Background:The incidence of colorectal cancer(CRC)has been increasing in recent years.Thus,the discovery of factors that can assist in alleviating CRC is urgently warranted.Methods:To identify a potential factor invol...Background:The incidence of colorectal cancer(CRC)has been increasing in recent years.Thus,the discovery of factors that can assist in alleviating CRC is urgently warranted.Methods:To identify a potential factor involved in the development of CRC,we screened the upregulated genes in tumor tissues through four datasets from an online database.The expression of reticulocalbin 1(RCN1),a Ca2+-binding protein,was upregulated in the four datasets.Based on loss-offunction experiments,the effect of RCN1 on cell viability was assessed by Cell Counting Kit-8(CCK-8)assay.The regulatory effect of RCN1 on apoptosis was evaluated through Annexin V-fluorescein 5-isothiocyanate(FITC)/propidium iodide(PI)staining assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay in RKO and SW480 cells.Activation of endoplasmic reticulum(ER)stress signaling pathways was confirmed by estimating the phosphorylation and expression of PRKR-like ER kinase(PERK),inositol-requiring kinase-1(IRE1),transcription factor 6(ACT6),and CCAAT/enhancer-binding protein-homologous protein(CHOP).The intracellular Ca2+homeostasis regulated by RCN1 was determined through the detection of Ca2+concentration and mitochondrial membrane potential(MMP)measurement.Moreover,whether inositol 1,4,5-trisphosphate receptor type 1(IP3R1)was involved in the regulation of RCN1 in CRC was verified through the depletion of IP3R1 in RKO cells.Results:Knockdown of RCN1 reduced cell viability and facilitated apoptosis in RKO and SW480 cells.Phosphorylation of PERK and IRE1,activation of ATF6,and upregulation of CHOP were induced by the absence of RCN1,suggesting that the unfolded protein response(UPR)was activated in CRC cells.The concentration of Ca2+in mitochondria was increased after RCN1 depletion,followed by reduction in the MMP and release of cytochrome c from mitochondria to the cytoplasm in RKO and SW480 cells.Moreover,it was demonstrated that IP3R1 mediates the effect of RCN1 on apoptosis induced by ER stress in CRC cells.The downregulation of IP3R1 restored the RCN1 loss-induced apoptosis and the increased Ca2+concentration.Conclusion:Taken together,our results confirmed that silencing of RCN1 disrupted intracellular Ca2+homeostasis and promoted cell apoptosis caused by TG-induced ER stress by regulating IP3R1 and activating the UPR signaling pathways.展开更多
Objective:To investigate the effect of Wujiadan Recipe on PLC-β1,IP3,IP3R,Cam,PLC-β1,IP3R1 mRNA and GnRH,Cam protein from hypothalamus tissue of ovariectomized female rats,and to further clarify whether Wujiadan Rec...Objective:To investigate the effect of Wujiadan Recipe on PLC-β1,IP3,IP3R,Cam,PLC-β1,IP3R1 mRNA and GnRH,Cam protein from hypothalamus tissue of ovariectomized female rats,and to further clarify whether Wujiadan Recipe plays a role through PLC-β/IP3/CaM signal pathway.Methods:We randomly divided 64 SD adult female rats into NG group(normal group),MG group(the model group),TMC group(the control group with traditional Chinese medicine),WMC group(the control group with western medicine)with 10 rats in each,and Wujiadan Recipe high(WH),Wujiadan Recipe medium(WM)and Wujiadan Recipe low dose group(WL)with 8 rats in each.Except the NG group,we removed the ovarian tissues of other six groups to establish the ovariectomized model.When the model is successfully replicated,we give the medicine to the rats in each group once a day.The TMC and WMC group were given the corresponding dose of Gengnian'an tablet suspension and Bujiale suspension by gavage respectively.The NG and MG group were given equal measure of normal saline.After 4 weeks,the rats were killed.We use ELISA to detecte the levels of PLC-β1,IP3,IP3R and Cam;and we use PCR to detecte the mRNA expression levels of PLC-βand IP3R1;and use WesternBlot to detecte the protein expression levels of Cam and GnRH in rat hypothalamus tissue.Results:The test results of PLC-β1,IP3,IP3R and Cam in the hypothalamus homogenate of the model group were significantly increased than the NG group(P<0.01).The results of PLC-β1 and IP3R1 and GnRHmRNA were significantly increased(P<0.01),and the expressions of GnRH and Cam protein were higher than NG(P<0.01).Wujiadan Recipe could reduce the results of PLC-β1,IP3,IP3R and Cam in hypothalamus of ovariectomized rats,and reduce the expression of PLC-β1 and IP3R1 and GnRH mRNA.Conclusion:Wujiadan Recipe down-regulates GnRH expression,which may be caused by acting on PLC-β1,IP3,IP3R,CaM in hypothalamus of ovariectomized rats,so as to coordinate the function of reproductive axis and improve the reproductive endocrine level of patients with menopausal syndrome.展开更多
