Vitamin B3 inhibits apoptosis and promotes autophagy of islet β cells under high glucose stress

Background: Hyperglycemia is a typical symptom of diabetes. High glucose induces apoptosis of islet β cells. While autophagy functions in cytoprotection and autophagic cell death. The interaction between autophagy and apoptosis is important in the modulation of the function of islet β cells. Vitamin B3 can induce autophagy and inhibit islet β apoptosis.Method: The mechanism of vitamin B3-mediated protective effect on the function of islet β cells was explored by the method of western blot, immunofluorescence and flow cytometry.Results: In the present study, high glucose stress increased the apoptosis rate, while vitamin B3 reduced the apoptosis rate. The effect of vitamin B3 on autophagy flux under normal and high glucose stress was also investigated. Vitamin B3 increased the number of autophagosomes and increased the light chain (LC)3-II/LC3-I ratio. In contrast, vitamin B3 decreased sequestosome 1 (SQSTM1)/p62 protein expression and inhibited the phosphorylation of mammalian ribosomal protein S6 kinase β-1 (p70S6K/S6K1), which was a substrate of mammalian target of rapamycin (mTOR) under normal and high glucose stress. To further verify the protective effect of vitamin B3 on apoptosis, we treated islet β cell RIN-m5F with autophagy inhibitor 3-methyladenine (3-MA). Vitamin B3 decreased the apoptosis rate under high glucose stress, while the inhibition of apoptosis by vitamin B3 was blocked after adding 3-MA.Conclusion: Our data suggested that vitamin B3 reduced the apoptosis rate of β cells, possibly through inducing autophagy under high glucose stress.     

Bushen Yizhi Formula regulates the IRE1α pathway to alleviate endoplasmic reticulum stress in an Alzheimer’s disease rat model

Background: While the Bushen Yizhi Formula can treat Alzheimer’s disease (AD), the yet to be ascertainedspecific mechanism of action was explored in this work. Methods: Different concentrations of the Bushen YizhiFormula and amyloid-beta peptide (Aβ) were used to treat rat pheochromocytoma cells (P12) and humanneuroblastoma cells (SH-SY5Y). Cell morphological changes were observed to determine the in vitro cell damage. CellCounting Kit (CCK)-8 assay and flow cytometry were employed to identify cell viability and apoptosis/cell cycle,respectively. Western blotting and immunohistochemistry were employed to measure the expressions of endoplasmicreticulum stress (ERS)-related proteins (GRP78 and CHOP), p-IRE1α, IRE1α, ASK1, p-JNK, JNK, Bax, Bcl-2, XBP-1,and Bim. Fura 2-acetoxymethyl ester (Fura-2/AM) was used to determine the intracellular calcium (Ca²⁺)concentration. Also, an AD model was constructed by injecting Aβ into the CA1 area of the hippocampus in SpragueDawley rats. AD model rats were gavaged with different concentrations of Bushen Yizhi Formula for 14 consecutivedays. The Morris water maze experiment was conducted to test the learning and memory of rats. Hematoxylin &Eosin (H&E) and Terminal-deoxynucleotidyl Transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL)staining were done to determine histopathological changes in the brain. Results: Bushen Yizhi Formula relieved theAβ-induced effects including cell injury, decreased viability, increased apoptosis, G0/G1 phase cell cycle arrest, upregulation of GRP78, CHOP, p-IRE1α, p-JNK, Bax, XBP-1 and Bim, as well as down-regulation of Bcl-2. Theseresults were also seen with IRE1α silencing. While Aβ suppressed the learning and memory abilities of rats, theBushen Yizhi Formula alleviated these effects of Aβ. Brain nerve cell injury induced by Aβ could also be treated withBushen Yizhi Formula. Conclusion: Bushen Yizhi Formula could influence ERS through the IRE1α signaling pathwayto achieve its therapeutic effects on AD.     

