miR‑423 sponged by lncRNA NORHA inhibits granulosa cell apoptosis

Background Atresia and degeneration,a follicular developmental fate that reduces female fertility and is triggered by granulosa cell(GC)apoptosis,have been induced by dozens of miRNAs.Here,we report a miRNA,miR-423,that inhibits the initiation of follicular atresia(FA),and early apoptosis of GCs.Results We showed that miR-423 was down-regulated during sow FA,and its levels in follicles were negatively correlated with the GC density and the P4/E2 ratio in the follicular fluid in vivo.The in vitro gain-of-function experiments revealed that miR-423 suppresses cell apoptosis,especially early apoptosis in GCs.Mechanically speaking,the miR-423 targets and interacts with the 3’-UTR of the porcine SMAD7 gene,which encodes an apoptosis-inducing factor in GCs,and represses its expression and pro-apoptotic function.Interestingly,FA and the GC apoptosis-related lncRNA NORHA was demonstrated as a ceRNA of miR-423.Additionally,we showed that a single base deletion/insertion in the miR-423 promoter is significantly associated with the number of stillbirths(NSB)trait of sows.Conclusion These results demonstrate that miR-423 is a small molecule for inhibiting FA initiation and GC early apoptosis,suggesting that treating with miR-423 may be a novel approach for inhibiting FA initiation and improving female fertility.

Dihydroartemisinin enhances cell apoptosis in diffuse large B cell lymphoma by inhibiting the STAT3 activity

Background:Dihydroartemisinin(DHA)is reported to be a potential anticancer agent,and the mechanisms underlying the effects of DHA on diffuse large B cell lymphoma however are still obscure.This study aimed to assess the antitumor effect of DHA on diffuse large B cell lymphoma cells and to determine the potential underlying mechanisms of DHA-induced cell apoptosis.Methods:Here,the Cell Counting Kit 8 assay was conducted to study cell proliferation.We performed Annexin V-FITC/propidium iodide staining,real-time polymerase chain reaction,and western blot analysis to analyze cell apoptosis and potential molecular mechanisms.Results:The results showed that DHA substantially suppressed cell proliferation and induced cell apoptosis in vitro in a time-and concentration-dependent fashion.Moreover,STAT3 activity could be inhibited after stimulation with DHA.Conclusion:These results imply that the underlying anti-tumoral effect of DHA may increase apoptosis in diffuse large B cell lymphoma cells via the STAT3 signaling pathway.In addition,DHA might be an effective drug for diffuse large B cell lymphoma therapy.

MiR-96-5p inhibition induces cell apoptosis in gastric adenocarcinoma

BACKGROUND Gastric adenocarcinoma(GAC)mortality rates have remained relatively changed over the past 30 years,and it continues to be one of the leading causes of cancerrelated death.AIM To search for novel miRNAs related to GAC prognosis and further investigate the effect of miR-96-5p on MGC-803 cells.METHODS The miRNA expression profile data of GAC based on The Cancer Genome Atlas were obtained and used to screen differently expressed miRNAs(DEMs)and DEMs related to GAC prognosis.Then,the expression of DEMs related to GAC prognosis was identified in GAC tumor samples and adjacent normal samples by qRT-PCR.The target gene,ZDHHC5,of miR-96-5p was predicted using TargetScan,miRTarBase,and miRDB databases and confirmed by luciferase reporter assay.Furthermore,MGC-803 cells were transfected with inhibitor NC,miR-96-5p inhibitor,si-ZDHHC5,or miR-96-5p inhibitor+si-ZDHHC5,and then cell apoptosis was detected by flow cytometry.The expression of ZDHHC5,Bcl-2,and COX-2 was detected using western blotting.RESULTS A total of 299 DEMs and 35 DEMs related to GAC prognosis were screened based on The Cancer Genome Atlas.Then compared with adjacent normal samples,the levels of miR-96-5p,miR-222-5p,and miR-652-5p were remarkably increased,while miR-125-5p,miR-145-3p,and miR-379-3p levels were reduced in GAC tumor samples(P<0.01),which were consistent with bioinformatics analysis.Furthermore,ZDHHC5 was defined as a direct target gene of miR-96-5p.miR-96-5p inhibition increased the number of apoptotic cells as well as promoted the expression of ZDHHC5,Bcl-2,and COX-2 in MGC-803 cells(P<0.01).After ZDHHC5 inhibition,the number of apoptotic cells and the expression of ZDHHC5,Bcl-2,and COX-2 were reduced.The addition of an miR-96-5p inhibitor partly reversed these effects(P<0.01).CONCLUSION Our findings identified six miRNAs related to GAC prognosis and suggested that downregulated miR-96-5p might induce cell apoptosis via upregulating ZDHHC5 expression in MGC-803 cells.