Objective:To investigate the regulatory effect of Qiliqiangxin Capsule on mitochondrial Ca^(2+)related genes in rats with myocardial infarction(MI).Methods:The rat model of MI was established by ligation of the left a...Objective:To investigate the regulatory effect of Qiliqiangxin Capsule on mitochondrial Ca^(2+)related genes in rats with myocardial infarction(MI).Methods:The rat model of MI was established by ligation of the left anterior descending coronary artery.After operation,the rats were randomly assigned to the model group,the Qiliqiangxin group and the captopril group;a sham-operated group was also available as a control.After four weeks of treatment,the extent of infarction in rats was observed by gross cardiac structure and the morphological changes of myocardial histopathology were observed by HE staining.Detection of mitochondrial Ca^(2+)transport-related genes such as inositol-1,4,5-trisphosphate receptor 2(IP3R2),glucose regulated protein 75(GRP75),voltage-dependent anion channel 1(VDAC1),and mitofusion 2(Mfn2)and mitochondrial apoptosis-related genes such as B-cell lymphoma-2(Bcl-2)and Bcl-2 related X protein(Bax)mRNA expression changes was measured by RT-PCR in the infarct margins of the heart;Western blot was used to detect changes in Bcl-2,Bax protein expression in myocardial tissue.The rate of apoptosis in cardiac myocardial tissue was detected by TUNEL staining.Results:Compared with the sham group,the anterior left ventricular wall of the model group showed a large area of infarction,and the structure of myocardial tissue was disordered.The mRNA expression level of mitochondrial Ca^(2+)transport-related genes such as IP3R2,GRP75,VDAC1,and Mfn2 were significantly increased(P<0.05,P<0.01);The mRNA and protein expression of Bcl-2,a molecule related to mitochondrial apoptosis,were significantly decreased(P<0.01),while the mRNA and protein expression of Bax were significantly increased(P<0.01);and apoptosis rate was significantly increased(P<0.01).Compared with the model group,the infarct size of cardiac gross specimens in the Qiliqiangxin group and the captopril group was reduced and myocardial fibers were relatively well ordered;The mRNA expression of mitochondrial Ca^(2+)transport-related genes such as IP3R2,GRP75,VDAC1,and Mfn2 were significantly reduced(P<0.01);the mRNA and protein expression of Bcl-2,a molecule related to mitochondrial apoptosis,were increased(P<0.05,P<0.01),and the mRNA and protein expression of Bax were significantly decreased(P<0.05,P<0.01).and apoptosis rate was significantly decreased(P<0.01).Conclusion:Qiliqiangxin Capsule can improve the morphological structure of the heart of rats with MI,and its mechanism is related to regulation of the gene expression of mitochondrial Ca^(2+)transport complex IP3R2/GRP75/VDAC1,thereby inhibiting apoptosis.展开更多
文摘Background:The incidence of colorectal cancer(CRC)has been increasing in recent years.Thus,the discovery of factors that can assist in alleviating CRC is urgently warranted.Methods:To identify a potential factor involved in the development of CRC,we screened the upregulated genes in tumor tissues through four datasets from an online database.The expression of reticulocalbin 1(RCN1),a Ca2+-binding protein,was upregulated in the four datasets.Based on loss-offunction experiments,the effect of RCN1 on cell viability was assessed by Cell Counting Kit-8(CCK-8)assay.The regulatory effect of RCN1 on apoptosis was evaluated through Annexin V-fluorescein 5-isothiocyanate(FITC)/propidium iodide(PI)staining assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay in RKO and SW480 cells.Activation of endoplasmic reticulum(ER)stress signaling pathways was confirmed by estimating the phosphorylation and expression of PRKR-like ER kinase(PERK),inositol-requiring kinase-1(IRE1),transcription factor 6(ACT6),and CCAAT/enhancer-binding protein-homologous protein(CHOP).The intracellular Ca2+homeostasis regulated by RCN1 was determined through the detection of Ca2+concentration and mitochondrial membrane potential(MMP)measurement.Moreover,whether inositol 1,4,5-trisphosphate receptor type 1(IP3R1)was involved in the regulation of RCN1 in CRC was verified through the depletion of IP3R1 in RKO cells.Results:Knockdown of RCN1 reduced cell viability and facilitated apoptosis in RKO and SW480 cells.Phosphorylation of PERK and IRE1,activation of ATF6,and upregulation of CHOP were induced by the absence of RCN1,suggesting that the unfolded protein response(UPR)was activated in CRC cells.The concentration of Ca2+in mitochondria was increased after RCN1 depletion,followed by reduction in the MMP and release of cytochrome c from mitochondria to the cytoplasm in RKO and SW480 cells.Moreover,it was demonstrated that IP3R1 mediates the effect of RCN1 on apoptosis induced by ER stress in CRC cells.The downregulation of IP3R1 restored the RCN1 loss-induced apoptosis and the increased Ca2+concentration.Conclusion:Taken together,our results confirmed that silencing of RCN1 disrupted intracellular Ca2+homeostasis and promoted cell apoptosis caused by TG-induced ER stress by regulating IP3R1 and activating the UPR signaling pathways.
