阿魏酸对糖尿病小鼠视网膜和高糖诱导的人RPE细胞损伤的抑制作用及其机制

目的探讨阿魏酸对糖尿病小鼠视网膜和高糖诱导的人视网膜色素上皮(RPE)细胞损伤的抑制作用及其机制。方法选取SPF级雄性8周龄2型糖尿病db/db小鼠30只,采用随机数字表法将小鼠分为模型组和阿魏酸组,每组15只,另选取15只同周龄db/m小鼠作为对照组。模型组和对照组每日采用生理盐水灌胃(5 ml/kg),阿魏酸组采用阿魏酸溶液灌胃(0.05 g/kg),治疗后2个月处死各组小鼠并摘除眼球。采用苏木精-伊红染色观察小鼠视网膜组织形态学变化;采用免疫荧光染色法和Western blot法检测各组小鼠视网膜组织线粒体钙离子单向转运蛋白(MCU)、p38丝裂原活化蛋白激酶(p38MAPK)、磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)蛋白荧光强度和表达水平。取人RPE细胞,将其分为对照组、二甲基亚砜(DMSO)组、高糖组和高糖+阿魏酸组,其中对照组不做任何处理,其余各组用相应试剂培养24 h。采用活性氧簇(ROS)检测试剂盒检测各组RPE细胞ROS水平;采用线粒体膜电位检测试剂盒(JC-1)检测各组RPE细胞线粒体膜电位水平;采用微丝绿色荧光探针检测各组RPE细胞MCU和微丝荧光强度;通过慢病毒转染技术沉默和过表达MCU蛋白水平探讨MCU与p38 MAPK、p-p38MAPK间的调控关系;采用免疫荧光染色法和Western blot法检测各组RPE细胞MCU、p38 MAPK、p-p38MAPK蛋白荧光强度和表达水平。结果与对照组比较,模型组小鼠视网膜组织外核层、内核层和神经节细胞层细胞间隙增大、排列紊乱,阿魏酸组小鼠视网膜组织明显改善。与对照组比较,模型组、阿魏酸组小鼠视网膜组织MCU、p-p38 MAPK和MCU+p-p38 MAPK蛋白荧光强度显著升高,差异均有统计学意义(均P<0.05);与模型组比较,阿魏酸组小鼠视网膜组织MCU、p-p38 MAPK和MCU+p-p38 MAPK蛋白荧光强度显著降低,差异均有统计学意义(均P<0.05)。与对照组比较,模型组小鼠视网膜组织MCU、p38 MAPK和p-p38 MAPK蛋白相对表达量显著升高,差异均有统计学意义(均P<0.05);与模型组比较,阿魏酸组小鼠视网膜组织MCU、p38 MAPK和p-p38 MAPK蛋白相对表达量显著降低,差异均有统计学意义(均P<0.05)。对照组、DMSO组、高糖组和高糖+阿魏酸组细胞ROS荧光强度分别为0.22±0.02、0.22±0.03、0.30±0.02和0.24±0.02,总体比较差异均有统计学意义(F=7.845,P<0.01),其中高糖组细胞ROS荧光强度明显高于对照组和DMSO组,高糖+阿魏酸组细胞ROS荧光强度明显低于高糖组,差异均有统计学意义(均P<0.05)。高糖组、高糖+阿魏酸组细胞线粒体膜电位水平明显低于对照组和DMSO组,高糖+阿魏酸组细胞线粒体膜电位水平明显高于高糖组,差异均有统计学意义(均P<0.05)。与对照组、DMSO组比较,高糖组MCU荧光强度较高,并伴随着细胞微丝减少和变细;高糖+阿魏酸组MCU蛋白荧光强度明显下降,细胞微丝数量明显增加。与对照组、DMSO组比较,高糖组细胞MCU、p38 MAPK和p-p38 MAPK蛋白荧光强度和相对表达量显著升高,差异均有统计学意义(均P<0.05);与高糖组比较,高糖+阿魏酸组细胞MCU、p38 MAPK和p-p38 MAPK蛋白荧光强度和相对表达量显著降低,差异均有统计学意义(均P<0.05)。与对照组和空载体组比较,MCU过表达组细胞MCU、p38 MAPK和p-p38 MAPK蛋白相对表达量显著升高,MCU shRNA组、MCU过表达+阿魏酸组细胞MCU、p38 MAPK和p-p38 MAPK蛋白相对表达量显著降低,差异均有统计学意义(均P<0.05);与MCU过表达组比较,MCU shRNA组、MCU过表达+阿魏酸组细胞MCU、p38 MAPK和p-p38 MAPK蛋白相对表达量显著降低,差异均有统计学意义(均P<0.05)。结论阿魏酸能够调控氧化应激和线粒体功能障碍,进而改善糖尿病小鼠视网膜和高糖诱导的RPE细胞损伤,其可能通过MCU及p38MAPK信号通路发挥保护作用。

