To investigate whether microRNA (miR)-34a mediates oxaliplatin (OXA) resistance of colorectal cancer (CRC) cells by inhibiting macroautophagy via the transforming growth factor (TGF)-β/Smad4 pathway.METHODSmiR-34a ex...To investigate whether microRNA (miR)-34a mediates oxaliplatin (OXA) resistance of colorectal cancer (CRC) cells by inhibiting macroautophagy via the transforming growth factor (TGF)-β/Smad4 pathway.METHODSmiR-34a expression levels were detected in CRC tissues and CRC cell lines by quantitative real-time polymerase chain reaction. Computational search, functional luciferase assay and western blotting were used to demonstrate the downstream target of miR-34a in CRC cells. Cell viability was measured with Cell Counting Kit-8. Apoptosis and macroautophagy of CRC cells were analyzed by flow cytometry and transmission electron microscopy, and expression of beclin I and LC3-II was detected by western blotting.RESULTSExpression of miR-34a was significantly reduced while expression of TGF-β and Smad4 was increased in CRC patients treated with OXA-based chemotherapy. OXA treatment also resulted in decreased miR-34a levels and increased TGF-β and Smad4 levels in both parental cells and the OXA-resistant CRC cells. Activation of macroautophagy contributed to OXA resistance in CRC cells. Expression levels of Smad4 and miR-34a in CRC patients had a significant inverse correlation and overexpressing miR-34a inhibited macroautophagy activation by directly targeting Smad4 through the TGF-β/Smad4 pathway. OXA-induced downregulation of miR-34a and increased drug resistance by activating macroautophagy in CRC cells.CONCLUSIONmiR-34a mediates OXA resistance of CRC by inhibiting macroautophagy via the TGF-β/Smad4 pathway.展开更多
AIM: To investigate the antifibrotic effects of bone morphogenetic protein-7 (BMP-7) on Schistosoma japonicum (S. japonicum )-induced hepatic fibrosis in BALB/C mice. METHODS: Sixty BALB/C mice were randomly divided i...AIM: To investigate the antifibrotic effects of bone morphogenetic protein-7 (BMP-7) on Schistosoma japonicum (S. japonicum )-induced hepatic fibrosis in BALB/C mice. METHODS: Sixty BALB/C mice were randomly divided into three groups, including a control group (group A, n = 20), model group (group B, n = 20) and BMP-7 treated group (group C, n = 20). The mice in group B and group C were abdominally infected with S. japonicum cercariae to induce a schistosomal hepatic fibrosis model. The mice in group C were administered human recombinant BMP-7. Liver samples were extracted from mice sacrificed at 9 and 15 wk after modeling. Hepatic histopathological changes were assessed using Masson's staining. Transforming growth factor-beta 1 (TGF-β1), alpha-smooth muscle actin (α-SMA), phosphorylated Smad2/3 (pSmad2/3) and Smad7 protein levels and localization were measured by Western blotting and immunohistochemistry, respectively, and their mRNA expressions were detected by reverse transcriptionpolymerase chain reaction (RT-PCR). RESULTS: The schistosomal hepatic fibrosis mouse model was successfully established, as the livers of mice in group B and group C showed varying degrees of typical schistosomal hepatopathologic changes such as egg granuloma and collagen deposition. The degree of collagen deposition in group C was higher than that in group A (week 9: 22.95±6.66vs 2.02±0.76; week 15: 12.84±4.36 vs 1.74±0.80; P<0.05), but significantly lower than that in group B (week 9: 22.95±6.66 vs 34.43±6.96; week 15: 12.84±4.36 vs 18.90±5.07;P<0.05) at both time points. According to immunohistochemistry data, the expressions of α-SMA, TGF-β1 and pSmad2/3 protein in group C were higher than those in group A (α-SMA: week 9: 21.24±5.73 vs 0.33±0.20; week 15: 12.42±4.88 vs 0.34±0.27; TGF-β1: week 9: 37.00±13.74 vs 3.73±2.14; week 15: 16.71±9.80 vs 3.08±2.35; pSmad2/3: week 9: 12.92±4.81 vs 0.83±0.48; week 15: 7.87±4.09 vs 0.90±0.45; P<0.05), but significantly lower than those in group B (α-SMA: week 9: 21.24±5.73 vs 34.39±5.74; week 15: 12.42±4.88 vs 25.90±7.01; TGF-β1: week 9: 37.00±13.74 vs 55.66±14.88; week 15: 16.71±9.80 vs 37.10±12.51; pSmad2/3: week 9: 12.92±4.81 vs 19.41±6.87; week 15: 7.87±4.09vs 