Objective:To investigate the biological functions and molecular regulatory mechanisms of kinesin family member 11(KIF11)in colorectal cancer(CRC).Methods:The expression of KIF11 in CRC was examined by qRT⁃PCR and publ...Objective:To investigate the biological functions and molecular regulatory mechanisms of kinesin family member 11(KIF11)in colorectal cancer(CRC).Methods:The expression of KIF11 in CRC was examined by qRT⁃PCR and public databases.Functional assays(CCK⁃8,colony formation,EdU,and Transwell)were employed to evaluate KIF11’s roles in CRC progression.Western blot,RIP⁃qPCR,MeRIP⁃qPCR,and RNA stability assays were performed to elucidate the molecular mechanism of N6⁃methyladenosine(m6A)modification for KIF11.RNA sequencing(RNA⁃seq)and correlation analysis were used to examine the downstream mechanism of KIF11 regulation.Results:KIF11 was highly expressed in CRC and promoted CRC proliferation and migration.Mechanistically,methyltransferase⁃like 3(METTL3)/insulin like growth factor 2 mRNA binding protein 2(IGF2BP2)enhanced KIF11 mRNA stability and expression in an m6A⁃dependent way.Furthermore,by means of the PROM1/PI3K/AKT pathway,KIF11 facilitated the progression of CRC.Conclusion:The m6A modification of KIF11 by METTL3/IGF2BP2 contributes to CRC progression via the PI3K/AKT signaling pathway,highlighting its potential as a prognostic biomarker and therapeutic target.展开更多
OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compoun...OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compound derived from traditional Chinese medicine,used in Asian countries for the prevention and treatment of hepatic disease.Therefore,the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs.METHODS The murine HSC(tHSC/Cl-6)cell line were incubated in 96-well plates and treated with TNF-αand CuE at various concentrations and indicated times.Cell viability was assessed with MTT assay.Another,t-HSC/Cl-6 were incubated in 6-well plates and also treated with TNF-α,CuE,AICAR or metformin for the indicated time and concentration.Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blotting and RT-PCR.RESULTS CuE inhibited cell proliferation of activated HSC/T-6cells in concentration-and time-dependent manner.CuE triggered the activation of caspase-3,cleaved the PARP,ration of bcl-2/bax,and cytochrom c protein in a time-and concentration-dependent manner.CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK.CuE inhibited the protein and mRNA expressions ofα-SMA,TIMP-1 and collagenⅠ in activated HSC-T6.CuE broadly blocked p-PI3 K,p-Akt,p-mTOR and p-p70S6 Kexpressions.CuE significantly increased phosphorylated AMPK expression as well as AICAR and metoformin.And metformin showed significantly higher activation of AMPK than AICAR.Ability of CuE on activation of AMPK was between AICAR and metformin.It′s also found that CuE significantly decreased p-mTOR as well as AICAR and metformin.CONCLUSION CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment.The findings demonstrate that CuE induced HSC apoptosis via ERK/MAPK and PI3K/Akt-AMPK-mTOR signaling.展开更多
Aim Middle cerebral artery occlusion/reperfusion （MCAO/R） is a widely used animal model for cere- bral ischemia-reperfusion injury, which causes great harm to human health. Salvianolic acid A （SalA） from Dansh- en...Aim Middle cerebral artery occlusion/reperfusion （MCAO/R） is a widely used animal model for cere- bral ischemia-reperfusion injury, which causes great harm to human health. Salvianolic acid A （SalA） from Dansh- en has been reported to possess various neuroprotection effects in lots of in vitro cell injury models. So in this study, the protection effects of SalA on MCAO/R injury were investigated and the protection mechanism was also dis- cussed. Methods MCAO for 1.5 h followed by 24 h of reperfusion induced severe brain injury. 10 mg · kg^-1 Sa- 1A was injected intravenously just after 1.5 h MCAO. The neurological deficit scores, infarct volume and cerebral edema were carried out after 24 h of reperfusion. Then the H＆E and Nissl staining methods were used to see wheth- er SalA could effectively ameliorated the pathological damages of brain. Then the Western blot assay was performed to study the possible signaling pathway stimulated by SalA. Results SalA （ 10 mg · kg^-1 ） could decrease the neurological deficit scores, infarct volume and cerebral edema of MCAO/R rats significantly （P 〈 0. 01, P 〈 0.05 and P 〈 0.01 ）. The brain pathological damages of MCAO/R rats could also be effectively ameliorated by the analy- sis of H＆E and Nissl staining results. Western blotting results showed that the upregulation of Caspase3 in MCAO/ R rat brain could be inhibited by SalA （P 〈 0.01）. With the administration of SalA, the Bcl-2 level in the brain was increased （ P 〈 0.01 ） and the Bax level was decreased （ P 〈 0.01 ） compared with model group. Furthermore, the phosphorylation levels of Akt, GSK3β and CREB decreased significantly in model group, while SalA treatment could significantly reverse the process （P 〈 0.01, P 〈 0.05 and P 〈 0. 05 ）. Conclusion These results indicated that SalA administration could protect rat brain against MCAO/R induced injury by ameliorating neurological deft-cits, decreasing the infarct volume and cerebral edema. And the anti-apoptosis effects of SalA may result from the regulation of PI3IC/Akt/GSK3β pathway.展开更多