JNK3 involvement in nerve cell apoptosis and neurofunctional recovery after traumatic brain injury

Increasing evidence has revealed that the activation of the JNK pathway participates In apoptosis o1 nerve cells and neurological function recovery after traumatic brain injury. However, which genes inI the JNK family are activated and their role in traumatic brain injury remain unclear. Therefore, in this study, in situ end labeling, reverse transcription-PCR and neurological function assessment were adopted to investigate the alteration of JNK1, JNK2 and JNK3 gene expression in cerebral injured rats, and their role in celt apoptosis and neurological function restoration. Results showed that JNK3 expression significantly decreased at 1 and 6 hours and 1 and 7 days post injury, but that JNK1 and JNK2 expression remained unchanged. In addition, the number of apoptotic nerve cells surrounding the injured cerebral cortex gradually reduced over time post injury. The Neurological Severity Scores gradually decreased over 1,3, 5, 14 and 28 days post injury. These findings suggested that JNK3 expression was downregulated at early stages of brain injury, which may be associated with apoptosis of nerve cells. Downregulation of JNK3 expression may promote the recovery of neurological function following traumatic brain injury.

Loss of the vitamin D receptor in human breast and prostate cancers strongly induces cell apoptosis through downregulation of Wnt/β-catenin signaling

Vitamin D co-regulates cell proliferation, differentiation and apoptosis in numerous tissues, including cancers. The known anti-proliferative and pro-apoptotic actions of the active metabolite of vitamin D, 1,25-dihydroxy-vitamin D [1,25（OH）2D] are mediated through binding to the vitamin D receptor （VDR）. Here, we report on the unexpected finding that stable knockdown of VDR expression in the human breast and prostate cancer cell lines, MDA-MB-231 and PC3, strongly induces cell apoptosis and inhibits cell proliferation in vitro. Implantation of these VDR knockdown cells into the mammary fat pad （MDA-MB-231）, subcutaneously （PC3） or intra-tibially （both cell lines） in immune-incompetent nude mice resulted in reduced tumor growth associated with increased apoptosis and reduced cell proliferation compared with controls. These growth-retarding effects of VDR knockdown occur in the presence and absence of vitamin D and are independent of whether cells were grown in bone or soft tissues. Transcriptome analysis of VDR knockdown and non-target control cell lines demonstrated that loss of the VDR was associated with significant attenuation in the Wnt/0-catenin signaling pathway. In particular, cytoplasmic and nuclear β-catenin protein levels were reduced with a corresponding downregulation of downstream genes such as Axin2, Cyclin D1, interleukin-6 （IL-6）, and IL-8. Stabilization of 0-catenin using the GSK-3β inhibitor BIO partly reversed the growth-retarding effects of VDR knockdown. Our results indicate that the unliganded VDR possesses hitherto unknown functions to promote breast and prostate cancer growth, which appear to be operational not only within but also outside the bone environment. These novel functions contrast with the known anti-proliferative nuclear actions of the liganded VDR and may represent targets for new diagnostic and therapeutic approaches in breast and prostate cancer.

