TdT-mediated dUTP Nick-end Labeling Assay Research Articles

Lithium intoxication induced pyroptosis via ROS/NF-κB/NLRP3 inflammasome regulatory networks in kidney of mice.

Humans and animals may be exposed to increasing contaminant lithium (Li) concentrations in the environment with the use and disposal of Li-containing products. Meanwhile, Li plays a key role in the treatment of human mental disorders, while the excessive accumulation of Li salts in the body can cause renal damage and nephrotic syndrome. In this study, the mechanism of renal inflammatory reaction induced by Li excessive intake was studied by establishing mice models in vitro and in vivo. The results of histopathology staining and TdT-mediated dUTP nick-end labeling assay showed that high Li condition (Lithium carbonate, 20 mg/kg/twice a day, i.e., for 30 consecutive days) caused inflammatory damage and apoptosis in kidney tissue cells. Western blot, qPCR, and immunohistochemical analysis were used to further study. In the vivo experiments, we found that Li reduced antioxidant enzyme capacity (glutathione peroxidase, total superoxide dismutase, total antioxidant capacity, and catalase) and induced the production of reactive oxygen species (ROS). Moreover, excessive Li activated nuclear factor kappa-B (NF-κB) signaling pathway and nucleotide-binding oligomerization domain-like receptors domains-containing protein 3 (NLRP3) inflammasome, resulting in activation of inflammatory factors tumor necrosis factor-α and IL-1β in the kidney of mice. In the vitro study, ROS as an upstream signal phosphorylated IκBα and NF-κB, up-regulated the NLRP3 inflammasome, increased caspase3, 6, 7, and 9 to exaggerate inflammation response, finally inducing pyroptosis in renal cells.

Resveratrol inhibited hepatocyte apoptosis and alleviated liver fibrosis through miR-190a-5p /HGF axis

BackgroundHepatocyte apoptosis plays an important role in the progression of liver fibrosis. Overexpression of HGF (Hepatocyte growth factor) can reduce hepatocyte apoptosis and alleviate liver fibrosis. Our study aims to investigate whether resveratrol (Res) can alleviate liver fibrosis through miR-190a-5p /HGF axis. MethodsThe TGF-β1 (transforming growth factor-β1)-induced primary hepatocyte model in vitro and CCl4-induced liver fibrosis model in vivo were established. Hepatocyte apoptosis was determined by flow cytometry and TdT-mediated dUTP nick-end labeling (TUNEL) assay. HE (hematoxylin and eosin) staining and Sirius red staining were performed to observe the pathological changes of the liver tissues. Western blotting was used to determine the expression of apoptosis-associated proteins and HGF. Quantitative Real-time PCR (QRT-PCR) was used to quantify miR-190a-5p expression. The dual-luciferase reporter assay was performed to verify the targeting relationship between miR-190a-5p and HGF. ResultsHepatocyte apoptosis and miR-190a-5p expression level were increased in TGF-β1-induced cell models in vitro while the results were reversed after treatment with Res. Besides, miR-190a-5p negatively regulated HGF expression, and miR-190a-5p regulated hepatocyte apoptosis by targeting HGF. Res reduced the apoptosis of hepatocytes and the effect was achieved by decreasing the expression levels of miR-190a-5p and increasing HGF expression in vitro. Moreover, experiments in vivo verified that Res reduced apoptosis of the hepatocytes reduced AST, and ALT levels, as well as raised Albumin levels in the serum. Apart from that, Res decreased the expression ofmiR-190a-5p and increased HGF levels at the same time, and further reduced liver fibrosis. ConclusionIn summary, our study indicated that Res could inhibit the up-regulation of miR-190a-5p caused by CCl4 or TGF-β1. And then the decreased miR-190a-5p could further lessen the hepatocyte apoptosis by increasing HGF levels and finally relieving liver fibrosis.

