Background Zearalenone(ZEA)widely exists in moldy grains,which seriously destroys the fertility of females.Isorhamnetin,a natural flavonoid,has extensive of pharmacological activities.However,the beneficial effect and...Background Zearalenone(ZEA)widely exists in moldy grains,which seriously destroys the fertility of females.Isorhamnetin,a natural flavonoid,has extensive of pharmacological activities.However,the beneficial effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced porcine oocyte damage have not been investigated.Methods Oocytes were treated with different concentrations of ZEA(3,5,8 and 10μmol/L)and isorhamnetin(5,10,20 and 30μmol/L)for 44 h at 39℃.ZEA(5μmol/L)and isorhamnetin(10μmol/L)were selected for subsequent studies.Polar body exclusion rate,apoptosis rate and apoptosis related proteins,ROS levels and SOD2 protein,mitochondrial membrane potential and distribution,endoplasmic reticulum distribution and proteins expression,and PI3K,Akt and p-Akt proteins expression of oocytes were detected.In addition,the effect of PI3K antagonist(LY294002)on oocyte nuclear maturation and apoptosis were used to determine the involvement of PI3K/Akt signaling pathway.Results Our findings showed that ZEA exposure damaged oocytes and isorhamnetin therapy restored the developmental capability of porcine oocytes.Isorhamnetin promoted polar body extrusion rate to rescue ZEA-induced meiotic arrest in porcine oocytes.Isorhamnetin alleviated ZEA-induced oxidative stress by stimulating SOD2 protein expression and inhibiting ROS production.Moreover,isorhamnetin enhanced normal mitochondrial distribution and mitochondrial membrane potential to prevent mitochondrial dysfunction induced by ZEA.Changing the expression of endoplasmic reticulum stress-related marker proteins(CHOP,GRP78)and the distribution rate of normal endoplasmic reticulum showed that isorhamnetin relieved ZEA-caused endoplasmic reticulum stress.Mechanistically,isorhamnetin decreased Bax/Bcl-2 protein expression and inhibited ZEA-induced apoptosis through PI3K/Akt signaling pathway.Conclusions Collectively,these results suggest that isorhamnetin protects oocytes from ZEA-caused damage through PI3K/Akt signaling pathway,which enhances meiotic maturation and mitochondrial function,and inhibits early apoptosis,oxidative stress and endoplasmic reticulum stress in porcine oocytes.Our study provides a new strategy for solving the reproductive toxicity induced by ZEA and treating woman infertility.展开更多
BACKGROUND Impaired wound healing can be associated with different pathological states.Burn wounds are the most common and detrimental injuries and remain a major health issue worldwide.Mesenchymal stem cells(MSCs)pos...BACKGROUND Impaired wound healing can be associated with different pathological states.Burn wounds are the most common and detrimental injuries and remain a major health issue worldwide.Mesenchymal stem cells(MSCs)possess the ability to regenerate tissues by secreting factors involved in promoting cell migration,proliferation and differentiation,while suppressing immune reactions.Preconditioning of MSCs with small molecules having cytoprotective properties can enhance the potential of these cells for their use in cell-based therapeutics.AIM To enhance the therapeutic potential of MSCs by preconditioning them with isorhamnetin for second degree burn wounds in rats.METHODS Human umbilical cord MSCs(hU-MSCs)were isolated and characterized by surface markers,CD105,vimentin and CD90.For preconditioning,hU-MSCs were treated with isorhamnetin after selection of the optimized concentration(5μmol/L)by cytotoxicity analysis.The migration potential of these MSCs was analyzed by the in vitro scratch assay.The healing potential of normal,and preconditioned hU-MSCs was compared by transplanting these MSCs in a rat model of a second degree burn wound.Normal,and preconditioned MSCs(IH+MSCs)were transplanted after 72 h of burn injury and observed for 2 wk.Histological and gene expression analyses were performed on day 7 and 14 after cell transplantation to determine complete wound healing.RESULTS The scratch assay analysis showed a significant reduction in the scratch area in the case of IH+MSCs compared to the normal untreated MSCs at 24 h,while complete closure of the scratch area was observed at 48 h.Histological analysis showed reduced inflammation,completely remodeled epidermis and dermis without scar formation and regeneration of hair follicles in the group that received IH+MSCs.Gene expression analysis was time dependent and more pronounced in the case of IH+MSCs.Interleukin(IL)-1β,IL-6 and Bcl-2 associated X genes showed significant downregulation,while transforming growth factorβ,vascular endothelial growth factor,Bcl-2 and matrix metallopeptidase 9 showed significant upregulation compared to the burn wound,showing increased angiogenesis and reduced inflammation and apoptosis.CONCLUSION Preconditioning of hU-MSCs with isorhamnetin decreases wound progression by reducing inflammation,and improving tissue architecture and wound healing.The study outcome is expected to lead to an improved cell-based therapeutic approach for burn wounds.展开更多