Inflammatory response in gastrointestinal cancers:Overview of six transmembrane epithelial antigens of the prostate in pathophysiology and clinical implications

Chronic inflammation is known to increase the risk of gastrointestinal cancers(GICs),the common solid tumors worldwide.Precancerous lesions,such as chronic atrophic inflammation and ulcers,are related to inflammatory responses in vivo and likely to occur in hyperplasia and tumorigenesis.Unfortunately,due to the lack of effective therapeutic targets,the prognosis of patients with GICs is still unsatisfactory.Interestingly,it is found that six transmembrane epithelial antigens of the prostate(STEAPs),a group of metal reductases,are significantly associated with the progression of malignancies,playing a crucial role in systemic metabolic homeostasis and inflammatory responses.The structure and functions of STEAPs suggest that they are closely related to intracellular oxidative stress,responding to inflammatory reactions.Under the imbalance status of abnormal oxidative stress,STEAP members are involved in cell transformation and the development of GICs by inhibiting or activating inflammatory process.This review focuses on STEAPs in GICs along with exploring their potential molecular regulatory mechanisms,with an aim to provide a theoretical basis for diagnosis and treatment strategies for patients suffering from these types of cancers.

Lutein-stevioside nanoparticle attenuates H_(2)O_(2)-induced oxidative damage in ARPE cells

In order to improve the bioavailability of lutein(LUT),a novel lutein-stevio side nanoparticle(LUT-STE)were prepared previously,but the information about LUT-STE on protecting of eye health was limited.This study investigated the effect of LUT-STE on antioxidant activity of H_(2)O_(2)-induced human retinal pigment epithelial(ARPE)cells.LUT and LUT-STE(final concentration of 5μg/mL)significantly enhanced cell viability from(74.84±5.10)%to(81.92±10.01)%(LUT)and(89.33±4.34)%(LUT-STE),and inhibited the cell apoptosis(P<0.05).After pretreatment with LUT-STE in ARPE cells,the levels of superoxide dismutase(SOD),catalase(CAT)and glutathion peroxidase(GSH-Px)in ARPE cells were significantly increased(P<0.05),the contents of reactive oxygen species(ROS)and malondialdehyde(MDA)were decreased.In addition,the vascular endothelial growth factor(VEGF)levels were inhibited by 13.61%and 17.39%,respectively,pretreatment with LUT and LUT-STE.Western blotting results showed that the pretreatment with LUT-STE inhibited the expression of caspase-9 and caspase-3 and up-regulated Bcl-2/Bax pathway to inhibit H_(2)O_(2)-induced apoptosis.In summary,the novel delivery LUT-STE had more pronounced inhibitory effect on H_(2)O_(2)-induced damage in human ARPE cells.

Hesperidin ameliorates H_(2)O_(2)-induced bovine mammary epithelial cell oxidative stress via the Nrf2 signaling pathway

Background Hesperidin is a citrus flavonoid with anti-inflammatory and antioxidant potential. However, its protective effects on bovine mammary epithelial cells(b MECs) exposed to oxidative stress have not been elucidated.Results In this study, we investigated the effects of hesperidin on H_(2)O_(2)-induced oxidative stress in b MECs and the underlying molecular mechanism. We found that hesperidin attenuated H_(2)O_(2)-induced cell damage by reducing reactive oxygen species(ROS) and malondialdehyde(MDA) levels, increasing catalase(CAT) activity, and improving cell proliferation and mitochondrial membrane potential. Moreover, hesperidin activated the Keap1/Nrf2/ARE signaling pathway by inducing the nuclear translocation of Nrf2 and the expression of its downstream genes NQO1 and HO-1, which are antioxidant enzymes involved in ROS scavenging and cellular redox balance. The protective effects of hesperidin were blocked by the Nrf2 inhibitor ML385, indicating that they were Nrf2 dependent.Conclusions Our results suggest that hesperidin could protect b MECs from oxidative stress injury by activating the Nrf2 signaling pathway, suggesting that hesperidin as a natural antioxidant has positive potential as a feed additive or plant drug to promote the health benefits of bovine mammary.

Effect of atractylenolide Ⅲ on zearalenone-induced Snail1-mediated epithelial–mesenchymal transition in porcine intestinal epithelium

Background The intestinal epithelium performs essential physiological functions,such as nutrient absorption,and acts as a barrier to prevent the entry of harmful substances.Mycotoxins are prevalent contaminants found in ani-mal feed that exert harmful effects on the health of livestock.Zearalenone(ZEA)is produced by the Fusarium genus and induces gastrointestinal dysfunction and disrupts the health and immune system of animals.Here,we evaluated the molecular mechanisms that regulate the effects of ZEA on the porcine intestinal epithelium.Results Treatment of IPEC-J2 cells with ZEA decreased the expression of E-cadherin and increased the expression of Snai1 and Vimentin,which induced Snail1-mediated epithelial-to-mesenchymal transition(EMT).In addition,ZEA induces Snail-mediated EMT through the activation of TGF-βsignaling.The treatment of IPEC-J2 cells with atractyle-nolideⅢ,which were exposed to ZEA,alleviated EMT.Conclusions Our findings provide insights into the molecular mechanisms of ZEA toxicity in porcine intestinal epi-thelial cells and ways to mitigate it.