8-hydroxy-2-(di-n-propylamino)tetralin intervenes with neural cell apoptosis following diffuse axonal injury

Previous studies have reported a neuroprotective effect of 8-hydroxy-2-（di-n-propylamino）tetralin （8-OH-DPAT） against traumatic brain injury. In accordance with the Marmarou method, rat models of diffuse axonal injury were established. 8-OH-DPAT was intraperitoneally injected into model rats. 8-OH-DPAT treated rats maintained at constant temperature served as normal temperature controls TUNEL results revealed that neural cell swelling, brain tissue necrosis and cell apoptosis occurred around the injured tissue. Moreover, the number of Bax-, Bcl-2- and caspase-3-positive cells increased at 6 hours after diffuse axonal injury, and peaked at 24 hours. However, brain injury was attenuated, the number of apoptotic cells reduced, Bax and caspase-3 expression decreased, and Bcl-2 expression increased at 6, 12, 24, 72 and 168 hours after diffuse axonal injury in normal temperature control and in 8-OH-DPAT-intervention rats. The difference was most significant at 24 hours. All indices in 8-OH-DPAT-intervention rats were better than those in the constant temperature group. These results suggest that 8-OH-DPAT inhibits Bax and caspase-3 expression, increases Bcl-2 expression, and reduces neural cell apoptosis, resulting in neuroprotection against diffuse axonal injury. This effect is associated with a decrease in brain temperature.

Brain cell apoptosis and enhancement of nervous excitability in pregnant rats with high plasma levels of homocysteine

Hyperhomocysteinemia is an important risk factor for preeclampsia-eclampsia. This study established a pregnant rat model of hyperhomocysteinemia, in which blood plasma homocysteine concentrations were twice or three times greater than that of normal pregnant rats. TUNEL revealed an increase in the number of apoptotic cells in the frontal cortex of pregnant rats with hyperhomocysteinemia. In addition, immunohistochemical staining detected activated nuclear factor-KB-positve cells in the frontal cortex. Reverse transcription-PCR detected that mRNA expression of the anti-apoptotic gene bcl-2 diminished in the frontal cortex. In situ hybridization and western blotting revealed that N-methyi-D- aspartate receptor 1 mRNA and protein expression was upregulated in the frontal cortex and hippocampus. These results indicate that hyperhomocysteinemia can induce brain cell apoptosis, increase nerve excitability, and promote the occurrence of preeclampsia in pregnant rats.

Nerve growth factor precursor and sortilin effects on perihematomal brain tissue and the relationship to secondary cell apoptosis

BACKGROUND： Neuronal apoptosis in perihematomal brain tissues following intracerebral hemorrhage is strongly related to the formation of a compound signal pathway between nerve growth factor precursor （proNGF）, p75NTR, and sortilin receptor. Sortilin acts as a co-receptor and molecular switch governing the p75NTR-mediated pro-apoptotic signal induced by proNGF. OBJECTIVE： To investigate proNGF and sortilin expressions in perihematomal brain tissues following intracerebral hemorrhage, and to study the effects of proNGF and sortilin on secondary cell apoptosis. DESIGN, TIME AND SETTING： A paired, comparison study was performed at the Laboratory of Histology and Embryology, Xi＇an Jiaotong University from October 2007 to September 2008. MATERIALS： Brain tissue samples were obtained from 15 patients with intracerebral hemorrhage, who were treated at the Department of Neurosurgery, First Affiliated Hospital, Medical College of Xi＇an Jiaotong University from October 2007 to March 2008. Rabbit anti-proNGF polyclonal antibody was provided by Chemicon, USA; rabbit anti-sortilin polyclonal antibody by Abcam, UK; and TUNEL kit by Promega, USA. METHODS： Perihematomal brain tissues selected 0.5 cm from the hemorrhage area were considered to be the hemorrhage group, while brain tissues from the middle temporal gyrus served as the control group. MAIN OUTCOME MEASURES： Histopathological changes were detected using hematoxylin-eosin staining, cell apoptosis was determined using the TUNEL method, and proNGF and sortilin expressions were determined using immunohistochemistry. RESULTS： Edema was clearly observed in perihematomal brain tissues, and infiltration of inflammatory cells was visible, with the presence of irregular and necrotic bodies. The apoptotic rate in the hemorrhage group was significantly greater than in the control group （P 〈 0.01）. Moreover, sortilin expression significantly increased （P 〈 0.01）, but proNGF expression remained unchanged （P 〉 0.05）. Correlation analysis suggested that sortilin expression positively correlated with apoptosis （rs = 0.648, P 〈 0.01）. CONCLUSION： proNGF expression was stable, but sortilin expression increased in perihematomal brain tissues following intracerebral hemorrhage, suggesting that sortilin acted as a co-receptor and molecular switch to govern p75NTR-mediated cell apoptosis.