13.00±4.98;P<0.05) at both time points; the expression of Smad7 protein in group B was higher than that in group A and group C at week 9 (8.46±3.95 vs 1.00±0.40 and 8.46±3.95 vs 0.77±0.42; P<0.05), while there were no differences in Smad7 expression between the three groups at week 15 (1.09±0.38 vs 0.97±0.42 vs 0.89±0.39; P>0.05). Although minor discrepancies were observed, the results of RT-PCR and Western blotting were mainly consistentwith the immunohistochemical results. CONCLUSION: Exogenous BMP-7 significantly decreased the degree of hepatic fibrosis in both the acute and chronic stages of hepato-schistosomiasis, and the regulatory mechanism may involve the TGF-β/Smad signaling pathway.展开更多
Summary： The activation of hepatic stellate cells （HSCs） and their transformation to myofibroblasts are the key steps in the pathological progress of liver fibrosis. The transforming growth factor-J3 （TGFβ）/Smad...Summary： The activation of hepatic stellate cells （HSCs） and their transformation to myofibroblasts are the key steps in the pathological progress of liver fibrosis. The transforming growth factor-J3 （TGFβ）/Smad pathway is involved in the proliferation and collagen synthesis of HSCs. This study aimed to examine the effect of the protease inhibitor MG132 on the signaling pathway of TGFβ/Smad in HSC-T6 cells and seek a novel therapeutic approach for liver fibrosis. The HSC-T6 cells were treated with MG132 at different concentrations （0-10 maol/L）. Cell proliferation was detected by MTT method. The mRNA and protein expression levels of TGFI31, Smad3 and Smad7 were determined in HSC-T6 cells by real-time PCR and Western blotting, respectively, after treatment with MG132 at different con- centrations （1, 2, 3 μtmol/L） or RPMI1640 alone （serving as control）. The results showed that MG132 could inhibit the proliferation of HSC-T6 cells in a dose-dependent manner, and the IC50 of MG132 was 6.84 μmol/L. After treatment with MG132 at 1, 2 or 3 nol/L for 24 h, the mRNA expression levels of TGF-β1 and Smad3 were significantly decreased （P〈0.05）, but the Smad7 mRNA expression had no significant change （P〉0.05）. There was also a significant decrease in the protein expression level of TGF-β1 and Smad3 （P〈0.05）. However, the expression of Smad7 protein was substantially increased when compared with the control group （P〈0.05）. It was concluded that the inhibition of TGFi/Smad pathway in HSC-T6 cells by MG132 can reduce the production of profibrosis factors （TGFI31, Smad3） and promote the expression of anti-fibrosis factor （Smad7）, suggesting that MG132 may become a po- tential therapeutic alternative for liver fibrosis.展开更多
OBJECTIVE:To explore the role and mechanism of Qufeng Jiejing(祛风解痉,QFJJ)formula in the asthma progression.METHODS:The Bagg Albino/c mice treated with Ovalbumin and AL(OH)3,and airway smooth muscle cells(ASMCs)trea...OBJECTIVE:To explore the role and mechanism of Qufeng Jiejing(祛风解痉,QFJJ)formula in the asthma progression.METHODS:The Bagg Albino/c mice treated with Ovalbumin and AL(OH)3,and airway smooth muscle cells(ASMCs)treated with platelet-derived growth factor(PDGF)-BB to establish a asthma model in vivo and in vitro.The cell morphology was observed with microscope and immunofluorescence staining.The cell viability was assessed with methyl thiazolyl tetrazolium assay.The tumor necrosis factor-αlpha(TNF-α),interleukin-1beta(IL-1β),laminin,fibronectin and collagen IV levels in the ASMCs were detected with corresponding enzyme linked immunosorbent assay kits.Transwell and wound healing assays were conducted to test the cell migration.The TGF-β1,Smad2 and Smad3 levels were measured with Western blot.RESULTS:We found that QFJJ formula treatment dramatically decreased the cell viability,TNF-α,IL-1β,laminin,fibronectin and collagen IV levels in the PDGFBB stimulated ASMCs.Additionally,the protein levels of TGF-β1,Smad2 and Smad3 in the PDGF-BB stimulated ASMCs were prominently depleted after QFJJ formula treatment.Besides,SRI treatment neutralized the role of QFJJ formula in the PDGF-BB stimulated ASMCs.CONCLUSION:QFJJ formula effectively relieved the asthma progression through ameliorate the ASMCs function,which was achieved through suppressing the TGF-β1/Smads signaling pathway.展开更多