Objective To explore the influence of different concentrations of isorhamnetin on C6 glioma cell morphology.Methods Set the blank control group,blank solvent control group and reagent group of four concentration,the g...Objective To explore the influence of different concentrations of isorhamnetin on C6 glioma cell morphology.Methods Set the blank control group,blank solvent control group and reagent group of four concentration,the growth of cells were observed under microscope;MTT assay was used to test the effect of isorhamnetin on cultured C6 glioma cells,as well as calculate the cell inhibition rate and survival rate;flow cytometry was used to check the detection peak and detection rate of Isorhamnetin group and negative control apoptosis group,and analyzed the relationship between different concentrations of isorhamnetin and C6 glioma cell apoptosis rate;total protein was extracted from cells,and used Western blotting to detected total AKT protein and Ser473 AKT protein loci in cells;used SD rats to construct brain glioma model,feed isorhamnetin plain to them for five days,and then used HPLC to detect plasma,liver,brain tissue content.Results Under the observation of inverted microscope and image analysis,after using Isorhamnetin,tumor cells appear apoptosis and necrosis change.Display with different Isorhamnetin MTT colorimetric method shows that the higher the concentration of added Isorhamnetin,the worse the growth rate of C6 glioma cells in vitro,and the higher the Inhibitory rate,the lower survival rate.The flow cytometric detection shows the C6 glioma cells which is added 40 ug/ul Isorhamnetin have the highest rate of apoptosis.After adding 80μg/μl concentration of the isorhamnetin,C6 glioma cells have the lowest survival rate.Western blot test shows the AKT protein and Ser473 total site AKT protein density is in reverse proportion to the increase of the concentration of the isorhamnetin.High performance liquid chromatographic method has determined that there are isorhamnetin in both the rat plasma and brain tissue,which shows that the plasma and tissue all have different isorhamnetin distribution,and isorhamnetin mainly exist in the brain tissue.Conclusion Low concentration of isorhamnetin can induce apoptosis of C6 glioma cells,and high concentration of isorhamnetin can lead to apoptosis and necrosis of C6 glioma cells in vitro,which has obvious inhibitory effect on the growth of glioma cells,and the mechanism is closely related to PI3K/AKT pathway,and in SD rat brain glioma model,the high performance liquid chromatography was used to detect the content of plasma and brain tissue,which indicated the isorhamnetin has target in brain tissue,which provided experimental evidence for the development and utilization of isorhamnetin in mice.展开更多
Background:Influenza A viruses(IAVs)are the major pathogens associated with respiratory infections which can result in extensive pathological damage in lungs and serious complications.Isorhamnetin,an abundant natural ...Background:Influenza A viruses(IAVs)are the major pathogens associated with respiratory infections which can result in extensive pathological damage in lungs and serious complications.Isorhamnetin,an abundant natural flavonoid in fruits and medicinal plants,has recently been shown to have strong antioxidative,anti-inflammatory,and antiviral effects.Objective:This study investigated the pharmacological effects of isorhamnetin on viral pneumonia and explored the underlying mechanisms by in vivo and in vitro experiments.Materials and methods:In the present study,the protective effect of isorhamnetin against IAV was evaluated by the cytopathogenic effect assay,cell counting kit-8 assay,real-time polymerase chain reaction,and immunofluorescence assay in vitro.Then the pathological damage associated with pneumonia was examined by calculating