Involvement of the Wnt signaling pathway and cell apoptosis in the rat hippocampus following cerebral ischemia/reperfusion injury

We investigated the role of the Wnt signaling pathway in cerebral ischemia/reperfusion injury by examining β-catenin and glycogen synthase kinase-3β protein expression in the rat hippocampal CA1 region following acute cerebral ischemia/reperfusion. Our results demonstrate that cell apoptosis increases in the CA1 region following ischemia/reperfusion. In addition, β-catenin and glycogen synthase kinase-3β protein expression gradually increases, peaking at 48 hours following reperfusion. Dickkopf-1 administration, after cerebral ischemia/reperfusion injury, results in decreased cell apoptosis, and β-catenin and glycogen synthase kinase-3β expression, in the CA1 region. This suggests that β-catenin and glycogen synthase kinase-3β, both components of the Wnt signaling pathway, participate in cell apoptosis following cerebral ischemia/reperfusion injury.

NORHA,a novel follicular atresia-related lncRNA,promotes porcine granulosa cell apoptosis via the miR-183-96-182 cluster and FoxO1 axis

Background:Follicular atresia has been shown to be strongly associated with a low follicle utilization rate and female infertility,which are regulated by many factors such as microRNAs(miRNAs),which constitute a class of noncoding RNAs(ncRNAs).However,little is known about long noncoding RNAs(lncRNAs),which constitute another ncRNA family that regulate follicular atresia.Results:A total of 77 differentially expressed lncRNAs,including 67 upregulated and 10 downregulated lncRNAs,were identified in early atretic follicles compared to healthy follicles by RNA-Sequencing.We characterized a noncoding RNA that was highly expressed in atretic follicles(NORHA).As an intergenic lncRNA,NORHA was one of the upregulated lncRNAs identified in the atretic follicles.To determine NORHA function,RT-PCR,flow cytometry and western blotting were performed,and the results showed that NORHA was involved in follicular atresia by influencing GC apoptosis with or without oxidative stress.To determine the mechanism of action,bioinformatics analysis,luciferase reporter assay and RNA immunoprecipitation assay were performed,and the results showed that NORHA acted as a‘sponge’,that directly bound to the miR-183-96-182 cluster,and thus prevented its targeted inhibition of FoxO1,a major sensor and effector of oxidative stress.Conclusions:We provide a comprehensive perspective of lncRNA regulation of follicular atresia,and demonstrate that NORHA,a novel lncRNA related to follicular atresia,induces GC apoptosis by influencing the activities of the miR-183-96-182 cluster and FoxO1 axis.

Effects of glossy privet fruit on neural cell apoptosis in the cortical parietal lobe and hippocampal CA1 region of vascular dementia rats