Peripheral nerve defect repair is a complex process that involves multiple cell types;perineurial cells play a pivotal role.Hair follicle neural crest stem cells promote perineurial cell proliferation and migration vi...Peripheral nerve defect repair is a complex process that involves multiple cell types;perineurial cells play a pivotal role.Hair follicle neural crest stem cells promote perineurial cell proliferation and migration via paracrine signaling;however,their clinical applications are limited by potential risks such as tumorigenesis and xenogeneic immune rejection,which are similar to the risks associated with other stem cell transplantations.The present study therefore focuses on small extracellular vesicles derived from hair follicle neural crest stem cells,which preserve the bioactive properties of the parent cells while avoiding the transplantation-associated risks.In vitro,small extracellular vesicles derived from hair follicle neural crest stem cells significantly enhanced the proliferation,migration,tube formation,and barrier function of perineurial cells,and subsequently upregulated the expression of tight junction proteins.Furthermore,in a rat model of sciatic nerve defects bridged with silicon tubes,treatment with small extracellular vesicles derived from hair follicle neural crest stem cells resulted in higher tight junction protein expression in perineurial cells,thus facilitating neural tissue regeneration.At 10 weeks post-surgery,rats treated with small extracellular vesicles derived from hair follicle neural crest stem cells exhibited improved nerve function recovery and reduced muscle atrophy.Transcriptomic and micro RNA analyses revealed that small extracellular vesicles derived from hair follicle neural crest stem cells deliver mi R-21-5p,which inhibits mothers against decapentaplegic homolog 7 expression,thereby activating the transforming growth factor-β/mothers against decapentaplegic homolog signaling pathway and upregulating hyaluronan synthase 2 expression,and further enhancing tight junction protein expression.Together,our findings indicate that small extracellular vesicles derived from hair follicle neural crest stem cells promote the proliferation,migration,and tight junction protein formation of perineurial cells.These results provide new insights into peripheral nerve regeneration from the perspective of perineurial cells,and present a novel approach for the clinical treatment of peripheral nerve defects.展开更多
基金Supported by Science Foundation of Education Department of Heilongjiang Province,China,no.12541430
文摘To investigate whether microRNA (miR)-34a mediates oxaliplatin (OXA) resistance of colorectal cancer (CRC) cells by inhibiting macroautophagy via the transforming growth factor (TGF)-β/Smad4 pathway.METHODSmiR-34a expression levels were detected in CRC tissues and CRC cell lines by quantitative real-time polymerase chain reaction. Computational search, functional luciferase assay and western blotting were used to demonstrate the downstream target of miR-34a in CRC cells. Cell viability was measured with Cell Counting Kit-8. Apoptosis and macroautophagy of CRC cells were analyzed by flow cytometry and transmission electron microscopy, and expression of beclin I and LC3-II was detected by western blotting.RESULTSExpression of miR-34a was significantly reduced while expression of TGF-β and Smad4 was increased in CRC patients treated with OXA-based chemotherapy. OXA treatment also resulted in decreased miR-34a levels and increased TGF-β and Smad4 levels in both parental cells and the OXA-resistant CRC cells. Activation of macroautophagy contributed to OXA resistance in CRC cells. Expression levels of Smad4 and miR-34a in CRC patients had a significant inverse correlation and overexpressing miR-34a inhibited macroautophagy activation by directly targeting Smad4 through the TGF-β/Smad4 pathway. OXA-induced downregulation of miR-34a and increased drug resistance by activating macroautophagy in CRC cells.CONCLUSIONmiR-34a mediates OXA resistance of CRC by inhibiting macroautophagy via the TGF-β/Smad4 pathway.
