Pyrroloquinoline quinone promotes human mesenchymal stem cell-derived mitochondria to improve premature ovarian insufficiency in mice through the SIRT1/ATM/p53 pathway.

BACKGROUND: DNA damage and oxidative stress induced by chemotherapy are important factors in the onset of premature ovarian insufficiency (POI). Studies have shown that mitochondria derived from mesenchymal stem cells (MSC-Mito) are beneficial for age-related diseases, but their efficacy alone is limited. Pyrroloquinoline quinone (PQQ) is a potent antioxidant with significant antiaging and fertility enhancement effects. This study aimed to investigate the therapeutic effect of MSC-Mito in combination with PQQ on POI and the underlying mechanisms involved. METHODS: A POI animal model was established in C57BL/6J mice by cyclophosphamide and busulfan. The effects of MSC-Mito and PQQ administration on the estrous cycle, ovarian pathological damage, sex hormone secretion, and oxidative stress in mice were evaluated using methods such as vaginal smears and ELISAs. Western blotting and immunohistochemistry were used to assess the expression of SIRT1, PGC-1α, and ATM/p53 pathway proteins in ovarian tissues. A cell model was constructed using KGN cells treated with phosphoramide mustard to investigate DNA damage and apoptosis through comet assays and flow cytometry. SIRT1 siRNA was transfected into KGN cells to further explore the role of the SIRT1/ATM/p53 pathway in combination therapy with MSC-Mito and PQQ for POI. RESULTS: The combined treatment of MSC-Mito and PQQ significantly restored ovarian function and antioxidant capacity in mice with POI. This treatment also reduced the loss of follicles at various stages, improving the disrupted estrous cycle. In vitro experiments demonstrated that PQQ facilitated the proliferation of MitoTracker-labelled MSC-Mito, synergistically restoring mitochondrial function and inhibiting oxidative stress in combination with MSC-Mito. Both in vivo and in vitro, the combination of MSC-Mito and PQQ increased mitochondrial biogenesis mediated by SIRT1 and PGC-1α while inhibiting the activation of ATM and p53, consequently reducing DNA damage-mediated cell apoptosis. Furthermore, pretreatment of KGN cells with SIRT1 siRNA reversed nearly all the aforementioned changes induced by the combined treatment. CONCLUSIONS: Our research findings indicate that PQQ facilitates MSC-Mito proliferation and, in combination with MSC-Mito, ameliorates chemotherapy-induced POI through the SIRT1/ATM/p53 signaling pathway.

OPN silencing reduces hypoxic pulmonary hypertension via PI3K-AKT-induced protective autophagy.

Hypoxic pulmonary hypertension (HPH) is a pulmonary vascular disease primarily characterized by progressive pulmonary vascular remodeling in a hypoxic environment, posing a significant clinical challenge. Leveraging data from the Gene Expression Omnibus (GEO) and human autophagy-specific databases, osteopontin (OPN) emerged as a differentially expressed gene, upregulated in cardiovascular diseases such as pulmonary arterial hypertension (PAH). Despite this association, the precise mechanism by which OPN regulates autophagy in HPH remains unclear, prompting the focus of this study. Through biosignature analysis, we observed significant alterations in the PI3K-AKT signaling pathway in PAH-associated autophagy. Subsequently, we utilized an animal model of OPNfl/fl-TAGLN-Cre mice and PASMCs with OPN shRNA to validate these findings. Our results revealed right ventricular hypertrophy and elevated mean pulmonary arterial pressure (mPAP) in hypoxic pulmonary hypertension model mice. Notably, these effects were attenuated in conditionally deleted OPN-knockout mice or OPN-silenced hypoxic PASMCs. Furthermore, hypoxic PASMCs with OPN shRNA exhibited increased autophagy compared to those in hypoxia alone. Consistent findings from in vivo and in vitro experiments indicated that OPN inhibition during hypoxia reduced PI3K expression while increasing LC3B and Beclin1 expression. Similarly, PASMCs exposed to hypoxia and PI3K inhibitors had higher expression levels of LC3B and Beclin1 and suppressed AKT expression. Based on these findings, our study suggests that OPNfl/fl-TAGLN-Cre effectively alleviates HPH, potentially through OPN-mediated inhibition of autophagy, thereby promoting PASMCs proliferation via the PI3K-AKT signaling pathway. Consequently, OPN emerges as a novel therapeutic target for HPH.

The curious case of the disappearing piRNAs.