BACKGROUND： Glossy privet fruit inhibits neural cell apoptosis following the onset of vascular dementia. OBJECTIVE： To confirm glossy privet fruit effects on neural cell apoptosis in the cortical parietal lobe and hippocampal CAI region of rat models of vascular dementia using molecular biology techniques. DESIGN, TIME AND SETTING： The neural cell morphology experiment was performed at the Laboratory of Flow Cells and Biochemistry, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, and the Basic Room of Pathology, Academy of Chinese Medicine from December 2006 to May 2008. MATERIALS： A total of 60 Wistar rats were used to establish vascular dementia models using a photochemical reaction method. Glossy privet fruit was purchased from Fujian, China. Hydergine was co-produced by Sandoz, Switzerland and Huajin, China. METHODS： The 60 Wistar rats were randomly divided into 6 equal sized groups （n = 10）, i.e. model, blank, high, moderate and low doses of Chinese medicine, and hydergine control groups. Rats in the model group were treated with distilled water （1 mL/100 g） by gavage following model establishment. Rats in the blank group underwent experimental procedures as for the model group, except that rat models were created without illumination. Rats in the high, moderate and low doses of Chinese medicine groups, and the hydergine control group respectively received high, moderate and low doses of glossy privet fruit, and hydergine suspension （1 mL/100 g） by gavage, once a day, for 30 days. MAIN OUTCOME MEASURES： Morphology of neural cells from the rat cortical parietal lobe and hippocampal CA1 region of all groups was observed with an electron microscope. Positive neural cells in the injury site of the rat cortical parietal lobe and hippocampal CA1 region were investigated using the Fas immunohistochemieal method. Absorbance of Fas-positive neurons was detected by the MPIAS-500 multimedia color imaging analysis system. RESULTS： Neural cells were normal, and nuclei were regular in the right cortical parietal lobe and hippoeampal CA1 region in the blank group. Karyopyknosis, an integral nuclear membrane, vacuole and apoptotic bodies were presented in the model group. The quantity and morphology of neural cells were normal in all doses of Chinese medicine groups, and the hydergine control group. Compared with the model group, absorbance was reduced at the injury site of rat cortical parietal lobe and hippocampal CA1 region in the blank, high, moderate and low doses of Chinese medicine, and hydergine control groups （P 〈 0.05）. The decrease was particularly significant in the blank group （P 〈 0.01 ）, followed by the high dose of Chinese medicine group （P 〈 0.01）. Compared with the model group, the percentage of apoptosis was decreased at the injury site of the rat cortical parietal lobe and hippocampal CAI region in the blank, high, moderate and low doses of Chinese medicine, and hydergine control groups （P 〈 0.01） and this decrease was significant in the high dose of Chinese medicine group （P 〈 0.01）. CONCLUSION： Glossy privet fruit, a kidney-tonifying Chinese herbal medicine, can inhibit cell apoptosis by reducing apoptotic signals induced by cerebral ischemia/hypoxia.

LncRNA LUCAT1 Activation Mediated by theDown-regulation of DNMT1 Is Involved inCell Apoptosis Induced by PM2.5

Particulate matter （PM）, which is a great environmental concern, has been classified as a Group 1 human carcinogen by the International Agency for Research on Cancer （IARC）;.Epidemiological and experimental studies have indicated that chronic exposure to PM, especially PM;(particles with an aerodynamic diameter less than

Morphological and Hormonal Identifi cation of Porcine Atretic Follicles and Relationship Analysis of Hormone Receptor Levels During Granulosa Cell Apoptosis In vivo

Recent reports have demonstrated that follicular atresia is initiated or caused by granulosa cell apoptosis followed by theca cell degeneration in mammalian ovaries, but the mechanism of follicular atresia is still to be elucidated. Therefore, our present study was designed to examine our hypothesis that the changes of follicular microenvironment induce the granulosa cell apoptosis during pocrine follicular atresia in vivo. We firstly isolated intact porcine antral follicles and identified them into three groups, healthy follicles （HF）, early atretic follicles （EAF） and progressed atretic follicles （PAF） through morphology and histology. To further confirm their status, we detected hormone levels in follicular fluids and the expression level of apoptosis gene Bax in granulosa cells. The rate of progesterone （P） and estradiol （E2） was increased with the expression of Bax, indicating hormone can be used as a marker of granulosa cell apoptosis or follicular atresia. Finally, we analyzed the expression level of hormone receptor genes in granulosa cells and their relationship with follicular atresia. In PAF, the expression of Progesterone receptor （PGR） was increased significantly while estradiol receptor （ER） had no notable changes, which suggesting the increased-PGR accelerated the effect of P-stimulated granulosa cell apoptosis. The dramatic increasing of androgen receptor （AR） expression in PAF and the obvious increase of tumor necrosis factor-u receptor （TNFR） in EAF indicated that there are different pathways regulating granulosa cell apoptosis during follicular atresia. Together, our results suggested that different pathways of granulosa cell apoptosis was induced by changing the follicular microenvironment during follicular atresia.