the pulmonary index and performing micro-CT and hematoxylin-eosin staining in vivo.Thereafter,the related protein or gene levels of factors in the mitogen-activated protein kinase(MAPK)and nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathways were determined by Western blot and immunofluorescence staining.Results:Isorhamnetin exerted significant anti-influenza effects and inhibited the expression of viral RNA in A549 cells,counteracting oxidative stress and apoptosis by suppressing the production of reactive oxygen species and caspase-3.The in vivo experiment results showed that isorhamnetin(20 and 40 mg/kg)caused a significant decrease in the pulmonary index,ameliorated pathological damage in the lung tissue,decreased viral load and NA activity,and reduced cytokines and nuclear factors.Furthermore,isorhamnetin could counteract the B cell lymphoma-2/B cell lymphoma-2-associated X protein(Bax)imbalance induced by PR8,suppress activation of the MAPK pathway,and upregulate the expression of Nrf2 and HO-1.Conclusions:Isorhamnetin can protect against viral pneumonia by activating theNrf2/HO-1 pathway and suppressing theMAPK pathway.This study deciphers the pharmacological mechanism of isorhamnetin in alleviating pathological damage in viral pneumonia and provides rationale for the application of isorhamnetin in influenza treatment.展开更多
Objective To investigate the effect of isorhamnetin on the pathology of rheumatoid arthritis(RA).Methods Tumor necrosis factor(TNF)-α-induced fibroblast-like synoviocytes(FLS)was exposed to additional isorhamnetin(10...Objective To investigate the effect of isorhamnetin on the pathology of rheumatoid arthritis(RA).Methods Tumor necrosis factor(TNF)-α-induced fibroblast-like synoviocytes(FLS)was exposed to additional isorhamnetin(10,20 and 40µmol/L).Overexpression vectors for matrix metalloproteinase-2(MMP2)or MMP9 or SRC were transfected to explore their roles in isorhamnetin-mediated RA-FLS function.RA-FLS viability,migration,and invasion were evaluated.Moreover,a collagen-induced arthritis(CIA)rat model was established.Rats were randomly divided to sham,CIA,low-,medium-,and high-dosage groups using a random number table(n=5 in each group)and administed with normal saline or additional isorhamnetin[2,10,and 20 mg/(kg·day)]for 4 weeks,respectively.Arthritis index was calculated and synovial tissue inflammation was determined in CIA rats.The levels of MMP2,MMP9,TNF-α,interleukin-6(IL-6),and IL-1β,as well as the phosphorylation levels of SRC,extracellular regulated kinase(ERK),and cyclic adenosine monophosphate response element-binding(CREB),were detected in RA-FLS and synovial tissue.Molecular docking was also used to analyze the binding of isorhamnetin to SRC.Results In in vitro studies,isorhamnetin inhibited RA-FLS viability,migration and invasion(P<0.05).Isorhamnetin downregulated the levels of MMP2,MMP9,TNF-α,IL-6,and IL-1βin RA-FLS(P<0.05).The overexpression of either MMP2 or MMP9 reversed isorhamnetin-inhibited RA-FLS migration and invasion,as well as the levels of TNF-α,IL-6,and IL-1β(P<0.05).Furthermore,isorhamnetin bound to SRC and reduced the phosphorylation of SRC,ERK,and CREB(P<0.05).SRC overexpression reversed the inhibitory effect of isorhamnetin on RA-FLS viability,migration and invasion,as well as the negative regulation of MMP2 and MMP9(P<0.05).In in vivo studies,isorhamnetin decreased arthritis index scores(P<0.05)and alleviated synovial inflammation.Isorhamnetin reduced the levels of MMP2,MMP9,TNF-α,IL-6,and IL-1β,as well as the phosphorylation of SRC,ERK,and CREB in synovial tissue(P<0.05).Notably,the inhibitory effect of isorhamnetin was more pronounced at higher concentrations(P<0.05).Conclusion Isorhamnetin exhibited anti-RA effects through modulating SRC/ERK/CREB and MMP2/MMP9 signaling pathways,suggesting that isorhamnetin may be a potential therapeutic agent for RA.展开更多