Small non-coding RNAs are key regulators of gene expression across eukaryotes. Piwi-interacting small RNAs (piRNAs) are a specific type of small non-coding RNAs, conserved across animals, which are best known as regulators of genome stability through their ability to target transposable elements for silencing. Despite the near ubiquitous presence of piRNAs in animal lineages, there are some examples where the piRNA pathway has been lost completely, most dramatically in nematodes where loss has occurred in at least four independent lineages. In this perspective I will provide an evaluation of the presence of piRNAs across animals, explaining how it is known that piRNAs are missing from certain organisms. I will then consider possible explanations for why the piRNA pathway might have been lost and evaluate the evidence in favor of each possible mechanism. While it is still impossible to provide definitive answers, these theories will prompt further investigations into why such a highly conserved pathway can nevertheless become dispensable in certain lineages. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

[Metformin suppresses hypoxia-inducible factor-1α expression in cancer-associated fibroblasts to block tumor-stromal cross-talk in breast cancer].

OBJECTIVE: To investigate the mechanism of metformin for regulating tumor-stromal cell cross-talk in breast cancer. METHODS: Tumor associated fibroblasts (CAFs) co-cultured with breast cancer cells were treated with metformin, and the changes in expressions of hypoxia-inducible factor-1α (HIF-1α), p-AMPK, stroma-derived factor-1 (SDF-1) and interleukin-8 (IL-8) in the CAFs were detected using ELISA, RT-qPCR or Western blotting; Transwell assay was used to evaluate the invasiveness of the tumor cells and its changes following treatment with exogenous SDF-1, IL-8 and TGF-ß1. The effects of HIF-1α shRNA or overexpression plasmid, AMPK shRNA, and treatment with OG (a proline hydroxylase inhibitor) or 2-OXO (a proline hydroxylase activator) were examined on p-AMPK, HIF-1α, SDF-1 and IL-8 expressions and invasiveness of the CAFs. RESULTS: Metformin treatment significantly increased the expression levels of p-AMPK, SDF-1 and IL-8 (P<0.05) and decreased HIF-1α expression (P<0.05) without affecting AMPK expression level (P>0.05) in the CAFs. The invasion ability of metformintreated breast cancer cells was significantly decreased (P<0.05). Exogenous SDF-1 and IL-8, HIF-1α overexpression, and OGinduced upregulation of HIF-1α all significantly attenuated the inhibitory effects of metformin on breast cancer cell invasion (P<0.05) and HIF-1α, SDF-1 and IL-8 expressions in CAFs (P<0.05). Transfection with HIF-1α shRNA or treatment with 2-OXO significantly decreased the invasiveness of breast cancer cells (P<0.05). P-AMPK knockdown significantly suppressed the inhibitory effect of metformin on HIF-1α expression in CAFs and on invasion of breast cancer cells (P<0.05). Treatment with TGF-ß1 partially decreased the inhibitory effect of metformin on HIF-1α expression in CAFs and invasiveness of the breast cancer cells (P<0.05). CONCLUSION: Metformin suppresses HIF-1α expression in CAFs to block tumor-stromal cross talk in breast cancer.

Excessive fat expenditure in MCT-induced heart failure rats is associated with BMAL1/REV-ERBα circadian rhythmic loop disruption.