Human Serum-derived Extracellular Vesicles Protect A549 from PM_(2.5)-induced Cell Apoptosis

Objective Epidemiological studies reveal that exposure to fine particulate matter(aerodynamic diameter≤2.5μm,PM_(2.5))increases the morbidity and mortality of respiratory diseases.Emerging evidence suggests that human circulating extracellular vesicles(EVs)may offer protective effects against injury caused by particulate matter.Currently,however,whether EVs attenuate PM_(2.5)-induced A549 cell apoptosis is unknown.Methods EVs were isolated from the serum of healthy subjects,quantified via nanoparticle tracking analysis,and qualified by the marker protein CD63.PM_(2.5)-exposed(50μg/mL)A549 cells were pretreated with 10μg/mL EVs for 24 h.Cell viability,cell apoptosis,and AKT activation were assessed via Cell Counting Kit-8,flow cytometry,and Western blot,respectively.A rescue experiment was also performed using MK2206,an AKT inhibitor.Results PM_(2.5)exposure caused a 100%in crease in cell apoptosis.EVs treatme nt reduced cell apoptosis by 10%,promoted cell survival,and inhibited the PM_(2.5)-induced upregulation of Bax/Bcl2 and cleaved caspase 3/caspase 3 in PM_(2.5)-exposed A549 cells.Moreover,EVs treatment reversed PM_(2.5)-induced reductions in p-AKT^(Thr308)and p-AKT^(Ser473).A KT inhibition attenuated the anti-apoptotic effect of EVs treatment on PM_(2.5)-exposed A549 cells.Conclusions EVs treatment promotes cell survival and attenuates PM_(2.5)-induced cell apoptosis via AKT phosphorylation.Human serum-derived EVs may be an efficacious novel therapeutic strategy in PM_(2.5)-induced lung injury.

Simulated Microgravity Conditions and Carbon Ion Irradiation Induce Spermatogenic Cell Apoptosis and Sperm DNA Damage

Objective To investigate the effect of simulated microgravity and carbon ion irradiation （CIR） on spermatogenic cell apoptosis and sperm DNA damage to the testis of male Swiss Webster mice, and assess the risk associated with space environment. Methods Sperm DNA damage indicated by DNA fragmentation index （DFI） and high DNA stainability （HDS） was measured by sperm chromatin structure assay （SCSA）. Apoptosis of spermatogenic cells was detected by annexin V-propidium iodide assay. Bax （the expression levels of p53） and proliferating cell nuclear antigen （PCNAI were measured by immunoblotting; p53 and PCNA were located by immunohistology. Results HDS, DFI, apoptosis index, and the expression levels of p53 and Bax were detected to be significantly higher in the experimental groups （P〈0.05） compared with those in the control group, however, the PCNA expression varied to a certain degree, p53- and PCNA- positive expression were detected in each group, mainly in relation to the spermatogonic cells and spermatocytes. Conclusion The findings of the present study demonstrated that simulated microgravity and CIR can induce spermatogenic cell apoptosis and sperm DNA damage. Sperm DNA damage may be one of the underlying mechanisms behind male fertility decline under space environment. These findings may provide a scientific basis for protectint~ astronauts and space traveler＇s health and safety.

Effects of chronic heat stress on granulosa cell apoptosis and follicular atresia in mouse ovary