Zearalenone(ZEA)is widely derived from moldy cereal grain,which has adverse effects on animal reproduction.In particular,pigs are more sensitive to ZEA-induced toxicity than other animals.Isorhamnetin has extensive ph...Zearalenone(ZEA)is widely derived from moldy cereal grain,which has adverse effects on animal reproduction.In particular,pigs are more sensitive to ZEA-induced toxicity than other animals.Isorhamnetin has extensive pharmacological activity.However,the role of isorhamnetin in ZEA-induced cytotoxicity remains unclear.This study was designed to investigate the therapeutic effect of isorhamnetin on ZEA-induced damage in porcine ovarian granulosa cells and elucidate its molecular mechanism.Two experiments were conducted,where a minimum of 3 biological replicates were used for each treatment.In Exp.1,ovarian granulosa cells were treated with different concentrations of isorhamnetin(1,5,10,20 and 30μmol/L)and ZEA(0,10,30,60,90 and 120μmol/L)for 24 h.Our results indicated that 60μmol/L ZEA(half-maximal inhibitory concentration value)and 20μmol/L isorhamnetin(the most effective concentration against ZEA-induced cytotoxicity)were optimum concentrations.In Exp.2,ovarian granulosa cells were treated with isorhamnetin(20μmol/L)for 2 h,before treatment with ZEA(60μmol/L)for 24 h.Apoptosis,endoplasmic reticulum stress,oxidative stress,proliferation and hormone secretion of ovarian granulosa cells were detected.Our findings showed that isorhamnetin suppressed(P<0.05)ZEA-induced apoptosis by altering mitochondrial membrane potential and apoptosis-related proteins(B-cell lymphoma-2[Bcl-2],Bcl2-associated x[Bax]and cleaved caspase-3[C-Casp3]).Changes in intracellular Ca2+levels and C/EBP homologous protein(CHOP),recombinant activating transcription factor 6(ATF6),glucose regulated protein78 k D(GRP78)indicated that isorhamnetin rescued(P<0.05)ZEA-induced endoplasmic reticulum stress.Furthermore,isorhamnetin prevented(P<0.05)ZEA-induced oxidative stress via the mitogen-activated protein kinase(P38)signaling pathway.Mechanistically,isorhamnetin stimulated(P<0.05)the expression of proliferating cell nuclear antigen(PCNA)and cyclin D,thereby increasing the ratio of S phase cells in response to ZEAinduced apoptosis via phosphatidylinositol 3 kinase/protein kinase B(PI3K/Akt)signaling pathway.Isorhamnetin also recovered(P<0.05)ZEA-induced steroidogenesis disorder by regulating steroidogenic enzyme gene and proteins(follicle-stimulating hormone receptor[FSHR]and cytochrome P450 family 19subfamily a member 1[CYP19A1]).Collectively,these findings show that isorhamnetin protects ovarian granulosa cells from ZEA-induced damage,which promotes proliferation,alleviates apoptosis,endoplasmic reticulum stress,oxidative stress,and steroidogenesis disorder.展开更多
基金supported Key Industry Innovation Chain of Shaanxi Province (2018ZDCXL-NY-02-06)。
文摘Background Zearalenone(ZEA)widely exists in moldy grains,which seriously destroys the fertility of females.Isorhamnetin,a natural flavonoid,has extensive of pharmacological activities.However,the beneficial effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced porcine oocyte damage have not been investigated.Methods Oocytes were treated with different concentrations of ZEA(3,5,8 and 10μmol/L)and isorhamnetin(5,10,20 and 30μmol/L)for 44 h at 39℃.ZEA(5μmol/L)and isorhamnetin(10μmol/L)were selected for subsequent studies.Polar body exclusion rate,apoptosis rate and apoptosis related proteins,ROS levels and SOD2 protein,mitochondrial membrane potential and distribution,endoplasmic reticulum distribution and proteins expression,and PI3K,Akt and p-Akt proteins expression of oocytes were detected.In addition,the effect of PI3K antagonist(LY294002)on oocyte nuclear maturation and apoptosis were used to determine the involvement of PI3K/Akt signaling pathway.Results Our findings showed that ZEA exposure damaged oocytes and isorhamnetin therapy restored the developmental capability of porcine oocytes.Isorhamnetin promoted polar body extrusion rate to rescue ZEA-induced meiotic arrest in porcine oocytes.Isorhamnetin alleviated ZEA-induced oxidative stress by stimulating SOD2 protein expression and inhibiting ROS production.Moreover,isorhamnetin enhanced normal mitochondrial distribution and mitochondrial membrane potential to prevent mitochondrial dysfunction induced by ZEA.Changing the expression of endoplasmic reticulum stress-related marker proteins(CHOP,GRP78)and the distribution rate of normal endoplasmic reticulum showed that isorhamnetin relieved ZEA-caused endoplasmic reticulum stress.Mechanistically,isorhamnetin decreased Bax/Bcl-2 protein expression and inhibited ZEA-induced apoptosis through PI3K/Akt signaling pathway.Conclusions Collectively,these results suggest that isorhamnetin protects oocytes from ZEA-caused damage through PI3K/Akt signaling pathway,which enhances meiotic maturation and mitochondrial function,and inhibits early apoptosis,oxidative stress and endoplasmic reticulum stress in porcine oocytes.Our study provides a new strategy for solving the reproductive toxicity induced by ZEA and treating woman infertility.