文摘AIM: To investigate the antifibrotic effects of bone morphogenetic protein-7 (BMP-7) on Schistosoma japonicum (S. japonicum )-induced hepatic fibrosis in BALB/C mice. METHODS: Sixty BALB/C mice were randomly divided into three groups, including a control group (group A, n = 20), model group (group B, n = 20) and BMP-7 treated group (group C, n = 20). The mice in group B and group C were abdominally infected with S. japonicum cercariae to induce a schistosomal hepatic fibrosis model. The mice in group C were administered human recombinant BMP-7. Liver samples were extracted from mice sacrificed at 9 and 15 wk after modeling. Hepatic histopathological changes were assessed using Masson's staining. Transforming growth factor-beta 1 (TGF-β1), alpha-smooth muscle actin (α-SMA), phosphorylated Smad2/3 (pSmad2/3) and Smad7 protein levels and localization were measured by Western blotting and immunohistochemistry, respectively, and their mRNA expressions were detected by reverse transcriptionpolymerase chain reaction (RT-PCR). RESULTS: The schistosomal hepatic fibrosis mouse model was successfully established, as the livers of mice in group B and group C showed varying degrees of typical schistosomal hepatopathologic changes such as egg granuloma and collagen deposition. The degree of collagen deposition in group C was higher than that in group A (week 9: 22.95±6.66vs 2.02±0.76; week 15: 12.84±4.36 vs 1.74±0.80; P<0.05), but significantly lower than that in group B (week 9: 22.95±6.66 vs 34.43±6.96; week 15: 12.84±4.36 vs 18.90±5.07;P<0.05) at both time points. According to immunohistochemistry data, the expressions of α-SMA, TGF-β1 and pSmad2/3 protein in group C were higher than those in group A (α-SMA: week 9: 21.24±5.73 vs 0.33±0.20; week 15: 12.42±4.88 vs 0.34±0.27; TGF-β1: week 9: 37.00±13.74 vs 3.73±2.14; week 15: 16.71±9.80 vs 3.08±2.35; pSmad2/3: week 9: 12.92±4.81 vs 0.83±0.48; week 15: 7.87±4.09 vs 0.90±0.45; P<0.05), but significantly lower than those in group B (α-SMA: week 9: 21.24±5.73 vs 34.39±5.74; week 15: 12.42±4.88 vs 25.90±7.01; TGF-β1: week 9: 37.00±13.74 vs 55.66±14.88; week 15: 16.71±9.80 vs 37.10±12.51; pSmad2/3: week 9: 12.92±4.81 vs 19.41±6.87; week 15: 7.87±4.09vs 13.00±4.98;P<0.05) at both time points; the expression of Smad7 protein in group B was higher than that in group A and group C at week 9 (8.46±3.95 vs 1.00±0.40 and 8.46±3.95 vs 0.77±0.42; P<0.05), while there were no differences in Smad7 expression between the three groups at week 15 (1.09±0.38 vs 0.97±0.42 vs 0.89±0.39; P>0.05). Although minor discrepancies were observed, the results of RT-PCR and Western blotting were mainly consistentwith the immunohistochemical results. CONCLUSION: Exogenous BMP-7 significantly decreased the degree of hepatic fibrosis in both the acute and chronic stages of hepato-schistosomiasis, and the regulatory mechanism may involve the TGF-β/Smad signaling pathway.
基金supported by a grant from the Natural Science Foundation of Hubei Province(No.2010CHB00401)
文摘Summary： The activation of hepatic stellate cells （HSCs） and their transformation to myofibroblasts are the key steps in the pathological progress of liver fibrosis. The transforming growth factor-J3 （TGFβ）/Smad pathway is involved in the proliferation and collagen synthesis of HSCs. This study aimed to examine the effect of the protease inhibitor MG132 on the signaling pathway of TGFβ/Smad in HSC-T6 cells and seek a novel therapeutic approach for liver fibrosis. The HSC-T6 cells were treated with MG132 at different concentrations （0-10 maol/L）. Cell proliferation was detected by MTT method. The mRNA and protein expression levels of TGFI31, Smad3 and Smad7 were determined in HSC-T6 cells by real-time PCR and Western blotting, respectively, after treatment with MG132 at different con- centrations （1, 2, 3 μtmol/L） or RPMI1640 alone （serving as control）. The results showed that MG132 could inhibit the proliferation of HSC-T6 cells in a dose-dependent manner, and the IC50 of MG132 was 6.84 μmol/L. After treatment with MG132 at 1, 2 or 3 nol/L for 24 h, the mRNA expression levels of TGF-β1 and Smad3 were significantly decreased （P〈0.05）, but the Smad7 mRNA expression had no significant change （P〉0.05）. There was also a significant decrease in the protein expression level of TGF-β1 and Smad3 （P〈0.05）. However, the expression of Smad7 protein was substantially increased when compared with the control group （P〈0.05）. It was concluded that the inhibition of TGFi/Smad pathway in HSC-T6 cells by MG132 can reduce the production of profibrosis factors （TGFI31, Smad3） and promote the expression of anti-fibrosis factor （Smad7）, suggesting that MG132 may become a po- tential therapeutic alternative for liver fibrosis.