circRNA3669 promotes goat endometrial epithelial cells proliferation via miR-26a/RCN2 to activate PI3K/AKT-mTOR and MAPK pathways

The development of receptive endometrium(RE) from pre-receptive endometrium(PE) for successful embryo implantation is a complex dynamic process in which the morphology and physiological states of the endometrial epithelium undergo a series of significant changes, including cell proliferation and apoptosis. However, the molecular mechanisms are not yet fully understood. In this study, a higher circRNA3669 level was observed in PE than in RE of goats. Functional assays revealed that this overexpression promoted the proliferation of goat endometrial epithelial cells(GEECs) by activating the expression of genes related to the PI3K/AKT-mTOR and MAPK pathways,thereby inhibiting apoptosis in vitro. Furthermore, circRNA3669 functioned as a competing endogenous RNA(ceRNA) to upregulate Reticulocalbin-2(RCN2) expression at the post-transcriptional level by interacting with and downregulating miR-26a in GEECs. In addition, RCN2, which is highly expressed in the PE of goats, was found to be regulated by β-estradiol(E2) and progesterone(P4). Our results demonstrated that RCN2 also affected the key proteins PI3K, AKT, mTOR, JNK, and P38 in the PI3K/AKT-mTOR and MAPK pathways, thereby facilitating GEECs proliferation and suppressing their apoptosis in vitro. Collectively, we constructed a new circRNA3669-miR-26aRCN2 regulatory network in GEECs, which further provides strong evidence that circRNA could potentially play a crucial regulatory role in the development of RE in goats.

Effect of acacetin on inhibition of apoptosis in Helicobacter pyloriinfected gastric epithelial cell line

BACKGROUND Helicobacter pylori(H.pylori)infection can cause extensive apoptosis of gastric epithelial cells,serving as a critical catalyst in the progression from chronic gastritis,gastrointestinal metaplasia,and atypical gastric hyperplasia to gastric carcinoma.Prompt eradication of H.pylori is paramount for ameliorating the pathophysiological conditions associated with chronic inflammation of the gastric mucosa and the primary prevention of gastric cancer.Acacetin,which has multifaceted pharmacological activities such as anti-cancer,anti-inflammatory,and antioxidative properties,has been extensively investigated across various domains.Nevertheless,the impact and underlying mechanisms of action of acacetin on H.pylori-infected gastric mucosal epithelial cells remain unclear.AIM To explore the defensive effects of acacetin on apoptosis in H.pylori-infected GES-1 cells and to investigate the underlying mechanisms.METHODS GES-1 cells were treated with H.pylori and acacetin in vitro.Cell viability was assessed using the CCK-8 assay,cell mortality rate via lactate dehydrogenase assay,alterations in cell migration and healing capacities through the wound healing assay,rates of apoptosis via flow cytometry and TUNEL staining,and expression levels of apoptosis-associated proteins through western blot analysis.RESULTS H.pylori infection led to decreased GES-1 cell viability,increased cell mortality,suppressed cell migration,increased rate of apoptosis,increased expressions of Bax and cle-caspase3,and decreased Bcl-2 expression.Conversely,acacetin treatment enhanced cell viability,mitigated apoptosis induced by H.pylori infection,and modulated the expression of apoptosis-regulatory proteins by upregulating Bcl-2 and downregulating Bax and cleaved caspase-3.CONCLUSION Acacetin significantly improved GES-1 cell viability and inhibited apoptosis in H.pylori-infected GES-1 cells,thereby exerting a protective effect on gastric mucosal epithelial cells.

Overexpression of TRPV1 activates autophagy in human lens epithelial cells under hyperosmotic stress through Ca^(2+)-dependent AMPK/mTOR pathway