Fat loss predicts adverse outcomes in advanced heart failure (HF). Disrupted circadian clocks are a primary cause of lipid metabolic issues, but it's unclear if this disruption affects fat expenditure in HF. To address this issue, we investigated the effects of disruption of the BMAL1/REV-ERBα circadian rhythmic loop on adipose tissue metabolism in HF.50 Wistar rats were initially divided into control (n = 10) and model (n = 40) groups. The model rats were induced with HF via monocrotaline (MCT) injections, while the control group received equivalent solvent injections. After establishing the HF model, the model group was further subdivided into four groups: normal rhythm (LD), inverted rhythm (DL), lentivirus vector carrying Bmal1 short hairpin RNA (LV-Bmal1 shRNA), and empty lentivirus vector control (LV-Control shRNA) groups, each with 10 rats. The DL subgroup was exposed to a reversed light-dark cycle of 8 h: 16 h (dark: light), while the rest adhered to normal light-dark conditions (light: dark 12 h: 12 h). Histological analyses were conducted using H&E, Oil Red O, and Picrosirius red stains to examine adipose and liver tissues. Immunohistochemical staining, RT-qPCR, and Western blotting were performed to detect markers of lipolysis, lipogenesis, and beiging of white adipose tissue (WAT), while thermogenesis indicators were detected in brown adipose tissue (BAT). The LD group rats exhibited decreased levels of BMAL1 protein, increased levels of REV-ERBα protein, and disrupted circadian circuits in adipose tissue compared to controls. Additionally, HF rats showed reduced adipose mass and increased ectopic lipid deposition, along with smaller adipocytes containing lower lipid content and fibrotic adipose tissue. In the LD group WAT, expression of ATGL, HSL, PKA, and p-PKA proteins increased, alongside elevated mRNA levels of lipase genes (Hsl, Atgl, Peripilin) and FFA ß-oxidation genes (Cpt1, acyl-CoA). Conversely, lipogenic gene expression (Scd1, Fas, Mgat, Dgat2) decreased, while beige adipocyte markers (Cd137, Tbx-1, Ucp-1, Zic-1) and UCP-1 protein expression increased. In BAT, HF rats exhibited elevated levels of PKA, p-PKA, and UCP-1 proteins, along with increased expression of thermogenic genes (Ucp-1, Pparγ, Pgc-1α) and lipid transportation genes (Cd36, Fatp-1, Cpt-1). Plasma NT-proBNP levels were higher in LD rats, accompanied by elevated NE and IL-6 levels in adipose tissue. Remarkably, morphologically, the adipocytes in the DL and LV-Bmal1 shRNA groups showed reduced size and lower lipid content, while lipid deposition in the liver was more pronounced in these groups compared to the LD group. At the gene/protein level, the BMAL1/REV-ERBα circadian loop exhibited severe disruption in LV-Bmal1 shRNA rats compared to LD rats. Additionally, there was increased expression of lipase genes, FFA ß oxidation genes, and beige adipocyte markers in WAT, as well as higher expression of thermogenic genes and lipid transportation genes in BAT. Furthermore, plasma NT-proBNP levels and adipose tissue levels of NE and IL-6 were elevated in LV-Bmal1 shRNA rats compared with LD rats. The present study demonstrates that disruption of the BMAL1/REV-ERBα circadian rhythmic loop is associated with fat expenditure in HF. This result suggests that restoring circadian rhythms in adipose tissue may help counteract disorders of adipose metabolism and reduce fat loss in HF.

Identification and expression patterns of somatic piRNAs and PIWI genes in Riptortus pedestris (Hemiptera: Alydidae).

RNA interference (RNAi)-based gene silencing is a feasible and sustainable technology for the management of hemipteran pests by double-stranded RNA involvement, including small-interfering RNA, microRNA, and Piwi-interacting RNA (piRNA) pathways, that may help to decrease the usage of chemical insecticides. However, only a few data are available on the somatic piRNAs and their biogenesis genes in Riptortus pedestris, which serves as a significant pest of soybean (Glycine max). In this study, two family members of the PIWI gene were identified and characterized in R. pedestris, containing Argonaute3 (RpAgo3) and Aubergine (RpAub) genes with conserved protein domains, and their clusters were validated by phylogenetic analysis. In addition, they were widely expressed in all developmental stages of the whole body of R. pedestris and had lower expression levels in R. pedestris guts under different rearing conditions based on previous transcriptome sequencing. Furthermore, abundant clean reads were filtered to a total number of 45,998 piRNAs with uridine bias at the first nucleotide (nt) position and 26-32 nt in length by mapping onto the reference genome of R. pedestris according to our previous whole-transcriptome sequencing. Finally, our data revealed that gut bacterial changes were significantly positively or negatively associated with differentially expressed piRNAs among the five comparison groups with Pearson correlation analysis. In conclusion, these findings paved new avenues for the application of RNAi-based biopesticides for broad-spectrum hemipteran pest control.

Copper-mediated siRNA activation for conditional control of gene expression.

Copper plays a crucial role in maintaining biological redox balance in living organisms, with elevated levels observed in cancer cells. Short interfering RNAs (siRNAs) are effective in gene silencing and find applications as both research tools and therapeutic agents. A method to regulate RNA interference using copper is especially advantageous for cancer-specific therapy. We present a chemical approach of selective siRNA activation triggered by intracellular copper ions. We designed and synthesized nucleotides containing copper-responsive moieties, which were incorporated into siRNAs. These copper-responsive siRNAs effectively silenced the target cyclin B1 mRNA in living cells. This pioneering study introduces a novel method for conditionally controlling gene silencing using biologically relevant metal ions in human cells, thereby expanding the repertoire of chemical knockdown tools.