Astaxanthin （ATX） , the most abundant flavonoids in propolis, has been proven to exert neuroprotective property against cerebral ischemia-induced apoptosis. However, the mechanisms by which ATX mediates its thera- p...Astaxanthin （ATX） , the most abundant flavonoids in propolis, has been proven to exert neuroprotective property against cerebral ischemia-induced apoptosis. However, the mechanisms by which ATX mediates its thera- peutic effects in vitro are unclear. In the present study, the article explored the underlying mechanisms involved in the protective effects of ATX via the PI3IC/Akt/GSK3β/Nrf2 signaling pathway in SH-SY5Y cells. For study of mechanism, the phosphoinositide 3 kinase （PI3K）-Akt inhibitor LY294002, Glycogen synthase kinase 3β （GSK313） inhibitor LiC1 were used. Pre-treatmentwith ATX for 24h significantly reduced the OGD induced viability loss, apoptotic rate and attenuated OGD-mediated ROS production. In addition, ATX inhibited OGD-induced mito- chondrial membrane potential, decreased Bcl-2/Bax ratio. PI3 IC/Akt/GSK3β/Nrf2 signaling pathway activation in SH-SY5Y was tested by Western blot. Nrf2 expression was increasing by ATX and counteracted by PI3IC/Akt in- hibitor LY294002, GSK3β inhibitor LiC1 in SH-SY5Y. Nrf2 Immunocytochemistry showed Nrf2 nuclear transloca- tion was increasing by ATX and counteracted by LY294002 or LiC1 in SH-SY5Y, respectively. It may be suggested that astaxanthin against cerebral ischemia-induced apoptosis in vitro via a programmed PI3 IC/Akt/GSK3β/Nrf2 sig- naling pathway in vitro.展开更多
OBJECTIVE To investigate the pharmacological effect and mechanism of Sanguisorba officinalis L.(SOL)in non-small cell lung cancer(NSCLC)in vitro and in vivo based on network pharmacology.METHODS Network pharmacology w...OBJECTIVE To investigate the pharmacological effect and mechanism of Sanguisorba officinalis L.(SOL)in non-small cell lung cancer(NSCLC)in vitro and in vivo based on network pharmacology.METHODS Network pharmacology was used to analyze the improving effect of SOL on NSCLC and possible targets.Cell counting kit 8(CCK-8)and 5-ethynyl-2′-deoxyuridine(EdU)staining,Western blotting,flow cytometry of AnnexinⅤ/PI,Hoechst 33342/PI staining detection and immunofluorescence were utilized in vitro.H&E staining,immunohistochemistry staining and Western blotting were performed in vivo.RESULTS Based on network prediction,we analyzed the 208 common targets of SOL and NSCLC.36 core targets in 208 common targets were obtained through cytoscape analysis.And the top 10 core targets included Akt,mTOR,EGFR,etc..KEGG analysis showed that PI3K-Akt signaling pathway was the most likely pathway.Furthermore,the mechanism study found that SOL could activate the PI3K/Akt/mTOR signaling pathway in vitro and in vivo.The anti-proliferative effect of SOL in A549 and H1299 cells was measured and validated by CCK-8 and EdU assay.Immunohistochemical results of Ki67 showed that SOL effectively inhibited tumor growth in vivo.SOL also significantly inhibited the migration and invasion of A549 and H1299 cells.SOL significantly increased the percentage of cells with PI signal in A549 and H1299,and the process of cell death of A549 cells indicated that SOL induced apoptosis.The PARP-1 and caspase-3 in A549 and H1299 were found to be activated in a dose manner.The results in vivo were consistent with those in vitro.CONCLUSION SOL-induced,caspase-3-mediated apoptosis via the induction of the PI3K/Akt/mTOR signaling pathway in NSCLC,which further clarified the mechanism of SOL in the inhibition of NSCLC,and thereby provided a possibility for SOL to serve as a novel therapeutic agent for NSCLC in the future.展开更多
基金江苏省卫生健康委员会医学科研重点项目(K2023024)789 Outstanding Talent Program of SAHNMU(789ZYRC202090147)。