文摘BACKGROUND Impaired wound healing can be associated with different pathological states.Burn wounds are the most common and detrimental injuries and remain a major health issue worldwide.Mesenchymal stem cells(MSCs)possess the ability to regenerate tissues by secreting factors involved in promoting cell migration,proliferation and differentiation,while suppressing immune reactions.Preconditioning of MSCs with small molecules having cytoprotective properties can enhance the potential of these cells for their use in cell-based therapeutics.AIM To enhance the therapeutic potential of MSCs by preconditioning them with isorhamnetin for second degree burn wounds in rats.METHODS Human umbilical cord MSCs(hU-MSCs)were isolated and characterized by surface markers,CD105,vimentin and CD90.For preconditioning,hU-MSCs were treated with isorhamnetin after selection of the optimized concentration(5μmol/L)by cytotoxicity analysis.The migration potential of these MSCs was analyzed by the in vitro scratch assay.The healing potential of normal,and preconditioned hU-MSCs was compared by transplanting these MSCs in a rat model of a second degree burn wound.Normal,and preconditioned MSCs(IH+MSCs)were transplanted after 72 h of burn injury and observed for 2 wk.Histological and gene expression analyses were performed on day 7 and 14 after cell transplantation to determine complete wound healing.RESULTS The scratch assay analysis showed a significant reduction in the scratch area in the case of IH+MSCs compared to the normal untreated MSCs at 24 h,while complete closure of the scratch area was observed at 48 h.Histological analysis showed reduced inflammation,completely remodeled epidermis and dermis without scar formation and regeneration of hair follicles in the group that received IH+MSCs.Gene expression analysis was time dependent and more pronounced in the case of IH+MSCs.Interleukin(IL)-1β,IL-6 and Bcl-2 associated X genes showed significant downregulation,while transforming growth factorβ,vascular endothelial growth factor,Bcl-2 and matrix metallopeptidase 9 showed significant upregulation compared to the burn wound,showing increased angiogenesis and reduced inflammation and apoptosis.CONCLUSION Preconditioning of hU-MSCs with isorhamnetin decreases wound progression by reducing inflammation,and improving tissue architecture and wound healing.The study outcome is expected to lead to an improved cell-based therapeutic approach for burn wounds.
文摘Objective To explore the influence of different concentrations of isorhamnetin on C6 glioma cell morphology.Methods Set the blank control group,blank solvent control group and reagent group of four concentration,the growth of cells were observed under microscope;MTT assay was used to test the effect of isorhamnetin on cultured C6 glioma cells,as well as calculate the cell inhibition rate and survival rate;flow cytometry was used to check the detection peak and detection rate of Isorhamnetin group and negative control apoptosis group,and analyzed the relationship between different concentrations of isorhamnetin and C6 glioma cell apoptosis rate;total protein was extracted from cells,and used Western blotting to detected total AKT protein and Ser473 AKT protein loci in cells;used SD rats to construct brain glioma model,feed isorhamnetin plain to them for five days,and then used HPLC to detect plasma,liver,brain tissue content.Results Under the observation of inverted microscope and image analysis,after using Isorhamnetin,tumor cells appear apoptosis and necrosis change.Display with different Isorhamnetin MTT colorimetric method shows that the higher the concentration of added Isorhamnetin,the worse the growth rate of C6 glioma cells in vitro,and the higher the Inhibitory rate,the lower survival rate.The flow cytometric detection shows the C6 glioma cells which is added 40 ug/ul Isorhamnetin have the highest rate of apoptosis.After adding 80μg/μl concentration of the isorhamnetin,C6 glioma cells have the lowest survival rate.Western blot test shows the AKT protein and Ser473 total site AKT protein density is in reverse proportion to the increase of the concentration of the isorhamnetin.High performance liquid chromatographic method has determined that there are isorhamnetin in both the rat plasma and brain tissue,which shows that the plasma and tissue all have different isorhamnetin distribution,and isorhamnetin mainly exist in the brain tissue.Conclusion Low concentration of isorhamnetin can induce apoptosis of C6 glioma cells,and high concentration of isorhamnetin can lead to apoptosis and necrosis of C6 glioma cells in vitro,which has obvious inhibitory effect on the growth of glioma cells,and the mechanism is closely related to PI3K/AKT pathway,and in SD rat brain glioma model,the high performance liquid chromatography was used to detect the content of plasma and brain tissue,which indicated the isorhamnetin has target in brain tissue,which provided experimental evidence for the development and utilization of isorhamnetin in mice.