●AIM:To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells(LECs)under hyperosmotic stress.●METHODS:LECs were treated with hyperosmotic stress at the concentration of 270,300,400,500,or 600 mOsm for 6,12,18,24h in vitro.Polymerase chain reaction(PCR)was employed for the mRNA expression of autophagyrelated genes,while Western blotting detected the targeted protein expression.The transfection of stub-RFP-sens-GFPLC3 autophagy-related double fluorescence lentivirus was conducted to detect the level of autophagy flux.Scanning electron microscopy was used to detect the existence of autolysosome.Short interfering RNA of autophagy-related gene(ATG)7,transient receptor potential vanilloid(TRPV)1 overexpression plasmid,related agonists and inhibitors were employed to their influence on autophagy related pathway.Flow cytometry was employed to test the apoptosis and intracellular Ca^(2+)level.Mitochondrial membrane potential was measured by JC-1 staining.The cell counting kit-8 assay was used to calculate the cellular viability.The wound healing assay was used to evaluate the wound closure rate.GraphPad 6.0 software was utilized to evaluate the data.●RESULTS:The hyperosmotic stress activated autophagy in a pressure-and time-dependent manner in LECs.Beclin 1 protein expression and conversion of LC3B II to LC3B I increased,whereas sequestosome-1(SQSTM1)protein expression decreased.Transient Ca^(2+)influx was stimulated caused by hyperosmotic stress,levels of mammalian target of rapamycin(mTOR)phosphorylation decreased,and the level of AMP-activated protein kinase(AMPK)phosphorylation increased in the early stage.Based on this evidence,autophagy activation through the Ca^(2+)-dependent AMPK/mTOR pathway might represent an adaptation process in LECs under hyperosmotic stress.Hyperosmotic stress decreased cellular viability and accelerated apoptosis in LECs and cellular migration decreased.Inhibition of autophagy by ATG7 knockdown had similar results.TRPV1 overexpression increased autophagy and might be crucial in the occurrence of autophagy promoted by hyperosmotic stress.●CONCLUSION:A combination of hyperosmotic stress and autophagy inhibition may be a promising approach to decrease the number of LECs in the capsular bag and pave the way for improving prevention of posterior capsular opacification and capsular fibrosis.

Semaphorin 7A impairs barrier function in cultured human corneal epithelial cells in a manner dependent on nuclear factor-kappa B

●AIM:To evaluate the role of semaphorin 7A(Sema7A)and its associated regulatory mechanisms in modulating the barrier function of cultured human corneal epithelial cells(HCEs).●METHODS:Barrier models of HCEs were treated with recombinant human Sema7A at concentrations of 0,125,250,or 500 ng/mL for 24,48,or 72h in vitro.Transepithelial electrical resistance(TEER)as well as Dextran-fluorescein isothiocyanate(FITC)permeability assays were conducted to assess barrier function.To quantify tight junctions(TJs)such as occludin and zonula occludens-1(ZO-1)at the mRNA level,reverse transcriptionpolymerase chain reaction(RT-PCR)analysis was performed.Immunoblotting was used to examine the activity of the nuclear factor-kappa B(NF-κB)signaling pathway and the production of TJs proteins.Immunofluorescence analyses were employed to localize the TJs.Enzyme-linked immunosorbent assay(ELISA)and RT-PCR were utilized to observe changes in interleukin(IL)-1βlevels.To investigate the role of NF-κB signaling activation and IL^(-1)βin Sema7A’s anti-barrier mechanism,we employed 0.1μmol/L IκB kinase 2(IKK2)inhibitor IV or 500 ng/mL IL^(-1)receptor(IL-1R)antagonist.●RESULTS:Treatment with Sema7A resulted in decreased TEER and increased permeability of Dextran-FITC in HCEs through down-regulating mRNA and protein levels of TJs in a time-and dose-dependent manner,as well as altering the localization of TJs.Furthermore,Sema7A stimulated the activation of inhibitor of kappa B alpha(IκBα)and expression of IL-1β.The anti-barrier function of Sema7A was significantly suppressed by treatment with IKK2 inhibitor IV or IL-1R antagonists.●CONCLUSION:Sema7A disrupts barrier function through its influence on NF-κB-mediated expression of TJ proteins,as well as the expression of IL-1β.These findings suggest that Sema7A could be a potential therapeutic target for the diseases in corneal epithelium.

LncRNA IDH1-AS1 sponges miR-518c-5p to suppress proliferation of epithelial ovarian cancer cell by targeting RMB47

Long noncoding RNA(lncRNA)IDH1 antisense RNA 1(IDH1-AS1)is involved in the progression of multiple cancers,but its role in epithelial ovarian cancer(EOC)is unknown.Therefore,we investigated the expression levels of IDH1-AS1 in EOC cells and normal ovarian epithelial cells by quantitative real-time PCR(qPCR).We first evaluated the effects of IDH1-AS1 on the proliferation,migration,and invasion of EOC cells through cell counting kit-8,colony formation,EdU,transwell,wound-healing,and xenograft assays.We then explored the downstream targets of IDH1-AS1 and verified the results by a dual-luciferase reporter,qPCR,rescue experiments,and Western blotting.We found that the expression levels of IDH1-AS1 were lower in EOC cells than in normal ovarian epithelial cells.High IDH1-AS1 expression of EOC patients from the Gene Expression Profiling Interactive Analysis database indicated a favorable prognosis,because IDH1-AS1 inhibited cell proliferation and xenograft tumor growth of EOC.IDH1-AS1 sponged miR-518c-5p whose overexpression promoted EOC cell proliferation.The miR-518c-5p mimic also reversed the proliferation-inhibiting effect induced by IDH1-AS1 overexpression.Furthermore,we found that RNA binding motif protein 47(RBM47)was the downstream target of miR-518c-5p,that upregulation of RBM47 inhibited EOC cell proliferation,and that RBM47 overexpressing plasmid counteracted the proliferation-promoting effect caused by the IDH1-AS1 knockdown.Taken together,IDH1-AS1 may suppress EOC cell proliferation and tumor growth via the miR-518c-5p/RBM47 axis.