GAS5 Suppresses Cell Proliferation, Migration, and Invasion via the miR-455-5p/CDKN1B Axis in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a common dermatologic malignancy characterized by aberrant proliferation of keratinocytes. Molecular targeted therapy appears to be a promising approach for advanced cSCC. Long noncoding RNA growth arrest specific 5 (GAS5) was reported to be downregulated in skin cancer tissues. This study aimed to explore the functions and underlying mechanism of GAS5 in the development of cSCC. GAS5 expression in cSCC cells was examined by reverse transcription quantitative polymerase chain reaction. The localization of GAS5 in cSCC cells was detected by subcellular fractionation assays. The viability, proliferation, apoptosis, migration, and invasion of cSCC cells after indicated transfection were examined by Cell Counting Kit-8 assays, colony formation assays, TdT-mediated dUTP Nick-End Labeling assays, wound healing assays, and Transwell assays. Western blot analysis was performed to quantify the protein level of messenger RNA cyclin-dependent kinase inhibitor 1B (CDKN1B). In addition, luciferase reporter assays, RNA pulldown assays and RNA immunoprecipitation assays were carried out to explore the binding relation between miR-455-5p and GAS5 (or CDKN1B). GAS5 was lowly expressed in cSCC cells and was primarily located in cytoplasm. GAS5 overexpression inhibited cell viability, proliferation, migration, and invasion while enhancing cell apoptosis. In addition, GAS5 interacted with miR-455-5p to positively regulate CDKN1B expression. CDKN1B is a direct target of miR-455-5p. Furthermore, CDKN1B knockdown rescued the suppressive effect of GAS5 overexpression on malignant behaviors of cSCC cells. GAS5 suppresses cSCC cell proliferation, migration, and invasion by targeting the miR-455-5p/CDKN1B axis. This study provides novel insight into the role of GAS5 in cSCC development.

Lingbao Huxin Pill Alleviates Apoptosis and Inflammation at Infarct Border Zone through SIRT1-Mediated FOXO1 and NF- κ B Pathways in Rat Model of Acute Myocardial Infarction

To investigate whether Lingbao Huxin Pill (LBHX) protects against acute myocardial infarction (AMI) at the infarct border zone (IBZ) of myocardial tissue by regulating apoptosis and inflammation through the sirtuin 1 (SIRT1)-mediated forkhead box protein O1 (FOXO1) and nuclear factor-κ B (NF-κ B) signaling pathways. Six-week-old Wistar rats with normal diet were randomized into the sham, the model, Betaloc (0.9 mg/kg daily), LBHX-L (0.45 mg/kg daily), LBHX-M (0.9 mg/kg daily), LBHX-H (1.8 mg/kg daily), and LBHX+EX527 (0.9 mg/kg daily) groups according to the method of random number table, 13 in each group. In this study, left anterior descending coronary artery (LADCA) ligation was performed to induce an AMI model in rats. The myocardial infarction area was examined using a 2,3,5-triphenyltetrazolium chloride solution staining assay. A TdT-mediated dUTP nick-end labeling (TUNEL) assay was conducted to assess cardiomyocyte apoptosis in the IBZ. The histopathology of myocardial tissue at the IBZ was assessed with Heidenhain, Masson and hematoxylineosin (HE) staining assays. The expression levels of tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-1 β, and intercellular adhesion molecule-1 were measured using enzyme-linked immunosorbent assays (ELISAs). The mRNA expressions of SIRT1 and FOXO1 were detected by real-time qPCR (RT-qPCR). The protein expressions of SIRT1, FOXO1, SOD2, BAX and NF- κ B p65 were detected by Western blot analysis. The ligation of the LADCA successfully induced an AMI model. The LBHX pretreatment reduced the infarct size in the AMI rats (P<0.01). The TUNEL assay revealed that LBHX inhibited cardiomyocyte apoptosis at the IBZ. Further, the histological examination showed that the LBHX pretreatment decreased the ischemic area of myocardial tissue (P<0.05), myocardial interstitial collagen deposition (P<0.05) and inflammation at the IBZ. The ELISA results indicated that LBHX decreased the serum levels of inflammatory cytokines in the AMI rats (P<0.05 or P<0.01). Furthermore, Western blot analysis revealed that the LBHX pretreatment upregulated the protein levels of SIRT1, FOXO1 and SOD2 (P<0.05) and downregulated NF- κ B p65 and BAX expressions (P<0.05). The RT-qPCR results showed that LBHX increased the SIRT1 mRNA and FOXO1 mRNA levels (P<0.05). These protective effects, including inhibiting apoptosis and alleviating inflammation in the IBZ, were partially abolished by EX527, an inhibitor of SIRT1. LBHX could protect against AMI by suppressing apoptosis and inflammation in AMI rats and the SIRT1-mediated FOXO1 and NF- κ B signaling pathways were involved in the cardioprotection effect of LBHX.