The neuroprotection of electro-acupuncture via the PGC-1α/TFAM pathway in transient focal cerebral ischemia rats

ATP depletion is one of the pathological bases in cerebral ischemia. Electro-acupuncture (EA) is widely used in clinical practice for ischemia. However, the mechanism of EA remains unclear. The purpose of this study was to investigate whether EA could activate the AMPK/PGC-1α/TFAM signaling pathway and, consequently, increase the preservation of ATP in rats with ischemia. In this study, 48 rats were randomly divided into four groups as a sham-operation control group (sham group), a middle cerebral artery occlusion group (MCAO group), an EA group, and an EA group blocked by the AMPK inhibitor compound C (EA + CC group) (N = 12/group). The rats of the EA group and EA + CC group received the EA treatment for 7 days. The rats that belonged in the two remaining groups were only grasped in the same condition. Then, their brain tissues were collected for further detection. When compared with other groups, EA significantly reduced neurological deficits score and increased motor function. The cerebral infarction volume was significantly reduced in the EA group according to TTC staining. With western blot, we found that EA improved the ratio of p-AMPKα/AMPKα (P < 0.05), however, there is no difference between the MCAO group and sham group (P > 0.05). In addition, EA also increased the expression of PGC-1α and TFAM (all P < 0.05). By Elisa, we observed that EA increased the preservation of ATP (P < 0.05) and mitochondrial respiratory enzymes, including Complex I (P < 0.05), Complex IV (P < 0.05), but not Complex III (P > 0.05). In summary, we conclude that EA may protect against ischemic damage in MCAO rats, improve the preservation of ATP and mitochondrial respiratory enzymes. This effect may be positively regulated by the activation of the PGC-1α/TFAM signaling pathway.     

Association of hypoxia-inducible factor-1α (HIF1α) 1772C/T gene polymorphism with susceptibility to renal cell carcinoma/prostate cancer

In this study, we used a meta-analysis method to evaluate the relationship between hypoxia-inducible factor-1α(HIF1α) 1772C/T gene polymorphism (rs 11549465) and renal cell carcinoma (RCC)/prostate cancer risk. We searchedfor relevant studies (before March 1, 2019) on Cochrane Library, Embase, and PubMed. Studies meeting the inclusioncriteria were recruited into this meta-analysis. The outcome of dichotomous data was showed in the way of oddsratios (OR), and 95% confidence intervals (CI) were also counted. In this investigation, there was no associationbetween HIF1α 1772C/T gene polymorphism and susceptibility to RCC in Caucasians, Asians as well as overallpopulations. In addition, HIF1α 1772C/T gene polymorphism was not found to be relevant to the survival in RCC.Interestingly, the T allele was relevant to prostate cancer risk in all populations, but not in Caucasians and Asians.However, the TT genotype and the CC genotype were not related to prostate cancer susceptibility in Asian,Caucasian, and all populations. In conclusion, the T allele of the HIF1α 1772C/T gene polymorphism was related toprostate cancer risk in the overall populations.     

Cardioprotective effect of ivabradine via the AMPK/SIRT1/PGC-1α signaling pathway in myocardial ischemia/reperfusion injury induced in H9c2 cell

Post-resuscitation myocardial dysfunction (PRMD) is the most severe myocardial ischemia-reperfusion injury(MIRI) and is characterized by difficult treatment and poor prognosis. Research has shown the protective effects of therational use of ivabradine (IVA) against PRMD; however, the molecular mechanisms of IVA remain unknown. In thisstudy, an ischemia-reperfusion injury (IRI) model was established using hypoxic chambers. The results demonstratedthat pretreatment with IVA reduced IRI-induced cytotoxicity and apoptosis. IVA attenuated mitochondrial damage,eliminated excess reactive oxygen species (ROS), suppressed IRI-induced ATP and NAD⁺, and increased theAMP/ATP ratio. We further found that IVA increased the mRNA levels of sirtuin 1 (SIRT1) and peroxisomeproliferator-activated receptor-γ coactivator 1α (PGC-1α) and upregulated the expression levels of phosphorylatedAMP-activated protein kinase (p-AMPK)/AMPK, SIRT1, and PGC-1α proteins. Interestingly, no change in AMPKmRNA levels was observed. Cardiomyocyte energy metabolism significantly changed after IRI. The aim of this studywas to demonstrate the cardioprotective effect of Ivabradine via the AMPK/SIRT1/PGC-1α signaling pathway inmyocardial ischemia/reperfusion injury-induced in H9c2 cell.     