基金Health Commission of Heilongjiang Province(2019-085)。
文摘Objective:To investigate the effect of Wujiadan Recipe on PLC-β1,IP3,IP3R,Cam,PLC-β1,IP3R1 mRNA and GnRH,Cam protein from hypothalamus tissue of ovariectomized female rats,and to further clarify whether Wujiadan Recipe plays a role through PLC-β/IP3/CaM signal pathway.Methods:We randomly divided 64 SD adult female rats into NG group(normal group),MG group(the model group),TMC group(the control group with traditional Chinese medicine),WMC group(the control group with western medicine)with 10 rats in each,and Wujiadan Recipe high(WH),Wujiadan Recipe medium(WM)and Wujiadan Recipe low dose group(WL)with 8 rats in each.Except the NG group,we removed the ovarian tissues of other six groups to establish the ovariectomized model.When the model is successfully replicated,we give the medicine to the rats in each group once a day.The TMC and WMC group were given the corresponding dose of Gengnian'an tablet suspension and Bujiale suspension by gavage respectively.The NG and MG group were given equal measure of normal saline.After 4 weeks,the rats were killed.We use ELISA to detecte the levels of PLC-β1,IP3,IP3R and Cam;and we use PCR to detecte the mRNA expression levels of PLC-βand IP3R1;and use WesternBlot to detecte the protein expression levels of Cam and GnRH in rat hypothalamus tissue.Results:The test results of PLC-β1,IP3,IP3R and Cam in the hypothalamus homogenate of the model group were significantly increased than the NG group(P<0.01).The results of PLC-β1 and IP3R1 and GnRHmRNA were significantly increased(P<0.01),and the expressions of GnRH and Cam protein were higher than NG(P<0.01).Wujiadan Recipe could reduce the results of PLC-β1,IP3,IP3R and Cam in hypothalamus of ovariectomized rats,and reduce the expression of PLC-β1 and IP3R1 and GnRH mRNA.Conclusion:Wujiadan Recipe down-regulates GnRH expression,which may be caused by acting on PLC-β1,IP3,IP3R,CaM in hypothalamus of ovariectomized rats,so as to coordinate the function of reproductive axis and improve the reproductive endocrine level of patients with menopausal syndrome.
基金This study was supported by Beijing University of Traditional Chinese Medicine Dongzhimen Hospital 2022 Science and Technology Innovation Special Project(DZMKJCX-2022-008)。
文摘Objective:To investigate the regulatory effect of Qiliqiangxin Capsule on mitochondrial Ca^(2+)related genes in rats with myocardial infarction(MI).Methods:The rat model of MI was established by ligation of the left anterior descending coronary artery.After operation,the rats were randomly assigned to the model group,the Qiliqiangxin group and the captopril group;a sham-operated group was also available as a control.After four weeks of treatment,the extent of infarction in rats was observed by gross cardiac structure and the morphological changes of myocardial histopathology were observed by HE staining.Detection of mitochondrial Ca^(2+)transport-related genes such as inositol-1,4,5-trisphosphate receptor 2(IP3R2),glucose regulated protein 75(GRP75),voltage-dependent anion channel 1(VDAC1),and mitofusion 2(Mfn2)and mitochondrial apoptosis-related genes such as B-cell lymphoma-2(Bcl-2)and Bcl-2 related X protein(Bax)mRNA expression changes was measured by RT-PCR in the infarct margins of the heart;Western blot was used to detect changes in Bcl-2,Bax protein expression in myocardial tissue.The rate of apoptosis in cardiac myocardial tissue was detected by TUNEL staining.Results:Compared with the sham group,the anterior left ventricular wall of the model group showed a large area of infarction,and the structure of myocardial tissue was disordered.The mRNA expression level of mitochondrial Ca^(2+)transport-related genes such as IP3R2,GRP75,VDAC1,and Mfn2 were significantly increased(P<0.05,P<0.01);The mRNA and protein expression of Bcl-2,a molecule related to mitochondrial apoptosis,were significantly decreased(P<0.01),while the mRNA and protein expression of Bax were significantly increased(P<0.01);and apoptosis rate was significantly increased(P<0.01).Compared with the model group,the infarct size of cardiac gross specimens in the Qiliqiangxin group and the captopril group was reduced and myocardial fibers were relatively well ordered;The mRNA expression of mitochondrial Ca^(2+)transport-related genes such as IP3R2,GRP75,VDAC1,and Mfn2 were significantly reduced(P<0.01);the mRNA and protein expression of Bcl-2,a molecule related to mitochondrial apoptosis,were increased(P<0.05,P<0.01),and the mRNA and protein expression of Bax were significantly decreased(P<0.05,P<0.01).and apoptosis rate was significantly decreased(P<0.01).Conclusion:Qiliqiangxin Capsule can improve the morphological structure of the heart of rats with MI,and its mechanism is related to regulation of the gene expression of mitochondrial Ca^(2+)transport complex IP3R2/GRP75/VDAC1,thereby inhibiting apoptosis.