文摘Objective:To investigate the biological functions and molecular regulatory mechanisms of kinesin family member 11(KIF11)in colorectal cancer(CRC).Methods:The expression of KIF11 in CRC was examined by qRT⁃PCR and public databases.Functional assays(CCK⁃8,colony formation,EdU,and Transwell)were employed to evaluate KIF11’s roles in CRC progression.Western blot,RIP⁃qPCR,MeRIP⁃qPCR,and RNA stability assays were performed to elucidate the molecular mechanism of N6⁃methyladenosine(m6A)modification for KIF11.RNA sequencing(RNA⁃seq)and correlation analysis were used to examine the downstream mechanism of KIF11 regulation.Results:KIF11 was highly expressed in CRC and promoted CRC proliferation and migration.Mechanistically,methyltransferase⁃like 3(METTL3)/insulin like growth factor 2 mRNA binding protein 2(IGF2BP2)enhanced KIF11 mRNA stability and expression in an m6A⁃dependent way.Furthermore,by means of the PROM1/PI3K/AKT pathway,KIF11 facilitated the progression of CRC.Conclusion:The m6A modification of KIF11 by METTL3/IGF2BP2 contributes to CRC progression via the PI3K/AKT signaling pathway,highlighting its potential as a prognostic biomarker and therapeutic target.
基金The project supported by National Natural Science Foundation of China(81260497,81460564)Science and Technology Department of Jilin Province Youth Scientific Research Fund Project(201201075)
文摘OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compound derived from traditional Chinese medicine,used in Asian countries for the prevention and treatment of hepatic disease.Therefore,the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs.METHODS The murine HSC(tHSC/Cl-6)cell line were incubated in 96-well plates and treated with TNF-αand CuE at various concentrations and indicated times.Cell viability was assessed with MTT assay.Another,t-HSC/Cl-6 were incubated in 6-well plates and also treated with TNF-α,CuE,AICAR or metformin for the indicated time and concentration.Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blotting and RT-PCR.RESULTS CuE inhibited cell proliferation of activated HSC/T-6cells in concentration-and time-dependent manner.CuE triggered the activation of caspase-3,cleaved the PARP,ration of bcl-2/bax,and cytochrom c protein in a time-and concentration-dependent manner.CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK.CuE inhibited the protein and mRNA expressions ofα-SMA,TIMP-1 and collagenⅠ in activated HSC-T6.CuE broadly blocked p-PI3 K,p-Akt,p-mTOR and p-p70S6 Kexpressions.CuE significantly increased phosphorylated AMPK expression as well as AICAR and metoformin.And metformin showed significantly higher activation of AMPK than AICAR.Ability of CuE on activation of AMPK was between AICAR and metformin.It′s also found that CuE significantly decreased p-mTOR as well as AICAR and metformin.CONCLUSION CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment.The findings demonstrate that CuE induced HSC apoptosis via ERK/MAPK and PI3K/Akt-AMPK-mTOR signaling.
文摘Aim Middle cerebral artery occlusion/reperfusion （MCAO/R） is a widely used animal model for cere- bral ischemia-reperfusion injury, which causes great harm to human health. Salvianolic acid A （SalA） from Dansh- en has been reported to possess various neuroprotection effects in lots of in vitro cell injury models. So in this study, the protection effects of SalA on MCAO/R injury were investigated and the protection mechanism was also dis- cussed. Methods MCAO for 1.5 h followed by 24 h of reperfusion induced severe brain injury. 10 mg · kg^-1 Sa- 1A was injected intravenously just after 1.5 h MCAO. The neurological deficit scores, infarct volume and cerebral edema were carried out after 24 h of reperfusion. Then the H＆E and Nissl staining methods were used to see wheth- er SalA could effectively ameliorated the pathological damages of brain. Then the Western blot assay was performed to study the possible signaling pathway stimulated by SalA. Results SalA （ 10 mg · kg^-1 ） could decrease the neurological deficit scores, infarct volume and cerebral edema of MCAO/R rats significantly （P 〈 0. 01, P 〈 0.05 and P 〈 0.01 ）. The brain pathological damages of MCAO/R rats could also be effectively ameliorated by the analy- sis of H＆E and Nissl staining results. Western blotting results showed that the upregulation of Caspase3 in MCAO/ R rat brain could be inhibited by SalA （P 〈 0.01）. With the administration of SalA, the Bcl-2 level in the brain was increased （ P 〈 0.01 ） and the Bax level was decreased （ P 〈 0.01 ） compared with model group. Furthermore, the phosphorylation levels of Akt, GSK3β and CREB decreased significantly in model group, while SalA treatment could significantly reverse the process （P 〈 0.01, P 〈 0.05 and P 〈 0. 05 ）. Conclusion These results indicated that SalA administration could protect rat brain against MCAO/R induced injury by ameliorating neurological deft-cits, decreasing the infarct volume and cerebral edema. And the anti-apoptosis effects of SalA may result from the regulation of PI3IC/Akt/GSK3β pathway.