Multiple factors and features dictate the selective production of ct-siRNA in Arabidopsis.

Coding transcript-derived siRNAs (ct-siRNAs) produced from specific endogenous loci can suppress the translation of their source genes to balance plant growth and stress response. In this study, we generated Arabidopsis mutants with deficiencies in RNA decay and/or post-transcriptional gene silencing (PTGS) pathways and performed comparative sRNA-seq analysis, revealing that multiple RNA decay and PTGS factors impede the ct-siRNA selective production. Genes that produce ct-siRNAs often show increased or unchanged expression and typically have higher GC content in sequence composition. The growth and development of plants can perturb the dynamic accumulation of ct-siRNAs from different gene loci. Two nitrate reductase genes, NIA1 and NIA2, produce massive amounts of 22-nt ct-siRNAs and are highly expressed in a subtype of mesophyll cells where DCL2 exhibits higher expression relative to DCL4, suggesting a potential role of cell-specific expression of ct-siRNAs. Overall, our findings unveil the multifaceted factors and features involved in the selective production and regulation of ct-siRNAs and enrich our understanding of gene silencing process in plants.

Inhibition of DAPK3 Suppresses Radiation-Induced Cellular Senescence by Activation of a PGC1α-Dependent Metabolism Pathway in Brain Endothelial Cells.

In the brain, environmental changes, such as neuroinflammation, can induce senescence, characterized by the decreased proliferation of neurons and dendrites and synaptic and vascular damage, resulting in cognitive decline. Senescence promotes neuroinflammatory disorders by senescence-associated secretory phenotypes and reactive oxygen species. In human brain microvascular endothelial cells (HBMVECs), we demonstrate that chronological aging and irradiation increase death-associated protein kinase 3 (DAPK3) expression. To confirm the role of DAPK3 in HBMVEC senescence, we disrupted DAPK3 activity using small interfering RNA (siRNA) or a dominant-negative mutant (DAPK3-P216S), which reduced cellular senescence phenotypes, as assessed by changes in tube formation, senescence-associated beta-galactosidase activity, and cell proliferation. In endothelial cells, DAPK3 promotes cellular senescence by regulating the phosphorylation and inactivation of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) via the protein kinase B pathway, resulting in the decreased expression of mitochondrial metabolism-associated genes, such as ATP5G1, BDNF, and COX5A. Our studies show that DAPK3 is involved in cellular senescence and PGC1α regulation, suggesting that DAPK3 regulation may be important for treating aging-related brain diseases or the response to radiation therapy.

Genome-wide view and characterization of natural antisense transcripts in Cannabis Sativa L.

Natural Antisense Transcripts (NATs) are a kind of complex regulatory RNAs that play crucial roles in gene expression and regulation. However, the NATs in Cannabis Sativa L., a widely economic and medicinal plant rich in cannabinoids remain unknown. In this study, we comprehensively predicted C. sativa NATs genome-wide using strand-specific RNA sequencing (ssRNA-Seq) data, and validated the expression profiles by strand-specific quantitative reverse transcription PCR (ssRT-qPCR). Consequently, a total of 307 NATs were predicted in C. sativa, including 104 cis- and 203 trans- NATs. Functional enrichment analysis demonstrated the potential involvement of the C. sativa NATs in DNA polymerase activity, RNA-DNA hybrid ribonuclease activity, and nucleic acid binding. Finally, 18 cis- and 376 trans- NAT-ST pairs were predicted to produce 621 cis- and 5,679 trans- small interfering RNA (nat-siRNAs), respectively. These nat-siRNAs were potentially involved in the biosynthesis of cannabinoids and cellulose. All these results will shed light on the regulation of NATs and nat-siRNAs in C. sativa.

Selective degradation of PL2L60 by metabolic stresses­induced autophagy suppresses multi­cancer growth.