基金National Natural Science Foundation of China(82104500,82141206,82305078).
文摘Background:Influenza A viruses(IAVs)are the major pathogens associated with respiratory infections which can result in extensive pathological damage in lungs and serious complications.Isorhamnetin,an abundant natural flavonoid in fruits and medicinal plants,has recently been shown to have strong antioxidative,anti-inflammatory,and antiviral effects.Objective:This study investigated the pharmacological effects of isorhamnetin on viral pneumonia and explored the underlying mechanisms by in vivo and in vitro experiments.Materials and methods:In the present study,the protective effect of isorhamnetin against IAV was evaluated by the cytopathogenic effect assay,cell counting kit-8 assay,real-time polymerase chain reaction,and immunofluorescence assay in vitro.Then the pathological damage associated with pneumonia was examined by calculating the pulmonary index and performing micro-CT and hematoxylin-eosin staining in vivo.Thereafter,the related protein or gene levels of factors in the mitogen-activated protein kinase(MAPK)and nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathways were determined by Western blot and immunofluorescence staining.Results:Isorhamnetin exerted significant anti-influenza effects and inhibited the expression of viral RNA in A549 cells,counteracting oxidative stress and apoptosis by suppressing the production of reactive oxygen species and caspase-3.The in vivo experiment results showed that isorhamnetin(20 and 40 mg/kg)caused a significant decrease in the pulmonary index,ameliorated pathological damage in the lung tissue,decreased viral load and NA activity,and reduced cytokines and nuclear factors.Furthermore,isorhamnetin could counteract the B cell lymphoma-2/B cell lymphoma-2-associated X protein(Bax)imbalance induced by PR8,suppress activation of the MAPK pathway,and upregulate the expression of Nrf2 and HO-1.Conclusions:Isorhamnetin can protect against viral pneumonia by activating theNrf2/HO-1 pathway and suppressing theMAPK pathway.This study deciphers the pharmacological mechanism of isorhamnetin in alleviating pathological damage in viral pneumonia and provides rationale for the application of isorhamnetin in influenza treatment.
基金Supported by the Natural Science Foundation of Hunan Province(Nos.2022JJ80086 and 2023JJ60342)the Project of Hunan Provincial Health and Health Commission(No.D202302078705)+4 种基金the Project of Hunan Provincial Student Innovation and Entrepreneurship Training Program(No.2022-5313)the Hunan Provincial Administration of Traditional Chinese Medicine Scientific Research Program(No.2021161)the Hunan University of Traditional Chinese Medicine Primary Discipline Open Fund Project in Chinese Medicine(No.2020ZYX01)the Key Discipline Project on Chinese Pharmacology of Hunan University of Chinese Medicine(No.202302)the Scientific Research Project of Hunan Provincial Administration of Traditional Chinese Medicine(No.B2023150)。
文摘Objective To investigate the effect of isorhamnetin on the pathology of rheumatoid arthritis(RA).Methods Tumor necrosis factor(TNF)-α-induced fibroblast-like synoviocytes(FLS)was exposed to additional isorhamnetin(10,20 and 40µmol/L).Overexpression vectors for matrix metalloproteinase-2(MMP2)or MMP9 or SRC were transfected to explore their roles in isorhamnetin-mediated RA-FLS function.RA-FLS viability,migration,and invasion were evaluated.Moreover,a collagen-induced arthritis(CIA)rat model was established.Rats were randomly divided to sham,CIA,low-,medium-,and high-dosage groups using a random number table(n=5 in each group)and administed with normal saline or additional isorhamnetin[2,10,and 20 mg/(kg·day)]for 4 weeks,respectively.Arthritis index was calculated and synovial tissue inflammation was determined in CIA rats.The levels of MMP2,MMP9,TNF-α,interleukin-6(IL-6),and IL-1β,as well as the phosphorylation levels of SRC,extracellular regulated kinase(ERK),and cyclic adenosine monophosphate response