文摘Astaxanthin （ATX） , the most abundant flavonoids in propolis, has been proven to exert neuroprotective property against cerebral ischemia-induced apoptosis. However, the mechanisms by which ATX mediates its thera- peutic effects in vitro are unclear. In the present study, the article explored the underlying mechanisms involved in the protective effects of ATX via the PI3IC/Akt/GSK3β/Nrf2 signaling pathway in SH-SY5Y cells. For study of mechanism, the phosphoinositide 3 kinase （PI3K）-Akt inhibitor LY294002, Glycogen synthase kinase 3β （GSK313） inhibitor LiC1 were used. Pre-treatmentwith ATX for 24h significantly reduced the OGD induced viability loss, apoptotic rate and attenuated OGD-mediated ROS production. In addition, ATX inhibited OGD-induced mito- chondrial membrane potential, decreased Bcl-2/Bax ratio. PI3 IC/Akt/GSK3β/Nrf2 signaling pathway activation in SH-SY5Y was tested by Western blot. Nrf2 expression was increasing by ATX and counteracted by PI3IC/Akt in- hibitor LY294002, GSK3β inhibitor LiC1 in SH-SY5Y. Nrf2 Immunocytochemistry showed Nrf2 nuclear transloca- tion was increasing by ATX and counteracted by LY294002 or LiC1 in SH-SY5Y, respectively. It may be suggested that astaxanthin against cerebral ischemia-induced apoptosis in vitro via a programmed PI3 IC/Akt/GSK3β/Nrf2 sig- naling pathway in vitro.
基金National Natural Science Foundation of China(81774013,81804221,82074129)and National Science and Technology Major Project of China(2018ZX09721004-006-004)。
文摘OBJECTIVE To investigate the pharmacological effect and mechanism of Sanguisorba officinalis L.(SOL)in non-small cell lung cancer(NSCLC)in vitro and in vivo based on network pharmacology.METHODS Network pharmacology was used to analyze the improving effect of SOL on NSCLC and possible targets.Cell counting kit 8(CCK-8)and 5-ethynyl-2′-deoxyuridine(EdU)staining,Western blotting,flow cytometry of AnnexinⅤ/PI,Hoechst 33342/PI staining detection and immunofluorescence were utilized in vitro.H&E staining,immunohistochemistry staining and Western blotting were performed in vivo.RESULTS Based on network prediction,we analyzed the 208 common targets of SOL and NSCLC.36 core targets in 208 common targets were obtained through cytoscape analysis.And the top 10 core targets included Akt,mTOR,EGFR,etc..KEGG analysis showed that PI3K-Akt signaling pathway was the most likely pathway.Furthermore,the mechanism study found that SOL could activate the PI3K/Akt/mTOR signaling pathway in vitro and in vivo.The anti-proliferative effect of SOL in A549 and H1299 cells was measured and validated by CCK-8 and EdU assay.Immunohistochemical results of Ki67 showed that SOL effectively inhibited tumor growth in vivo.SOL also significantly inhibited the migration and invasion of A549 and H1299 cells.SOL significantly increased the percentage of cells with PI signal in A549 and H1299,and the process of cell death of A549 cells indicated that SOL induced apoptosis.The PARP-1 and caspase-3 in A549 and H1299 were found to be activated in a dose manner.The results in vivo were consistent with those in vitro.CONCLUSION SOL-induced,caspase-3-mediated apoptosis via the induction of the PI3K/Akt/mTOR signaling pathway in NSCLC,which further clarified the mechanism of SOL in the inhibition of NSCLC,and thereby provided a possibility for SOL to serve as a novel therapeutic agent for NSCLC in the future.