It has been reported that PL2L60 proteins, a product of PIWIL2 gene which might be activated by an intragenic promoter, could mediate a common pathway specifically for tumorigenesis. In the present study, it was further identified by using western blot assay that the PL2L60 proteins could be degraded in cancer cells through a mechanism of selective autophagy in response to oxidative stress. The PL2L60 was downregulated in various types of cancer cells under the hypoxic condition independently of HIF­1α, resulting in apoptosis of cancer cells. Inhibition of autophagy by small interfering RNA targeting of either Beclin­1 (BECN1) or Atg5 resulted in restoration of PL2L60 expression in hypoxic cancer cell. The hypoxic degradation of PL2L60 was also blocked by the attenuation of the autophagosome membrane protein Atg8/microtubule­associated protein 1 light chain 3 (LC3) or autophagy cargo protein p62 expression. Surprisingly, Immunofluorescence analysis demonstrated that LC3 could be directly bound to PL2L60 and was required for the transport of PL2L60 from the nucleus to the cytoplasm for lysosomal flux under basal or activated autophagy in cancer cells. Moreover, flow cytometric analysis displayed that knocking down of PL2L60 mRNA but not PIWIL2 mRNA effectively inhibited cancer cell proliferation and promoted apoptosis of cancer cells. The similar results were obtained from in vivo tumorigenic experiment, in which PL2L60 downregulation in necroptosis areas was confirmed by immunohistochemistry. These results suggested that various cancer could be suppressed by promoting autophagy. The present study revealed a key role of autophagic degradation of PL2L60 in hypoxia­induced cancer cell death, which could be used as a novel therapeutic target of cancer.

[Research progress of piRNA as a biomarker for male infertility].

piRNA is a class of small non-coding RNA which specifically binds with PIWI protein. It is mainly expressed in germ cells and involved in the regulation of spermatogenesis. The role of piRNA pathway in the regulation of spermatogenesis mainly includes inhibition of transposons, induction of mRNA translation or degradation, and mediation of degradation of Miwi ubiquitination in late-stage sperm cells. With the detection of piRNA in seminal plasma, more attention has been attracted to whether piRNA can be used as a non-invasive molecular biomarker for the evaluation of spermatogenesis. This paper has reviewed recent studies on the mechanism of piRNA pathways mediating spermatogenesis and potential roles of piRNA disorders in the diagnosis and treatment of male infertility.

Dishevelled Segment Polarity Protein 3 (DVL3) Induced by Bacterial LPS Promotes the Proliferation and Migration of Prostate Cancer Cells through the TLR4 Pathway.

BACKGROUND: Chronic inflammation is associated with various malignant tumors. Bacterial lipopolysaccharides (LPSs) play a significant part in the event and development of prostate cancer. Dishevelled segment polarity protein 3 (DVL3) is a shared component of the Wnt/ß-catenin and Notch signaling pathways, which are involved in tumor progression, chemoresistance, and maintenance of stem cell-like properties. According to reports, prostatic cancer cell invasion and proliferation are mediated by toll-like receptor 4 (TLR4). However, the role and regulation of DVL3 in prostate cancer and its relationship with TLR4 remain unclear. METHODS: Survival curves were plotted to evaluate the relationship between DVL3 expression and prognosis in patients with prostate cancer. DVL3 was silenced in PC3 and DU145 cells using small interfering RNAs (siRNAs). Subsequently, cell counting kit-8 (CCK-8) assay, colony formation assay, transwell migration assay, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were performed to investigate the role of DVL3 in cell proliferation and migration in vitro. The protein markers of potential pathways were analyzed via western blotting. RESULTS: DVL3 expression was linked to prognosis in patients with prostate cancer; In particular, patients with high DVL3 expression had a poor prognosis. LPS stimulation increased (p < 0.01) the expression of DVL3 in PC3 cells. DVL3 regulated tumor cell proliferation and migration by mediating the increase (p < 0.01) in TLR4 expression. Knockout of TLR4 validated that TLR4 played a crucial role in LPS-induced DVL3 expression. Silencing of DVL3 decreased (p < 0.01) the LPS-induced proliferation and migration of PC3 cells. CONCLUSIONS: Bacterial LPS-induced DVL3 promoted the multiplication and migration of prostate cancer cells through the TLR4 pathway. This study offers a valuable reference for the development and clinical application of targeted drugs for prostate cancer.

[Downregulation of SETDB1 suppresses epithelial mesenchymal transition and invasion and metastasis in oral cancer via SOX7 methylation].

