcl- 2, p53. The positively stained cells were analyzed with CMIAS- Ⅱ image analysis system at a magnification 400. All data were analyzed by SPSS8.0 software. Results: The proliferation and viability of NIH- 3T3 cells were markedly inhibited and significant apoptosis was induced following exposure to EMP.EMP could increase the number of non- adherent cells, the highest ratio (33.9%) of non- adherent cells also occurred at 6h. The As70 value of MTT decreased immediately at 1 h, 6h following the cells were exposed as compared with the control (P ＜ 0.05). The highest ratio of apoptosis was 15.07% at 6h, then decreased gradually. Down - regulation of bcl - 2 and up - regulation of p53 were induced by EMP ( P ＜ 0.05). Conclusions: EMP could promote apoptosis of NIH - 3T3 fibroblasts. EMP could also down - regulate bcl - 2 level and up - regulate p53 level in NIH - 3T3 fibroblasts. Bcl - 2 and p53 gene may take part in the process of apoptosis.展开更多
Background:Wound healing has being extensively investigated over the world.Healing impairment is caused by many reasons including increasing of free-radicals-mediated damage,delaying in granulation tissue formation,re...Background:Wound healing has being extensively investigated over the world.Healing impairment is caused by many reasons including increasing of free-radicals-mediated damage,delaying in granulation tissue formation,reducing in angiogenesis and decreasing in collagen reorganization.These facts consequently lead to chronic wound healing.Piper betle Linn(Betle)leaves have been folklore used as an ingredient of drugs for cutaneous wound treatment.However,the effect of betle leaf on wound healing is not yet well elucidated.In this study,we aimed to investigate the healing efficacy of methanol leaf extract of Piper betle Linn on proliferation of fibroblast NIH3T3 cells as well as full-thickness burn and excision wounds in swiss mice.Methods:Scratch wound healing assays were conducted to examine the effects of betle leaf extract on healing activity of fibroblast cells.Burn and excision wounds on swiss mouse skins were created for investigating the wound healing progress caused by the betle leaf extract.Malondialdehyde(MDA)was also evaluated to examine the products of lipid hydroperoxide(LPO)under conditions of with or without betle leaf extract treatment.Results:The results of this study showed that Piper betle Linn leaf extract in methanol increased proliferation of NIH3T3 cells and promoted wound healing in vitro and in vivo with both burn wound and excision wound models.In addition,this extract significant decreased level of malondialdehyde(MDA)in liver of treated-mice compared with that in non-treated mice.Conclusions:Our results suggest that Piper betle Linn can be used as an ingredient in developing natural origin drugs for treatment of cutaneous wounds.展开更多
文摘AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10<sup>-4</sup>mol/L-10<sup>-7</sup>mol/L SA-A for 22 h.The cell viability wasassayed by[<sup>3</sup>H]proline incorporation,cellproliferation by[<sup>3</sup>H]TdR incorporation,cellcollagen synthetic rate was measured with[<sup>3</sup>H]proline impulse and collagenase digestionmethod.The total RNA was prepared from thecontrol cells and the drug treated cellsrespectively,and α（1）I pro-collagen mRNAexpression was semi-quantitatively analyzedwith RT-PCR.RESULTS 10<sup>-4</sup>mol/L SA-A decreased cellviability and exerted some cytotoxiciy,while10<sup>-5</sup>mol/L-10<sup>-7</sup>mol/L SA-A did not affect cellviability,but inhibited cell proliferationsignificantly,and 10<sup>-6</sup>mol/L SA-A had the besteffect on cell viability among theseconcentrations of drugs.10<sup>-5</sup>mol/L-10<sup>-6</sup>mol/LSA-A inhibited intracellular collagen syntheticrate,but no significant influence on extracellularcollagen secretion.Both 10<sup>-5</sup>mol/L and10<sup>-6</sup>mol/L SA-A could decrease α（1）I pro-collagen mRNA expression remarkably.CONCLUSION SA-A had potent action againstliver fibrosis.It inhibited NIH/3T3 fibroblastproliferation,intracellular collagen syntheticrate and type I pro-collagen gene expression,which may be one of the main mechanisms of thedrug.