Transcriptome and Metabolome Analyses Provide Insights into the Stomium Degeneration Mechanism in Lily

Lily (Lilium spp.) is a widely cultivated horticultural crop that has high ornamental and commercial value but also the serious problem of pollen pollution. However, mechanisms of anther dehiscence in lily remain largely unknown. In this study, the morphological characteristics of the stomium zone (SZ) from different developmental stages of ‘Siberia’ lily anthers were investigated. In addition, transcriptomic and metabolomic data were analyzed to identify the differentially expressed genes (DEGs) and secondary metabolites involved in stomium degeneration. According to morphological observations, SZ lysis occurred when flower buds were 6–8 cm in length and was completed in 9 cm. Transcriptomic analysis identified the genes involved in SZ degeneration, including those associated with hormone signal transduction, cell structure, reactive oxygen species (ROS), and transcription factors. A weighted co-expression network showed strong correlations between transcription factors. In addition, TUNEL (TdT-mediated dUTP nick-end labeling) assays showed that programmed cell death was important during anther SZ degeneration. Jasmonates might also have key roles in anther dehiscence by affecting the expression of the genes involved in pectin lysis, water transport, and cysteine protease. Collectively, the results of this study improve our understanding of anther dehiscence in lily and provide a data platform from which the molecular mechanisms of SZ degeneration can be revealed.

PM2.5 Aggravated OVA-Induced Epithelial Tight Junction Disruption Through Fas Associated via Death Domain-Dependent Apoptosis in Asthmatic Mice.

BackgroundExposure to air pollutants cause exacerbation of asthma, but the experimental evidence and the mechanisms still need to be collected and addressed.MethodsAsthma model was constructed by ovalbumin (OVA) combined with or without airborne fine particulate matter 2.5 (PM2.5) exposure. Lung sections were stained by hematoxylin-eosin staining (H&E) and Masson’s trichrome. RNA-seq and gene set enrichment analysis (GSEA) was performed to identify the key pathway. TdT mediated dUTP Nick End Labeling (TUNEL) assay, real-time qPCR, Western blot, immunofluorescence and lentivirus transfection were applied for mechanism discovery.ResultsIn this study, we found PM2.5 aggravated airway inflammation in OVA-induced asthmatic mice. RNA-seq analysis also showed that epithelial mesenchymal transition (EMT) was enhanced in OVA-induced mice exposed to PM2.5 compared with that in OVA-induced mice. In the meantime, we observed that apoptosis was significantly increased in asthmatic mice exposed to PM2.5 by using GSEA analysis, which was validated by TUNEL assay. By using bioinformatic analysis, Fas associated via death domain (FADD), a new actor in innate immunity and inflammation, was identified to be related to apoptosis, EMT and tight junction. Furthermore, we found that the transcript and protein levels of tight junction markers, E-cadherin, zonula occludens (ZO)-1 and Occludin, were decreased after PM2.5 exposure in vivo and in vitro by using RT-qPCR and immunofluorescence, with the increased expression of FADD. Moreover, down-regulation of FADD attenuated PM2.5-induced apoptosis and tight junction disruption in human airway epithelial cells.ConclusionTaken together, we demonstrated that PM2.5 aggravated epithelial tight junction disruption through apoptosis mediated by up-regulation of FADD in OVA-induced model.

The W9 peptide inhibits osteoclastogenesis and osteoclast activity by downregulating osteoclast autophagy and promoting osteoclast apoptosis.

The W9 peptide has been shown to act as a receptor activator for nuclear factor-κB ligand (RANKL) antagonist and tumor necrosis factor (TNF)-α antagonist, which can promote bone formation and inhibit bone resorption. Studies on the W9 peptide at the cellular level have mainly focused on osteoblasts, and little research on the mechanism by which the W9 peptide regulates osteoclasts has been reported, which was the aim of this work. In this study, a rat mandibular defect model was established in vivo and implanted with hydrogel containing the W9 peptide for 2weeks and 4weeks, and histochemical staining was used to evaluate the formation of new bone and the changes in osteoclasts. RAW264.7 cells were cultured in vitro for osteoclast induction, and different concentrations of W9 peptide were added. Tartrate resistant acid phosphatase staining, monodansylcadaverine staining, TdT-mediated dUTP Nick-End Labeling assay, real-time PCR and Western blot were used to detect osteoclast differentiation, autophagy and apoptosis. Our results showed that the W9 peptide could reduce osteoclastogenesis and osteoclast activity induced by RANKL, and these effects were partly due to the inhibition of osteoclast autophagy. On the other hand, the W9 peptide could promote mature osteoclast apoptosis, in which autophagy might play an antagonistic role. Taken together, these results suggest that the W9 peptide inhibits osteoclastogenesis and osteoclast activity by downregulating osteoclast autophagy and promoting osteoclast apoptosis. Our results will benefit the development and application of new small molecule peptides for the treatment of bone resorption diseases.