PURPOSE: To explore the mechanism of SETDB1 inhibiting epithelial mesenchymal transition (EMT),migration and invasion in oral cancer via SOX 7 methylation. METHODS: SETDB1 and SOX7 mRNA and protein expression levels in KB cells of oral cancer and oral mucosal epithelial ATCC cells were determined by qRT-PCR and Western blot (WB). SETDB1 si-RNA was structured, then transfect into KB cells of oral cancer by liposome-mediated method. siRNA-SETDB1 was the experimental group (si-S), siRNA empty vector was the negative control group (si-N), and untransfected KB cells were the blank control group(NC). SETDB1 mRNA and protein expression levels were detected by qRT-PCR and Western blot(WB), to verify the transfection effect. The methylation levels of SOX7 were determined by pyrosequencing. The expression of N-cadherin, Vimentin, ß-catenin, and Slug proteins was detected by WB. Cell viability was measured by MTT assay, migration ability was tested by scratch healing assay, and invasion ability was tested by Transwell chamber assay. Statistical analysis was performed with SPSS 21.0 software package. RESULTS: The results of Rt-qPCR and WB showed that the SETDB1 mRNA and protein expression decreased significantly in si-S group(P＜0.05). Pyrosequencing test results showed that the regulation of SETDB1 could significantly reduce the SOX7 methylation rate and increased the SOX7 protein expression. WB results showed that knockdown of SETDB1 significantly inhibited the expression of EMT-related proteins N-cadherin, Vimentin, ß-catenin and Slug in oral cancer KB cells (P＜0.05). The results of cell functology experiments showed that knockdown of SETDB1 could significantly inhibit survival, migration and invasion of KB cells. CONCLUSIONS: Downregulation of SETDB1 could suppress EMT, migration and invasion of oral cancer cells by regulating SOX7 methylation level, providing new ideas and targets for the diagnosis and treatment of oral cancer.

Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis.

Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline-deficient, high-fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.

Selenium protects against Pb-induced renal oxidative injury in weaning rats and human renal tubular epithelial cells through activating NRF2.

BACKGROUND: Lead (Pb) poisoning posing a crucial health risk, especially among children, causing devastating damage not only to brain development, but also to kidney function. Thus, an urgent need persists to identify highly effective, safe, and low-toxicity drugs for the treatment of Pb poisoning. The present study focused on exploring the protective effects of Se on Pb-induced nephrotoxicity in weaning rats and human renal tubular epithelial cells, and investigated the possible mechanisms. METHODS: Forty weaning rats were randomly divided into four groups in vivo: control, Pb-exposed, Pb+Se and Se. Serum creatinine (Cr), urea nitrogen (BUN) and hematoxylin and eosin (H&E) staining were performed to evaluate renal function. The activities of antioxidant enzymes in the kidney tissue were determined. In vitro experiments were performed using human renal tubular epithelial cells (HK-2 cells). The cytotoxicity of Pb and Se was detected by 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Inverted fluorescence microscope was used to investigate cell morphological changes and the fluorescence intensity of reactive oxygen species (ROS). The oxidative stress parameters were measured by a multi-detection reader. Nuclear factor-erythroid-2-related factor (NRF2) signaling pathways were measured by Western blot and reverse transcription polymerase chain reaction (RT-PCR) in HK-2 cells. RESULTS: We found that Se alleviated Pb-induced kidney injury by relieving oxidative stress and reducing the inflammatory index. Se significantly increased the activity of the antioxidant enzymes glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), whereas it decreased the excessive release of malondialdehyde (MDA) in the kidneys of weaning rats and HK-2 cells. Additionally, Se enhanced the antioxidant defense systems via activating the NRF2 transcription factor, thereby promoting the to downstream expression of heme oxygenase 1. Furthermore, genes encoding glutamate-cysteine ligase synthetase catalytic (GCLC), glutamate-cysteine ligase synthetase modifier (GCLM) and NADPH quinone oxidoreductase 1 (NQO1), downstream targets of NRF2, formed a positive feedback loop with NRF2 during oxidative stress responses. The MTT assay results revealed a significant decrease in cell viability with Se treatment, and the cytoprotective role of Se was blocked upon knockdown of NRF2 by small interfering RNA (siRNA). MDA activity results also showed that NRF2 knockdown inhibited the NRF2-dependent transcriptional activity of Se. CONCLUSIONS: Our findings demonstrate that Se ameliorated Pb-induced nephrotoxicity by reducing oxidative stress both in vivo and in vitro. The molecular mechanism underlying Se's action in Pb-induced kidney injury is related to the activation of the NRF2 transcription factor and the activity of antioxidant enzymes, ultimately suppressing ROS accumulation.

Depletion of CNOT4 modulates the DNA damage responses following ionizing radiation (IR).