STMN1 promotes the proliferation and inhibits the apoptosis of acute myeloid leukemiacells by activating the PI3K/Akt pathway

Recent studies have shown that the microtubule disrupting protein Stathmin 1 (STMN1) is differentiallyexpressed in AML patients and healthy control. The aim of this study was to explore the effects and molecularmechanism of STMN1 in AML. Here, the expression of STMN1 in peripheral blood cells (PBMCs) and bone marrowof AML patients and healthy volunteers was detected by RT-PCR and Western blot. STMN1 expression was regulatedby transfected with STMN1 overexpressed plasmid or shRNA in two human leukemia cell lines K562 and HL60. Cellproliferation was examined by CCK8 and Edu staining. Annexin V and TUNEL assays were applied to test cellapoptosis. Flow cytometry was used to test the cell cycle distribution. The activation of the PI3K signaling pathwayand the expression levels of cell cycle and cell apoptosis-related protein were determined by Western blot. In thisstudy, we found that STMN1 was overexpressed in PBMCs and bone marrow of AML patients. STMN1 expressionwas closely related to FAB subtypes, risk stratification, disease-free survival, and overall survival of AML. Functionalassays showed that overexpression of STMN1 in HL60 and K562 cells enhanced cell proliferation, decreased cellapoptosis, and caused G₁ phase arrest. In contrast, suppression of STMN1reduced cell proliferation and enhanced cellapoptosis in both HL60 and K562 cells. Moreover, the PI3K/Akt pathway was activated by STMN1, while suppressionof STMN1 dysregulated the PI3K/Akt pathway and upregulating the levels of caspases3 and Bax expression. Inconclusion, STMN1 was confirmed to promote the proliferation and inhibit the apoptosis of HL60 and K562 cells bymodulating the PI3K/Akt pathway. STMN1 might be a novel molecular target for treating AML.     

3-<i>epi</i>-bufotalin suppresses the proliferation in colorectal cancer cells through the inhibition of the JAK1/STAT3 signaling pathway

Traditional Chinese medicine (TCM) has been increasingly employed in the last decades in China for both preventing and treating a variety of cancers. 3-epi-bufotalin is an active ingredient of TCM “Chanpi” with anti-tumor potential. However, the effect and mechanism of 3-epi-bufotalin on colorectal cancers were not well disclosed. The present study demonstrated that 3-epi-bufotalin could reduce viability, trigger apoptosis, and block the cell cycle at the G₂/M stage in colorectal cancer cell lines HT29, RKO, and COLO205 in vitro. Moreover, 3-epi-bufotalin inhibited the JAK1/STAT3 signaling pathway. These results indicated the anti-proliferation ability of 3-epi-bufotalin in colorectal cancer cells.     

Hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen–glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy

In this study, we investigated the protective effect of hyperbaric oxygen (HBO) on PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion and its possible mechanism. PC12 and H9C2 cell oxygen-glucose deprivation/reperfusion model were established. Cells were divided into a control group, model group, hyperbaric air (HBA) group and HBO group. The cell viability was detected by the CCK8 assay. Hoechst 33342 and PI staining assays and mitochondrial membrane potential (MMP) assays were used to detect cell apoptosis. The ultrastructure of cells, including autophagosomes, lysosomes, and apoptosis, were examined using a transmission electron microscope. The expression of autophagy-related proteins was detected by cellular immunofluorescence and immunocytochemistry. Our results showed that HBO can significantly improve the vitality of damaged PC12 and H9C2 cells caused by oxygen–glucose deprivation/reperfusion. HBO can significantly inhibit apoptosis of PC12 and H9C2 cells caused by oxygen-glucose deprivation/reperfusion. Importantly, we found that the protective mechanism of PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion may be related to the inhibition of the autophagy pathway. In this study, the results of cellular immunofluorescence and immunocytochemistry experiments showed that the 4E-BP1, p-AKt and mTOR levels of PC12 and H9C2 cells in the model group decreased, while the levels of LC3B, Atg5 and p53 increased. However, after HBO treatment, these autophagy-related indexes were reversed. In addition, observation of the cell ultrastructure with transmission electron microscopy found that in the model group, a significant increase in the number of autophagic vesicles was observed. In the HBO group, a decrease in autophagic vesicles was observed. The study demonstrated that hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy.     