Regulation role of miR-204 on SIRT1/VEGF in metabolic memory induced by high glucose in human retinal pigment epithelial cells

AIM:To examine the regulatory role of microRNA-204(miR-204)on silent information regulator 1(SIRT1)and vascular endothelial growth factor(VEGF)under highglucose-induced metabolic memory in human retinal pigment epithelial(hRPE)cells.METHODS:Cells were cultured with either normal(5 mmol/L)or high D-glucose(25 mmol/L)concentrations for 8d to establish control and high-glucose groups,respectively.To induce metabolic memory,cells were cultured with 25 mmol/L D-glucose for 4d followed by culture with 5 mmol/L D-glucose for 4d.In addition,exposed in 25 mmol/L D-glucose for 4d and then transfected with 100 nmol/L miR-204 control,miR-204 inhibitor or miR-204 mimic in 5 mmol/L D-glucose for 4d.Quantitative reverse transcription-polymerase chain reaction(RT-qPCR)was used to detect miR-204 mRNA levels.SIRT1 and VEGF protein levels were assessed by immunohistochemical and Western blot.Flow cytometry was used to investigate apoptosis rate.RESULTS:It was found that high glucose promoted miR-204 and VEGF expression,and inhibited SIRT1 activity,even after the return to normal glucose culture conditions.Upregulation of miR-204 promoted apoptosis inhibiting SIRT1 and increasing VEGF expression.However,downregulation of miR-204 produced the opposite effects.CONCLUSION:The study identifies that miR-204 is the upstream target of SIRT1and VEGF,and that miR-204 can protect hRPE cells from the damage caused by metabolic memory through increasing SIRT1 and inhibiting VEGF expression.

Umbilical cord mesenchymal stem cell exosomes alleviate necrotizing enterocolitis in neonatal mice by regulating intestinal epithelial cells autophagy

BACKGROUND Necrotizing enterocolitis(NEC)is a severe gastrointestinal disease that affects premature infants.Although mounting evidence supports the therapeutic effect of exosomes on NEC,the underlying mechanisms remain unclear.AIM To investigate the mechanisms underlying the regulation of inflammatory response and intestinal barrier function by umbilical cord mesenchymal stem cell(UCMSCs)exosomes,as well as their potential in alleviating NEC in neonatal mice.METHODS NEC was induced in 5-d-old C57BL/6 pups through hypoxia and gavage feeding of formula containing lipopolysaccharide(LPS),after which the mice received human UCMSC exosomes(hUCMSC-exos).The control mice were allowed to breastfeed with their dams.Ileal tissues were collected from the mice and analyzed by histopathology and immunoblotting.Colon tissues were collected from NEC neonates and analyzed by immunofluorescence.Molecular biology and cell culture approaches were employed to study the related mechanisms in intestinal epithelial cells.RESULTS We found that autophagy is overactivated in intestinal epithelial cells during NEC,resulting in reduced expression of tight junction proteins and an increased inflammatory response.The ability of hUCMSC-exos to ameliorate NEC in a mouse model was dependent on decreased intestinal autophagy.We also showed that hUCMSC-exos alleviate the inflammatory response and increase migration ability in intestinal epithelial cells induced by LPS.CONCLUSION These results contribute to a better understanding of the protective mechanisms of hUCMSC-exos against NEC and provide a new theoretical and experimental foundation for NEC treatment.These findings also enhance our understanding of the role of the autophagy mechanism in NEC,offering potential avenues for identifying new therapeutic targets.

SIRT1 inhibits apoptosis of human lens epithelial cells through suppressing endoplasmic reticulum stress in vitro and in vivo