Background： Heat stress is known to alter follicular dynamics and granulosa cell function and may contribute to the diminished reproductive efficiency commonly observed in mammals during the summer. Although several investigators have studied heat-induced ovarian injury, few reports have focused on the effects of chronic heat stress on ovarian function and the molecular mechanisms through which it induces ovarian injury.Methods： In Exp. 1, 48 female mice were assigned to a control or heat-stressed treatment. After exposure to a constant temperature of 25 ℃ for 7, 14, 21 or 28 d（n = 6） or to 42 ℃ for 3 h per d for 7, 14, 21 or 28 d（n = 6）, the mice were euthanized and their ovaries were analyzed for follicular atresia, granulosa cell apoptosis, changes in the abundance of HSP70 protein and serum concentrations of estradiol. In Exp. 2, the expression of HSP70 and aromatase was quantified in antral follicles cultured in vitro at 37 or 42 ℃ for 24 h. In Exp. 3, granulosa cells from ovaries maintained at 37 or 41 ℃ for 2 h were analyzed for their expression of HSP70, Bim, caspase-3 and cleaved caspase-3.Results： In Exp. 1, body weight and food intake of heat-stressed mice decreased（P 〈 0.05） compared with control mice while the concentration of estradiol in serum was lower（P 〈 0.05） in heat-stressed mice than in control mice. Compared with control mice, the percentage of atretic follicles and the number of antral follicles with severe apoptotic signals were increased（P 〈 0.05） after 21 d of heat-stressed treatment. HSP70 protein was more abundant（P 〈 0.05） in heat-stressed mice than control mice. In Exp. 2, heat stress increased HSP70 and decreased aromatase proteins（P 〈 0.05） in antral follicles. In Exp. 3, TUNEL-positive granulosa cells from heat-stressed ovaries were observed concomitant with a significant increase in HSP70, Bim and cleaved caspase-3 protein.Conclusion： Heat-stress in mice decrease estradiol in serum and aromatase in antral follicles but increased number of atretic follicles and granulosa cell undergoing apoptosis which may explain the decreased fertility commonly observed in heat-stressed animals.

Integrin β5 subunit regulates hyperglycemia-induced vascular endothelial cell apoptosis through FoxO1-mediated macroautophagy

Background:Hyperglycemia frequently induces apoptosis in endothelial cells and ultimately contributes to microvascular dysfunction in patients with diabetes mellitus(DM).Previous research reported that the expression of integrins as well as their ligands was elevated in the diseased vessels of DM patients.However,the association between integrins and hyperglycemia-induced cell death is still unclear.This research was designed to investigate the role played by integrin subunit β5(ITGB5)in hyperglycemia-induced endothelial cell apoptosis.Methods:We used leptin receptor knockout(Lepr-KO)(db/db)mice as spontaneous diabetes animal model.Selective deletion of ITGB5 in endothelial cell was achieved by injecting vascular targeted adeno-associated virus via tail vein.Besides,we also applied small interfering RNA in vitro to study the mechanism of ITGB5 in regulating high glucose-induced cell apoptosis.Results:ITGB5 and its ligand,fibronectin,were both upregulated after exposure to high glucose in vivo and in vitro.ITGB5 knockdown alleviated hyperglycemia-induced vascular endothelial cell apoptosis and microvascular rarefaction in vivo.In vitro analysis revealed that knockdown of either ITGB5 or fibronectin ameliorated high glucose-induced apoptosis in human umbilical vascular endothelial cells(HUVECs).In addition,knockdown of ITGB5 inhibited fibronectin-induced HUVEC apoptosis,which indicated that the fibronectin-ITGB5 interaction participated in high glucose-induced endothelial cell apoptosis.By using RNA-sequencing technology and bioinformatic analysis,we identified Forkhead Box Protein O1(FoxO1)as an important downstream target regulated by ITGB5.Moreover,we demonstrated that the excessive macroautophagy induced by high glucose can contribute to HUVEC apoptosis,which was regulated by the ITGB5-FoxO1 axis.Conclusion:The study revealed that high glucose-induced endothelial cell apoptosis was positively regulated by ITGB5,which suggested that ITGB5 could potentially be used to predict and treat DM-related vascular complications.

SETDB1-mediated CD147-K71 di-methylation promotes cell apoptosis in non-small cell lung cancer

Protein post-translational modifications(PTMs)are at the heart status of cellular signaling events and broadly involved in tumor progression.CD147 is a tumor biomarker with various PTMs,promoting tumor metastasis and metabolism reprogramming.Nevertheless,the relationship between the PTMs of CD147 and apoptosis has not been reported.In our study,we produced a specific anti-CD147-K71 di-methylation(CD147-K71me2)antibody by immunizing with a di-methylated peptide and observed that the level of CD147-K71me2 in non-small cell lung cancer(NSCLC)tissues were lower than that in NSCLC adjacent tissues.SETDB1 was identified as the methyltransferase catalyzing CD147 to generate CD147-K71me2.RNA-seq showed that FOSB was the most significant differentially expressed gene(DEG)between wild-type CD147(CD147-WT)and K71-mutant CD147(CD147-K71R)groups.Subsequently,we found that CD147-K71me2 promoted the expression of FOSB by enhancing the phosphorylation of p38,leading to tumor cell apoptosis.In vivo experiments showed that CD147-K71me2 significantly inhibited tumor progression by promoting cell apoptosis.Taken together,our findings indicate the inhibitory role of CD147-K71me2 in tumor progression from the perspective of post-translational modification,which is distinct from the pro-cancer function of CD147 itself,broadening our perspective on tumor-associated antigen CD147.