Neural stem cell-conditioned medium upregulated the PCMT1 expression and inhibited the phosphorylation of MST1 in SH-SY5Y cells induced by Aβ_25-35

A progressive neurodegenerative disease, Alzheimer’s disease (AD). Studies suggest that highly expressedprotein isoaspartate methyltransferase 1 (PCMT1) in brain tissue. In the current study, we explored the effects ofneural stem cell-conditioned medium (NSC-CDM) on the PCMT1/MST1 pathway to alleviate Aβ_25-35-induceddamage in SH-SY5Y cells. Our data suggested that Aβ25-35 markedly inhibited cell viability. NSC-CDM or Neuralstem cell-complete medium (NSC-CPM) had a suppression effect on toxicity when treatment with Aβ_25-35, with agreater effect observed with NSC-CDM. Aβ_25-35 + NSC-CDM group exhibited an increase in PCMT1 expression.sh-PCMT1 markedly decreased cell proliferation and suppressed the protective role of NSC-CDM through theinduction of apoptosis and improved p-MST1 expression. Overexpression of PCMT1 reversed the Aβ_25-35-induceddecrease in cell proliferation and apoptosis. In summary, our findings suggest that NSC-CDM corrects the Aβ_25-35-induced damage to cells by improving PCMT1 expressions, which in turn reduces phosphorylation of MST1.     

Overexpression of inhibin α (1-32) fusion protein promotes apoptosis and cell cycle arrest in a cervical cancer cell model (Hela cells)

Inhibins play important roles in the reproductive system. To evaluate whether inhibin α (1-32) fusion protein plays a role in cervical cancer growth, the plasmid pVAX-inhα was constructed and its effect on proliferation and apoptosis of the human cervical cancer cell line (Hela) was checked by flow cytometry and real-time PCR. The expression and localization of inhibin α protein were detected by RT-PCR and confocal microscopy which showed that inhibin α protein was expressed and localized in the nucleus of Hela cells. Over expression of inhibin α gene significantly induced cell apoptosis and ceased S phase of cell cycle. Furthermore, cell proliferation was significantly suppressed 96 h post-transfection and mRNA level of anti-apoptosis genes (Bcl-2, NFκB) were decreased but pro-apoptosis genes (Bax, wild type p53) and inhibin co receptor (TGFβR3) were increased, indicating that inhibin, through its co-receptor, might activate apoptotic and cell growth cascades which regulate proliferation and apoptosis in Hela cells. These results suggest that inhibin α (1-32) fusion protein, located in the cell nucleus, can regulate Hela cells growth and apoptosis by induction of apoptotic pathways such as NFκB, Bcl-2 and p53 families. These findings may have a significant impact on future research regarding cervical cancer cell lines     

Effects of TGF‐β1 on mineralization mediated by rat calvaria‐derived osteogenic cells

In this study, we have analyzed the viability and cell growth, as well as, the mineralization of extracellular matrix (ECM) by alizarin red and von Kossa staining of calvaria‐derived osteogenic cultures, treated with TGF‐β1 alone or associated with Dex comparing with acid ascorbic (AA) + β‐glicerophosphate (βGP) (positive mineralization control). The expression of the noncollagenous proteins bone sialoprotein (BSP), osteopontin (OPN) and fibronectin (FN) were evaluated by indirect immunofluorescence. In addition, the main ultrastructural morphological findings were assessed by transmission electron microscopy. Osteogenic cells were isolated of calvaria bone from newborn (2‐day‐old) Wistar rats were treated with TGF‐β1 alone or with dexamethasone for 7, 10, and 14 days. As positive mineralization control, the cells were supplemented only with AA+ βGP. As negative control, the cells were cultured with basal medium (α‐MEM + 10%FBS + 1%gentamicin). The treatment with TGF‐β1, even when combined with Dex, decreased the viability and cell growth when compared with the positive control. Osteoblastic cell cultures were positive to alizarin red and von Kossa stainings after AA + βGP and Dex alone treatments. Positive immunoreaction was found for BSP, OPN and FN in all studied treatments. Otherwise, when the cell cultures were supplemented with TGF‐β1 and TGF‐β1 + Dex, no mineralization was observed in any of the studied periods. These present findings suggest that TGF‐β1, in the studied in vitro doses, inhibits the proliferation and differentiation of osteoblastic cells by impairment of nodule formation.     