BACKGROUND: The Ccr4-Not complex (CNOT complex in mammals) is a unique and highly conserved complex with numerous cellular functions. Until now, there has been relatively little known about the importance of the CNOT complex subunits in the DNA damage response (DDR) in mammalian cells. CNOT4 is a subunit of the complex with E3 ubiquitin ligase activity that interacts transiently with the CNOT1 subunit. Here, we attempt to investigate the role of human CNOT4 subunit in the DDR in human cells. MATERIAL AND METHODS: In this study, cell viability in the absence of CNOT4 was assessed using a Cell Titer-Glo Luminescence assay up to 4 days post siRNA transfection. In a further experiment, CNOT4-depleted HeLa cells were exposed to 3Gy ionizing radiation (IR). Ataxia telangiectasia-mutated (ATM) and ATM Rad3-related (ATR) signaling pathways were then investigated by western blotting for phosphorylated substrates. In addition, foci formation of histone 2A family member X (γH2AX), replication protein A (RPA), TP53 binding protein 1 (53BP1), and DNA repair protein RAD51 homolog 1 was also determined by immunofluorescence microscopy comparing control and CNOT4-depleted HeLa cells 0, 8, and 24 h post IR treatment. RESULTS: Our results from cell viability assays showed a significant reduction of cell growth activity at 24 (P value 0.02) and 48 h (P value 0.002) post siRNA. Western blot analysis showed slightly reduced or slightly delayed DDR signaling in CNOT4-depleted HeLa cells after IR. More significantly, we observed increased formation of γH2AX, RPA, 53BP1, and RAD51 foci after IR in CNOT4-depleted cells compared with the control cells. CONCLUSION: We conclude that depletion of CNOT4 affects various aspects of the cellular response to DNA damage.

[Inhibitory effect of downregulating G protein-coupled receptor class C group 5 member A expression on lipopolysaccharide-induced inflammatory response in human gingival fibroblasts].

Objective: To clarify the effect and the mechanism of G protein-coupled receptor class C group 5 member A (GPRC5A) on lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (GFs), thus to provide a foundation for delving into the role of G protein coupled receptor (GPCR) in periodontitis. Methods: Gingival tissue samples were collected from 3 individuals periodontally healthy (health group) and 3 patients with periodontitis (periodontitis group) in Shandong Stomatological Hospital from December 2022 to February 2023. The expressions of GPRC5A of the two groups were detected by immunohistochemistry staining. GFs used in this study were isolated from a portion of gingiva for the extraction of impacted teeth in School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University from December 2022 to February 2023. GFs were isolated with enzymic digestion and transfected with 30, 50 and 80 µmol/L small interfering RNA-GPRC5A (siGPRC5A) or small interfering RNA-negative control (siNC), regarded as the experimental group and the negative control one, respectively. The silencing efficiency of siGPRC5A was evaluated by real-time fluorescence quantitative PCR (RT-qPCR). Experiments were then conducted using these cells which were divided into four groups of negative control (NC), LPS, siGPRC5A+LPS and siGPRC5A. The mRNA and protein levels of GPRC5A in GFs under 1 mg/L LPS-induced GFs inflammatory state were evaluated by RT-qPCR and Western blotting analysis after GPRC5A knockdown. RT-qPCR was used to detect the gene expression levels of the inflammatory cytokines in GFs induced by LPS, namely, interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α, prostaglandin endoperoxide synthase 2 (PTGS2) after GPRC5A knockdown. Western blotting analysis and immunofluorescence staining were used to further investigate the activation of nuclear factor-kappa B (NF-κB) signaling pathway. Results: Immunohistochemistry staining showed that the expression of GPRC5A in gingival tissues of periodontitis group (0.132±0.006) increased compared with that in periodontally healthy group (0.036±0.019) (t=8.24, P=0.001). Meanwhile, RT-qPCR results showed that the gene expression levels of GPRC5A at different time point (2, 6, 12, 24 h) in LPS-induced GFs (0.026±0.002, 0.042±0.005, 0.004±0.000, 0.016±0.000) were upregulated compared with those in the NC group (0.004±0.000, 0.004±0.000, 0.002±0.000, 0.007±0.000) (all P<0.001), respectively, and peaked at 6 h. The 50 µmol/L group displayed the most significant decrease in siGPRC5A expression (31.16±3.29) compared with that of the siNC group (100.00±4.88) (F=297.98, P<0.001). The results of RT-qPCR and Western blotting analysis showed that siGPRC5A (0.27±0.03, 0.71±0.00) suppressed the expressions of GPRC5A at both gene and protein levels, while LPS (1.30±0.10, 1.43±0.03) was able to promote the expressions of GPRC5A compared with those of the NC group (1.00±0.01, 1.00±0.00)(all P<0.001). The siGPRC5A+LPS group (0.39±0.03, 1.06±0.16) also inhibited the increase of GPRC5A at both gene and protein levels induced by LPS (1.30±0.10, 1.43±0.03) (F=208.38, P<0.001; F=42.04, P<0.001). RT-qPCR results showed that the expressions of IL-8, IL-1ß, IL-6, TNF-α, and PTGS2 at the gene level in LPS group were highly increased compared with those in the NC group (all P<0.001). siGPRC5A significantly suppressed LPS-induced expressions of these inflammatory cytokines in GFs (all P<0.001). Western blotting analysis showed that the levels of p65 and IκBα protein phosphorylation in the LPS group were highly increased compared with those in the NC group, and siGPRC5A could effectively suppressed LPS-induced protein phosphorylation (all P<0.01). Furthermore, immunofluorescence staining showed that NF-κB p65 in the control group was mainly concentrated in the cytoplasm, and partially translocated to the nucleus under the stimulation of LPS. siGPRC5A was able to inhibit LPS-induced intranuclear translocation of p65 to a certain extent. Conclusions: GPRC5A expression was upregulated in periodontitis, and GPRC5A knockdown inhibited LPS-induced inflammation. Moreover, GPRC5A played a role in inflammation regulation by interacting with NF-κB signaling pathway.