AIM:To explore the effect of silent information regulator factor 2-related enzyme 1(SIRT1)on modulating apoptosis of human lens epithelial cells(HLECs)and alleviating lens opacification of rats through suppressing endoplasmic reticulum(ER)stress.METHODS:HLECs(SRA01/04)were treated with varying concentrations of tunicamycin(TM)for 24h,and the expression of SIRT1 and C/EBP homologous protein(CHOP)was assessed using real-time quantitative polymerase chain reaction(RT-PCR),Western blotting,and immunofluorescence.Cell morphology and proliferation was evaluated using an inverted microscope and cell counting kit-8(CCK-8)assay,respectively.In the SRA01/04 cell apoptosis model,which underwent siRNA transfection for SIRT1 knockdown and SRT1720 treatment for its activation,the expression levels of SIRT1,CHOP,glucose regulated protein 78(GRP78),and activating transcription factor 4(ATF4)were examined.The potential reversal of SIRT1 knockdown effects by 4-phenyl butyric acid(4-PBA;an ER stress inhibitor)was investigated.In vivo,age-related cataract(ARC)rat models were induced by sodium selenite injection,and the protective role of SIRT1,activated by SRT1720 intraperitoneal injections,was evaluated through morphology observation,hematoxylin and eosin(H&E)staining,Western blotting,and RT-PCR.RESULTS:SIRT1 expression was downregulated in TMinduced SRA01/04 cells.Besides,in SRA01/04 cells,both cell apoptosis and CHOP expression increased with the rising doses of TM.ER stress was stimulated by TM,as evidenced by the increased GRP78 and ATF4 in the SRA01/04 cell apoptosis model.Inhibition of SIRT1 by siRNA knockdown increased ER stress activation,whereas SRT1720 treatment had opposite results.4-PBA partly reverse the adverse effect of SIRT1 knockdown on apoptosis.In vivo,SRT1720 attenuated the lens opacification and weakened the ER stress activation in ARC rat models.CONCLUSION:SIRT1 plays a protective role against TM-induced apoptosis in HLECs and slows the progression of cataract in rats by inhibiting ER stress.These findings suggest a novel strategy for cataract treatment focused on targeting ER stress,highlighting the therapeutic potential of SIRT1 modulation in ARC development.

Bone morphogenetic protein-6 suppresses TGF-β_(2)-induced epithelial-mesenchymal transition in retinal pigment epithelium

AIM:To evaluate the effect of bone morphogenetic protein-6(BMP-6)on transforming growth factor(TGF)-β_(2)-induced epithelial-mesenchymal transition(EMT)in retinal pigment epithelium(RPE).METHODS:Adult retinal pigment epithelial cell line(ARPE-19)were randomly divided into control,TGF-β_(2)(5μg/L),and BMP-6 small interfering RNA(siRNA)group.The cell morphology was observed by microscopy,and the cell migration ability were detected by Transwell chamber.The EMT-related indexes and BMP-6 protein levels were detected by Western blotting.Furthermore,a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group,TGF-β_(2)+empty plasmid group,BMP-6 overexpression group,and TGF-β_(2)+BMP-6 overexpression group.The EMT-related indexes and extracellular regulated protein kinases(ERK)protein levels were detected.RESULTS:Compared with the control group,the migration of RPE cells in the TGF-β_(2) group was significantly enhanced.TGF-β_(2) increased the protein expression levels ofα-smooth muscle actin(α-SMA),fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6(P<0.05)in RPE.Similarly,the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced.BMP-6 siRNA increased the protein expression levels ofα-SMA,fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin(P<0.05).Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-β_(2) and prevented TGF-β_(2) from affecting EMT-related biomarkers(P<0.05).CONCLUSION:BMP-6 prevents the EMT in RPE cells induced by TGF-β_(2),which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

胰岛素样生长因子1对人RPE细胞分泌TGF-β2、MMP-2的影响及机制研究

目的研究胰岛素样生长因子1(IGF-1)对人视网膜色素上皮细胞(ARPE-19)表达转化生长因子β2(TGF-β2)、基质金属蛋白酶2(MMP-2)的影响,并探索其作用机制。方法ARPE-19细胞分别按不同浓度IGF-1和不同浓度LY294002培养6 h、12 h、24 h、48 h,采用CCK-8法检测细胞活力,确定IGF-1、LY294002的最佳作用浓度与时间。细胞划痕法检测细胞迁移活性。ELISA法检测细胞培养上清液中TGF-β2浓度。将ARPE-19细胞分为对照组、IGF-1组(80μg·L^(-1) IGF-1)、IGF-1+LY294002组(80μg·L^(-1) IGF-1+30 mmol·L^(-1) LY294002)、LY294002组(30 mmol·L^(-1) LY294002),使用无血清DMEM/F12培养基培养,对照组不做任何处理,分别采用RT-PCR、Western blot检测细胞中TGF-β2、MMP-2、磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶B(AKT)的mRNA和蛋白表达量。结果与0μg·L^(-1) IGF-1比较,80μg·L^(-1) IGF-1的细胞活力24 h变化显著(P<0.05),故确定其为IGF-1最佳作用浓度和时间。与0 mmol·L^(-1) LY294002比较,24 h的30 mmol·L^(-1) LY294002接近半数抑制浓度,故确定其为LY294002最佳作用时间和浓度。细胞划痕法检测结果显示,0μg·L^(-1) IGF-1组、40μg·L^(-1) IGF-1组、80μg·L^(-1) IGF-1组细胞迁移率整体比较及两两比较差异均有统计学意义(均为P<0.05)。ELISA检测结果显示,0μg·L^(-1) IGF-1组、40μg·L^(-1) IGF-1组、80μg·L^(-1) IGF-1组细胞上清液中TGF-β2浓度整体比较及两两比较差异均有统计学意义(均为P<0.05)。RT-PCR、Western blot检测结果显示,IGF-1、LY294002培养24 h,与对照组比较,IGF-1组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均升高,而LY294002组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均下降(均为P<0.05);与IGF-1组比较,IGF-1+LY294002组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均下降(均为P<0.05)。结论IGF-1能促进ARPE-19细胞增殖、迁移;IGF-1可能通过PI3K/AKT信号通路上调ARPE-19细胞中TGF-β2、MMP-2的表达,参与近视的发生与发展。