Uncoupling tumor necrosis factor-α and interleukin-10 at tumor immune microenvironment of breast cancer through miR-17-5p/MALAT-1/H19 circuit

Triple Negative Breast Cancer (TNBC) immunotherapy has recently shown promising approach. However, some TNBC patients presented with resistance. One of the reasons was attributed to the excessive release of cytokines at the tumor microenvironment (TME) such as Tumor necrosis factor alpha (TNF-α) and Interleukin-10 (IL-10). Fine regulation of these cytokines’ levels via non-coding RNAs (ncRNAs) might alleviate the immune quiescent nature of TME at TNBC tumors. However, the extrapolation of ncRNAs as therapeutic tools is highly challenging. Therefore, disentanglement the nature for the isolation of natural compounds that could modulate the ncRNAs and their respective targets is an applicable translational therapeutic approach. Hence, this study aimed to targeting the chief immune suppressive cytokines at the TME (TNF-α and IL-10) via ncRNAs and to examine the effects of Rosemary aerial parts extract on the expression levels of these ncRNAs in TNBC. Results revealed miR-17-5p as a dual regulator of TNF-α and IL-10. Moreover, an intricate interaction has been shown between miR-17-5p and the oncogenic lncRNAs: MALAT1 and H19. Knocking down of MALAT1 and/or H19 caused an induction in miR-17-5p and reduction in TNF-α and IL-10 expression levels. miR-17-5p was found to be down-regulated, while TNF-α, IL-10, MALAT1 and H19 were up-regulated in BC patients. Forced expression of miR-17-5p in MDA-MB-231 cells reduced TNF-α, IL-10, MALAT1 and H19 expression levels, as well as several BC hallmarks. In a translational approach, ursolic acid (UA) isolated from rosemary induced the expression of miR-17-5p, MALAT1 and decreased H19 expression levels. In conclusion, this study suggests miR-17-5p as a tumor suppressor and an immune-activator miRNA in BC through tuning up the immunological targets TNF-α, IL-10 at the TME and the oncological mediators MALAT1 and H19 lncRNAs.     

Inhibition of H₂O₂-induced apoptosis of GC2-spg cells by functionalized selenium nanoparticles with lentinan through ROS-mediated ERK/p53 signaling pathways

A H₂O₂-induced oxidative stress injury cell model was established to investigate the antioxidant effect of nano-selenium on mouse spermatocyte lines and the regulation mechanism of the expression level and activity of selenium-containing antioxidant enzymes induced by oxidative stress. A safe and effective nano-drug system of functionalized selenium-containing nanoparticles (SeNPs) was developed with lentinan (LNT) (SeNPs@LNT). Mice spermatocyte line GC2-spg cells were treated with SeNPs@LNT (1, 2, 4, 8, 16, 32 μM) for 24–72 h to evaluate the cytotoxicity of selenium. GC2-spg cells were randomly divided into the following groups: control, hydrogen peroxide (H₂O₂), SeNPs@LNT, and H₂O₂+SeNPs@LNT groups. H₂O₂+SeNPs@LNT group was pretreated with SeNPs@LNT 4 μM for 12 h, followed by H₂O₂ 600 μM for 8 h. The cell viability decreased in the H₂O₂ group and increased significantly in the SeNPs@LNT group. Compared with the H₂O₂ group, the SeNPs@LNT+H₂O₂ group exhibited obvious red fluorescence, indicating a higher level of mitochondrial membrane potential. The content of intracellular reactive oxygen species (ROS) in the SeNPs@LNT group reduced significantly, and the intensity of green fluorescence in the SeNPs@LNT+H₂O₂ group decreased significantly compared with the H₂O₂ group, indicating the inhibitory effect of SeNPs@LNT on the generation of ROS-induced oxidative stress. The activity of GPx and SOD increased significantly in the SeNPs@LNT group. The expression of p53 decreased significantly under the intervention of nano-selenium, and GPx1 expression increased. In the oxidative stress group, the expressions of DNA damage-related proteins and apoptosis-related proteins were higher than those in other groups. Thus, SeNPs@LNT can promote GC2-spg cell proliferation, improve GPx and SOD activities, remove intracellular ROS, and reduce mitochondrial damage and functional abnormalities caused by oxidative stress by regulating the ERK and p53 protein levels. SeNPs@LNT has good biological activity and antioxidant effect, which can be used to protect the male reproductive system from oxidative stress.