element-binding(CREB),were detected in RA-FLS and synovial tissue.Molecular docking was also used to analyze the binding of isorhamnetin to SRC.Results In in vitro studies,isorhamnetin inhibited RA-FLS viability,migration and invasion(P<0.05).Isorhamnetin downregulated the levels of MMP2,MMP9,TNF-α,IL-6,and IL-1βin RA-FLS(P<0.05).The overexpression of either MMP2 or MMP9 reversed isorhamnetin-inhibited RA-FLS migration and invasion,as well as the levels of TNF-α,IL-6,and IL-1β(P<0.05).Furthermore,isorhamnetin bound to SRC and reduced the phosphorylation of SRC,ERK,and CREB(P<0.05).SRC overexpression reversed the inhibitory effect of isorhamnetin on RA-FLS viability,migration and invasion,as well as the negative regulation of MMP2 and MMP9(P<0.05).In in vivo studies,isorhamnetin decreased arthritis index scores(P<0.05)and alleviated synovial inflammation.Isorhamnetin reduced the levels of MMP2,MMP9,TNF-α,IL-6,and IL-1β,as well as the phosphorylation of SRC,ERK,and CREB in synovial tissue(P<0.05).Notably,the inhibitory effect of isorhamnetin was more pronounced at higher concentrations(P<0.05).Conclusion Isorhamnetin exhibited anti-RA effects through modulating SRC/ERK/CREB and MMP2/MMP9 signaling pathways,suggesting that isorhamnetin may be a potential therapeutic agent for RA.
基金financially supported by Ministry of Agriculture Transgenic Major Projects(2018ZX0801013B)Key Industry Innovation Chain of Shaanxi Province(2018ZDCXL-NY-02-06)。
文摘Zearalenone(ZEA)is widely derived from moldy cereal grain,which has adverse effects on animal reproduction.In particular,pigs are more sensitive to ZEA-induced toxicity than other animals.Isorhamnetin has extensive pharmacological activity.However,the role of isorhamnetin in ZEA-induced cytotoxicity remains unclear.This study was designed to investigate the therapeutic effect of isorhamnetin on ZEA-induced damage in porcine ovarian granulosa cells and elucidate its molecular mechanism.Two experiments were conducted,where a minimum of 3 biological replicates were used for each treatment.In Exp.1,ovarian granulosa cells were treated with different concentrations of isorhamnetin(1,5,10,20 and 30μmol/L)and ZEA(0,10,30,60,90 and 120μmol/L)for 24 h.Our results indicated that 60μmol/L ZEA(half-maximal inhibitory concentration value)and 20μmol/L isorhamnetin(the most effective concentration against ZEA-induced cytotoxicity)were optimum concentrations.In Exp.2,ovarian granulosa cells were treated with isorhamnetin(20μmol/L)for 2 h,before treatment with ZEA(60μmol/L)for 24 h.Apoptosis,endoplasmic reticulum stress,oxidative stress,proliferation and hormone secretion of ovarian granulosa cells were detected.Our findings showed that isorhamnetin suppressed(P<0.05)ZEA-induced apoptosis by altering mitochondrial membrane potential and apoptosis-related proteins(B-cell lymphoma-2[Bcl-2],Bcl2-associated x[Bax]and cleaved caspase-3[C-Casp3]).Changes in intracellular Ca2+levels and C/EBP homologous protein(CHOP),recombinant activating transcription factor 6(ATF6),glucose regulated protein78 k D(GRP78)indicated that isorhamnetin rescued(P<0.05)ZEA-induced endoplasmic reticulum stress.Furthermore,isorhamnetin prevented(P<0.05)ZEA-induced oxidative stress via the mitogen-activated protein kinase(P38)signaling pathway.Mechanistically,isorhamnetin stimulated(P<0.05)the expression of proliferating cell nuclear antigen(PCNA)and cyclin D,thereby increasing the ratio of S phase cells in response to ZEAinduced apoptosis via phosphatidylinositol 3 kinase/protein kinase B(PI3K/Akt)signaling pathway.Isorhamnetin also recovered(P<0.05)ZEA-induced steroidogenesis disorder by regulating steroidogenic enzyme gene and proteins(follicle-stimulating hormone receptor[FSHR]and cytochrome P450 family 19subfamily a member 1[CYP19A1]).Collectively,these findings show that isorhamnetin protects ovarian granulosa cells from ZEA-induced damage,which promotes proliferation,alleviates apoptosis,endoplasmic reticulum stress,oxidative stress,and steroidogenesis disorder.