废旧塑料(RPE)改性沥青混合料性能优化研究

为了改善和提升改性沥青性能及混合料的路用性能,选取掺量为4%、6%、8%、10%的废旧聚乙烯(Recycle Polyethylene,RPE)对基质沥青进行了改性,并在不同混合温度(160、170、180℃)和搅拌时间(1.0、1.5、2.0 h)下进行了针入度、延度和软化点试验。研究了不同改性工艺条件对RPE改性沥青常规性能的影响,确定了最佳改性工艺条件,并比较了最佳改性条件下普通沥青混合料与RPE改性沥青混合料的力学性能。通过高温车辙、低温弯曲和水稳定性等试验评价了RPE改性沥青混合料的路用性能。研究结果表明:普通沥青混合料中最佳沥青掺量为5.16%,RPE改性沥青混合料中最佳RPE掺量为6.5%;在170℃的混合温度和1.5 h的搅拌时间下,RPE改性沥青混合料的性能最佳;RPE改性沥青混合料的力学性能优于普通沥青混合料,高温性能和水稳定性得到了提升,低温性能降低。

Six transmembrane epithelial antigens of the prostate to illustrate inflammatory response in gastrointestinal cancers

Gastrointestinal cancer(GIC)is a common and widespread form of tumor,with colonoscopy and upper gastrointestinal endoscopy available to detect relevant precancerous polyps and lesions.However,many patients are already in the late stages when first diagnosed with such cancer,resulting in a poor prognosis.Thus,it is necessary to explore new methods and research directions in order to improve the treatment of GIC.Given the specific nature of the gastrointestinal tract,research should focus on the mechanisms of various inflammations and the interactions between food entering and exiting from the gastrointestinal tract and cancer cells.Interestingly,six transmembrane epithelial antigens of the prostates(STEAPs)have been found to be significantly linked to the progression of malignant tumors,associated with intracellular oxidative stress and playing a major role in inflammation with their structure and function.This paper explores the mechanism of STEAPs in the inflammatory response of GIC,providing a theoretical basis for the prevention and early intervention of GIC.The basic properties of the STEAP family as metal reductase are also explained.When it comes to intervention for GIC prevention,STEAPs can affect the activity of Fe^(3+),Cu^(2+) reductase and regulate metal ion uptake in vivo,participating in inflammation-related iron and copper homeostasis.Thus,the mechanism of STEAPs on inflammation is of important value in the prevention of GIC.

红景天苷预处理对高糖诱导的ARPE-19细胞上皮-间充质转化的作用与机制研究

目的探讨红景天苷(SAL)对高糖诱导的ARPE-19细胞的上皮-间充质转化的作用及机制。方法采用MTT法筛选刺激ARPE-19细胞增殖的合适的葡萄糖浓度,确定为50 mmol·L^(-1)。将ARPE-19细胞分为正常对照组(Control组)(5 mmol·L^(-1)的D-葡萄糖和45 mmol·L^(-1)甘露醇培养)、高糖组(HG组)(50 mmol·L^(-1)葡萄糖培养)、HG+低SAL组、HG+中SAL组、HG+高SAL组,低、中、高SAL浓度分别为20μmol·L^(-1)、80μmol·L^(-1)、320μmol·L^(-1),SAL预处理4 h后,加50 mmol·L^(-1)葡萄糖,作用48 h。采用MTT法观察细胞增殖率;划痕实验观察细胞迁移活性;免疫荧光检测细胞α平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)的表达;Western blot法检测细胞中α-SMA、Vimentin、纤维粘连素(FN)、Ⅰ型胶原蛋白(Col I)、钙黏附蛋白E(E-Cadherin)、转化生长因子-β1(TGF-β1)、p-Smad2及p-Smad3蛋白的表达水平。结果与Control组相比,HG组ARPE-19细胞增殖率与伤口愈合百分比均增加,细胞内a-SMA、Vimentin、FN、Col I蛋白相对表达量均增加,E-cadherin蛋白相对表达量降低,细胞内TGF-β1、p-Smad2及p-Smad3蛋白相对表达量均增加,差异均有统计学意义(均为P<0.01);与HG组相比,HG+低SAL组、HG+中SAL组及HG+高SAL组ARPE-19细胞增殖率与伤口愈合百分比均降低,细胞内a-SMA、Vimentin、FN、Col I、TGF-β1、p-Smad2及p-Smad3蛋白相对表达量均呈浓度依赖性降低,E-cadherin蛋白相对表达量呈浓度依赖性增加,差异均有统计学意义(均为P<0.05)。结论SAL能够降低高糖诱导的ARPE-19细胞的增殖、迁移能力,以及干预其上皮-间充质转化进程,这可能与SAL抑制了ARPE-19细胞的TGF-β/Smads信号通路有关。