文摘Background The CREG is an important lysosomal protein involved in a variety of cellular functions including promoting cell differentiation,sustaining mature homeostasis, and antagonizing apoptosis.Deficiency of CREG in cell and tissue results in a pathologic apoptosis.The present study aimed to elucidate the mechanism of CREG regulation apoptosis.Methods We firstly generated stable NIH3T3 fibroblasts by transfection of pDS_shCREGs vectors.Furthermore, PI-Annexin V and TUNEL staining were used to identify that CREG knockdown promoted the cell apoptosis in NIH3T3 fibroblasts.Western blotting and immunofluorescence staining was used to identify the expression and localization of M6P/ IGF2R and cathepsin L in cytoplasm.Results pDS_shCREGs vector transfection produced an approximately 80%decrease in CREG levels both in the lysate and in the media.The expression and localization of M6P/IGF2R and cathepsin L in cytoplasma changed obviously associated with down-regulated of CREG.In addition,the retention and secretion of cathepsin L enhanced significantly.Using the specific inhibitor or siRNA to block cathepsin L activation attenuated the apoptosis mediated by CREG downregulation.Conclusions Our findings indicated that inhibition of CREG expression in NIH3T3 fibroblasts leads to impaired cathepsin L sorting function mediated by M6P/IGF2R and subsequently promotes pathological cell apoptosis.
基金supported by the project foundation of the 9th-Five Year Plan of the Logistics Department of the Chinese PLA (No. 39770697)
文摘Aim: To observe the apoptotic changes following exposure to EMP and to explore the possible injury mechanism in NIH - 3T3 fibroblasts. Methods: Following NIH - 3T3 cells were exposed to EMP,the proliferation and viability of NIH - 3T3 fibroblasts were estimated by hemacytometer and MTT Measurements. Apoptotic rate was measured by flow cytometer. The imnmohistochemical SP method was used to determine bcl- 2, p53. The positively stained cells were analyzed with CMIAS- Ⅱ image analysis system at a magnification 400. All data were analyzed by SPSS8.0 software. Results: The proliferation and viability of NIH- 3T3 cells were markedly inhibited and significant apoptosis was induced following exposure to EMP.EMP could increase the number of non- adherent cells, the highest ratio (33.9%) of non- adherent cells also occurred at 6h. The As70 value of MTT decreased immediately at 1 h, 6h following the cells were exposed as compared with the control (P ＜ 0.05). The highest ratio of apoptosis was 15.07% at 6h, then decreased gradually. Down - regulation of bcl - 2 and up - regulation of p53 were induced by EMP ( P ＜ 0.05). Conclusions: EMP could promote apoptosis of NIH - 3T3 fibroblasts. EMP could also down - regulate bcl - 2 level and up - regulate p53 level in NIH - 3T3 fibroblasts. Bcl - 2 and p53 gene may take part in the process of apoptosis.
基金supported by Grants-in-Aid for Scientific Research under grant number KLEPT-14-02.
文摘Background:Wound healing has being extensively investigated over the world.Healing impairment is caused by many reasons including increasing of free-radicals-mediated damage,delaying in granulation tissue formation,reducing in angiogenesis and decreasing in collagen reorganization.These facts consequently lead to chronic wound healing.Piper betle Linn(Betle)leaves have been folklore used as an ingredient of drugs for cutaneous wound treatment.However,the effect of betle leaf on wound healing is not yet well elucidated.In this study,we aimed to investigate the healing efficacy of methanol leaf extract of Piper betle Linn on proliferation of fibroblast NIH3T3 cells as well as full-thickness burn and excision wounds in swiss mice.Methods:Scratch wound healing assays were conducted to examine the effects of betle leaf extract on healing activity of fibroblast cells.Burn and excision wounds on swiss mouse skins were created for investigating the wound healing progress caused by the betle leaf extract.Malondialdehyde(MDA)was also evaluated to examine the products of lipid hydroperoxide(LPO)under conditions of with or without betle leaf extract treatment.Results:The results of this study showed that Piper betle Linn leaf extract in methanol increased proliferation of NIH3T3 cells and promoted wound healing in vitro and in vivo with both burn wound and excision wound models.In addition,this extract significant decreased level of malondialdehyde(MDA)in liver of treated-mice compared with that in non-treated mice.Conclusions:Our results suggest that Piper betle Linn can be used as an ingredient in developing natural origin drugs for treatment of cutaneous wounds.