TfR1 mediated iron metabolism dysfunction as a potential therapeutic target for osteoarthritis.

OBJECTIVE: Transferrin receptor-1 (TfR1) plays important roles in controlling cellular iron levels, but its role in OA pathology is unknown. Herein we aim to investigate the role of TfR1 in OA progression and its underlying mechanisms. METHODS: TfR1 expression in cartilage during OA development were examined both in vivo and in vitro. Then IL-1ß was used to induce chondrocytes degeneration in vitro and TfR1 siRNA was used for observing the effect of TfR1 in modulating iron homeostasis, mitochondrial function and degrading enzymes expression. Also the inhibitor of TfR1 was exploited to analyze the protective effect of TfR1 inhibition in vivo. RESULTS: TfR1 is elevated in OA cartilage and contributes to OA inflammation condition. Excess iron not only results in oxidative stress damage and sensitizes chondrocytes to ferroptosis, but also triggers c-GAS/STING-mediated inflammation by promoting mitochondrial destruction and the release of mtDNA. Silencing TfR1 using TfR1 siRNA not only reduced iron content in chondrocytes and inhibited oxidative stress, but also facilitated the mitophagy process and suppressed mtDNA/cGAS/STING-mediated inflammation. Importantly, we also found that Ferstatin II, a novel and selective TfR1 inhibitor, could substantially suppress TfR1 activity both in vivo and in vitro and ameliorated cartilage degeneration. CONCLUSION: Our work demonstrates that TfR1 mediated iron influx plays important roles in chondrocytes degeneration and OA pathogenesis, suggesting that maintaining iron homeostasis through the targeting of TfR1 may represent a novel therapeutic strategy for the treatment of OA.

Absence of Heme Oxygenase-1 Affects Trophoblastic Spheroid Implantation and Provokes Dysregulation of Stress and Angiogenesis Gene Expression in the Uterus.

The enzyme heme oxygenase-1 (HO-1) is pivotal in reproductive processes, particularly in placental and vascular development. This study investigated the role of HO-1 and its byproduct, carbon monoxide (CO), in trophoblastic spheroid implantation. In order to deepen our understanding of the role of HO-1 during implantation, we conducted in vivo experiments on virgin and pregnant mice, aiming to unravel the cellular and molecular mechanisms. Using siRNA, HO-1 was knocked down in JEG-3 and BeWo cells and trophoblastic spheroids were generated with or without CO treatment. Adhesion assays were performed after transferring the spheroids to RL-95 endometrial epithelial cell layers. Additionally, angiogenesis, stress, and toxicity RT2-Profiler™ PCR SuperArray and PCR analyses were performed in uterine murine samples. HO-1 knockdown by siRNA impeded implantation in the 3D culture model, but this effect could be reversed by CO. Uteruses from virgin Hmox1-/- females exhibited altered expression of angiogenesis and stress markers. Furthermore, there was a distinct expression pattern of cytokines and chemokines in uteruses from gestation day 14 in Hmox1-/- females compared to Hmox1+/+ females. This study strongly supports the essential role of HO-1 during implantation. Moreover, CO appears to have the potential to compensate for the lack of HO-1 during the spheroid attachment process. The absence of HO-1 results in dysregulation of angiogenesis and stress-related genes in the uterus, possibly contributing to implantation failure.