基金Supported by Science and Technology Innovation Project of China Academy of Chinese Medical Sciences of Study on the Mechanism of Qufeng Jiejing Formula in Regulating Mitogen-activated Protein Kinase Signaling Pathway to Inhibit Phenotypic Transformation of Airway Smooth Muscle Cells in Asthma(No.CI2021A01108)Cultivation Project of The National Natural Science Foundation of China of Xiyuan Hospital,China Academy of Chinese Medical Sciences of Research on the Role of Traditional Chinese Medicines-Qufeng Jiejing in the Proliferation,Migration and Phenotypic Transformation of Airway Smooth Muscle Cells in Asthma(No.XY20-17)。
文摘OBJECTIVE:To explore the role and mechanism of Qufeng Jiejing(祛风解痉,QFJJ)formula in the asthma progression.METHODS:The Bagg Albino/c mice treated with Ovalbumin and AL(OH)3,and airway smooth muscle cells(ASMCs)treated with platelet-derived growth factor(PDGF)-BB to establish a asthma model in vivo and in vitro.The cell morphology was observed with microscope and immunofluorescence staining.The cell viability was assessed with methyl thiazolyl tetrazolium assay.The tumor necrosis factor-αlpha(TNF-α),interleukin-1beta(IL-1β),laminin,fibronectin and collagen IV levels in the ASMCs were detected with corresponding enzyme linked immunosorbent assay kits.Transwell and wound healing assays were conducted to test the cell migration.The TGF-β1,Smad2 and Smad3 levels were measured with Western blot.RESULTS:We found that QFJJ formula treatment dramatically decreased the cell viability,TNF-α,IL-1β,laminin,fibronectin and collagen IV levels in the PDGFBB stimulated ASMCs.Additionally,the protein levels of TGF-β1,Smad2 and Smad3 in the PDGF-BB stimulated ASMCs were prominently depleted after QFJJ formula treatment.Besides,SRI treatment neutralized the role of QFJJ formula in the PDGF-BB stimulated ASMCs.CONCLUSION:QFJJ formula effectively relieved the asthma progression through ameliorate the ASMCs function,which was achieved through suppressing the TGF-β1/Smads signaling pathway.
基金supported by the National Natural Science Foundation of China,No.81571211(to FL)the Natural Science Foundation of Shanghai,No.22ZR1476800(to CH)。
文摘Peripheral nerve defect repair is a complex process that involves multiple cell types;perineurial cells play a pivotal role.Hair follicle neural crest stem cells promote perineurial cell proliferation and migration via paracrine signaling;however,their clinical applications are limited by potential risks such as tumorigenesis and xenogeneic immune rejection,which are similar to the risks associated with other stem cell transplantations.The present study therefore focuses on small extracellular vesicles derived from hair follicle neural crest stem cells,which preserve the bioactive properties of the parent cells while avoiding the transplantation-associated risks.In vitro,small extracellular vesicles derived from hair follicle neural crest stem cells significantly enhanced the proliferation,migration,tube formation,and barrier function of perineurial cells,and subsequently upregulated the expression of tight junction proteins.Furthermore,in a rat model of sciatic nerve defects bridged with silicon tubes,treatment with small extracellular vesicles derived from hair follicle neural crest stem cells resulted in higher tight junction protein expression in perineurial cells,thus facilitating neural tissue regeneration.At 10 weeks post-surgery,rats treated with small extracellular vesicles derived from hair follicle neural crest stem cells exhibited improved nerve function recovery and reduced muscle atrophy.Transcriptomic and micro RNA analyses revealed that small extracellular vesicles derived from hair follicle neural crest stem cells deliver mi R-21-5p,which inhibits mothers against decapentaplegic homolog 7 expression,thereby activating the transforming growth factor-β/mothers against decapentaplegic homolog signaling pathway and upregulating hyaluronan synthase 2 expression,and further enhancing tight junction protein expression.Together,our findings indicate that small extracellular vesicles derived from hair follicle neural crest stem cells promote the proliferation,migration,and tight junction protein formation of perineurial cells.These results provide new insights into peripheral nerve regeneration from the perspective of perineurial cells,and present a novel approach for the clinical treatment of peripheral nerve defects.