Reproductive toxicity of enrofloxacin in Caenorhabditis elegans involves oxidative stress-induced cell apoptosis

Fluoroquinolone antibiotics(FQs)that persist and bioaccumulate in the environment have aroused people’s great concern.Here,we studied the adverse effects of FQs in soil animals of Caenorhabditis elegans via food-chronically exposure.The result shows C.elegans exposed to FQs exhibited reproductive toxicity with small-brood size and low-egg hatchability.To study the underlying mechanism,we conduct a deep investigation of enrofloxacin(ENR),one of the most frequently detected FQs,on nematodes which is one of commonly used animal indicator of soil sustainability.The concentration-effect curves simulated by the Hill model showed that the half effect concentrations(EC50)of ENR were(494.3±272.9)μmol/kg and(107.4±30.9)μmol/kg for the brood size and the hatchability,respectively.Differential gene expression between the control and the ENR-exposure group enriched with the oxidative stress and cell apoptosis pathways.The results together with the enzyme activity in oxidative stress and the cell corpses suggested that ENR-induced reproductive toxicity was related to germ cell apoptosis under oxidative stress.The risk quotients of some soil and livestock samples were calculated based on the threshold value of EC10 for the egg hatchability(2.65μmol/kg).The results indicated that there was possible reproductive toxicity on the nematodes in certain agricultural soils for the FQs.This study suggested that chronic exposure to FQs at certain levels in environment would induce reproductive toxicity to the nematodes and might reduce the soil sustainability,alarming the environment risks of antibiotics abuse.

Dihydromyricetin Alleviates H9C2 Cell Apoptosis and Autophagy by Regulating CircHIPK3 Expression and PI3K/AKT/mTOR Pathway

Objective: To investigate the effect and potential mechanism of dihydromyricetin(Dmy) on H9C2 cell proliferation, apoptosis, and autophagy. Methods: H9C2 cells were randomly divided into 7 groups, namely control, model, EV(empty p CDH-CMV-MCS-EF1-Cop GFP-T2A-Puro vector), IV(circHIPK3 interference), Dmy(50 μmol/L), Dmy+IV, and Dmy+EV groups. Cell proliferation and apoptosis were detected by cell counting kit-8 assay and flow cytometry, respectivley. Western blot was used to evaluate the levels of light chain 3 Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ), phospho-phosphoinositide 3-kinase(p-PI3K), protein kinase B(p-AKT), and phospho-mammalian target of rapamycin(p-mTOR). The level of circHIPK3 was determined using reverse transcriptase polymerase chain reaction. Electron microscopy was used to observe autophagosomes in H9C2 cells. Results: Compared to H9C2 cells, the expression of circHIPK in H9C2 hypoxia model cells increased significantly(P<0.05). Compared to the control group, the cell apoptosis and autophagosomes increased, cell proliferation rate decreased significantly, and the expression of LC3Ⅱ/Ⅰ significantly increased(all P<0.05). Compared to the model group, the rate of apoptosis and autophagosomes in IV, Dmy, and Dmy+IV group decreased, the cell proliferation rate increased, and the expression of LC3Ⅱ/Ⅰ decreased significantly(all P<0.05). Compared to the control group, the expressions of p-PI3K, p-AKT, and p-mTOR in the model group significantly reduced(P<0.05), whereas after treatment with Dmy and sh-circHIPK3, the above situation was reversed(P<0.05). Conclusion: Dmy plays a protective role in H9C2 cells by inhibiting circHIPK expression and cell apoptosis and autophagy, and the mechanism may be related to PI3K/AKT/mTOR pathway.