非接触共培养体系下抑制ARPE-19中CAMKⅡ表达对HUVECs迁移和侵袭及管腔形成的影响

目的:探讨非接触共培养体系下抑制人视网膜色素上皮细胞(ARPE)中Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ(CAMKⅡ)表达对人脐静脉内皮细胞(HUVECs)迁移、侵袭、管腔形成的影响。方法:将过表达CAMKⅡ-δ的ARPE-19样本进行RNA测序,应用生物信息学分析差异基因参与的功能。使用transwell小室构建ARPE-19和HUVECs非接触共培养体系,根据实验干预措施分为:空白组:仅接种未共培养的HUVECs,无ARPE-19细胞;对照组:ARPE-19和HUVECs细胞均使用完全培养基进行共培养;AIP组(CAMKⅡ抑制组):ARPE-19使用含有AIP(160 nmol/L)的完全培养基,HUVECs使用完全培养基,进行共培养。检测HUVECs迁移、侵袭和管腔形成能力的变化,并通过Western blotting检测CAMKⅡ/AMPK/mTOR/VEGFA蛋白表达水平。结果:生信分析发现差异基因参与细胞生长与死亡和细胞运动等生物学过程。划痕和transwell迁移实验均表明AIP组的HUVECs相对迁移率均明显低于对照组(均P<0.05)。而侵袭和小管形成实验表明,AIP组的相对侵袭率和相对管腔形成率较对照组无明显改变(均P>0.05)。Western blotting结果表明AIP组CAMKⅡ、P-mTOR、VEGFA蛋白表达较对照组均明显下调,而P-AMPK蛋白表达较明显上调(均P<0.05)。结论:在非接触共培养体系下抑制ARPE-19细胞中CAMKⅡ表达可以显著降低HUVECs迁移能力,但不能改变侵袭和管腔形成能力,这可能是通过AMPK/mTOR/VEGFA信号通路实现的。

SC79对高糖诱导RPE细胞凋亡的拮抗作用及其对AKT-XIAP信号通路的调控机制

目的探讨特异性AKT激活剂SC79对体外高糖诱导人视网膜色素上皮(ARPE)-19细胞凋亡的拮抗作用及其潜在机制。方法将体外培养的ARPE-19细胞分别置于含5、10、20μg/ml SC79的高糖(30 mmol/L葡萄糖)培养液中培养6、12、24 h,根据细胞增生率探索最佳的作用浓度和作用时间。将细胞分为4个组,其中正常对照组、甘露醇组和高糖组、分别于含5.6 mmol/L葡萄糖正常培养液、含5.6 mmol/L葡萄糖和24.4 mmol/L甘露醇培养液、高糖培养液中培养48 h,高糖+SC79组细胞于含10μg/ml SC79正常培养液中培养12 h,然后于高糖培养液中继续培养36 h。采用MTS法检测细胞的增生率,采用流式细胞仪检测各组细胞凋亡率,采用Western blot法检测磷酸化的蛋白激酶B(p-Akt)、X连锁凋亡抑制蛋白(XIAP)、caspase-9、caspase-3及活化片段active-caspase-3的相对表达量。将细胞分为Neg-shRNA组、AKT shRNA组和空白对照组分别加入相应转染复合物和无血清培养基,采用实时荧光定量PCR检测各转染组Akt mRNA表达。将转染细胞分为Neg-shRNA+SC79组和AKT shRNA+SC79组,参照高糖+SC79组处理方式进行培养,采用流式细胞仪检测各组细胞凋亡率。结果不同浓度SC79处理不同时间细胞中以10μg/ml SC79预处理12 h细胞的增生率最高。高糖组细胞增生率明显低于正常对照组、甘露糖组和高糖+SC79组,差异具有统计学意义(均P<0.01)。高糖组细胞凋亡率为(52.27±3.21)%,明显高于正常对照组的(3.90±0.71)%和高糖+SC79组的(20.70±3.62)%,差异均有统计学意义(均P<0.01)。高糖组p-Akt、XIAP、caspase-9及caspase-3蛋白相对表达量明显低于正常对照组和高糖+SC79组,active-caspase-3蛋白相对表达量明显高于正常对照组和高糖+SC79组,差异均具有统计学意义(均P<0.05)。正常对照组、Neg-shRNA组和AKT shRNA组AKT mRNA相对表达量分别为0.60±0.07、0.59±0.03和0.11±0.10,总体比较差异有统计学意义(F=30.44,P<0.01)。AKT shRNA+SC79组细胞凋亡率明显高于高糖+SC79组和Neg-shRNA+SC79组,差异均有统计学意义(均P<0.001)。结论SC79可以部分拮抗高糖诱导的ARPE-19细胞凋亡,其机制可能与活化AKT/XIAP通路,抑制凋亡执行蛋白caspase家族有关。