LINC00265 promotes the viability, proliferation, and migration of bladder cancer cells via the miR-4677-3p/FGF6 axis.

Bladder cancer (BCa) is a common genitourinary malignancy with higher incidence in males. Long intergenic non-protein coding RNA 265 (LINC00265) is identified as an oncogene in many malignancies, while its role in BCa development remains unknown. To explore the functions and mechanism of LINC00265 in BCa. Reverse transcription quantitative polymerase chain reaction was performed to examine LINC00265 expression in BCa cells. Cell counting kit-8 assays, colony formation assays, TdT-mediated dUTP Nick-End Labeling assays, and Transwell assays were conducted to examine BCa cell viability, proliferation, apoptosis, and migration. Luciferase reporter assays and RNA immunoprecipitation assays were carried out to explore the binding capacity between miR-4677-3p and messenger RNA fibroblast growth factor 6 (FGF6) (or LINC00265). Xenograft tumor model was established to explore the role of LINC00265 in vivo. LINC00265 was highly expressed in BCa cells. LINC00265 knockdown inhibited xenograft tumor growth and BCa cell viability, proliferation and migration while enhancing cell apoptosis. Moreover, LINC00265 interacted with miR-4677-3p to upregulate the expression of FGF6. FGF6 overexpression reversed the suppressive effect of LINC00265 knockdown on malignant phenotypes of BCa cells. LINC00265 promotes the viability, proliferation, and migration of BCa cells by binding with miR-4677-3p to upregulate FGF6 expression.

Extract from Zanthoxylum piperitum Induces Apoptosis of AGS Gastric Cancer Cells Through Akt/MDM2/p53 Signaling Pathway

To determine the effect of Zanthoxylum piperitum extracet (ZPE) on apoptosis and analyze anticancer substances in ZPE, changes in proteins related to apoptosis, and pathological changes in tumors in mouse. Fifteen 4-week-old female BALB/c nu/nu mice were divided into 3 groups depending on ZPE dose, with 5 in each group. AGS gastric carcinoma cells (1 × 106 cells/200 µL) were subcutaneously injected into the flank of each mouse. One week after the injection of AGS cells, ZPE was administered to the skin tissue [10 or 50 mg/(kg·d)] in the low- and high-dose groups, respectively for 20 days. Control animals were injected with vehicle only. After 3 weeks, the tumor was extracted and carried out for immunohistochemistry, the tendency of apoptosis and p53 in the body was checked using TdT-mediated dUTP nick-end labeling (TUNEL) assay. For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V dead cell staining, cell cycle arrest and Western blotting, AGS gastric carcinoma cells were incubated with various concentrations of ZPE for 24 h. Cell survival rates were analyzed by MTT assays. Apoptosis was analyzed using annexin V dead cell staining and cell cycle arrest and measured using Muse cell analyzer. High performance liquid chromatography (HPLC) analysis showed that ZPE contained organic sulfur compounds such as alliin and S-allylcysteine. MTT assay results revealed that ZPE (10-85 µ g/mL) could effectively inhibit the growth of AGS gastric cancer cells at higher concentrations (P<0.05, P<0.01). The annexin V & dead cell staining assay and cell cycle arrest assay confirmed a dose-dependent increase in the apoptosis rate and G1 phase in ZPE (10-70 µ g/mL) groups. ZPE decreased the expression of anti-apoptotic proteins (p-Akt, p-MDM2, Bcl-2), while increased pro-apoptotic proteins (cleaved PARP, p53, pro-Caspase 3, Bax). TUNEL assays revealed an increase in cell apoptosis. Immunohistochemistry staining confirmed the involvement of p53. ZPE decreases AGS cell proliferation and induces apoptosis by inhibiting Akt and MDM2 expression.

Cadmium promotes apoptosis and inflammation via the circ08409/miR-133a/TGFB2 axis in bovine mammary epithelial cells and mouse mammary gland

Cadmium is a common environmental heavy metal pollutant that can accumulate over long periods of time and cause disease. Thus, analysis of the molecular mechanisms affected by cadmium in the body could be of great significance for the prevention and treatment of cadmium-related diseases. In this study, flow cytometry, immunofluorescence, transmission electron microscopy, H&E (Hematoxylin Eosin) staining and TUNEL (TdT-mediated dUTP Nick-End Labeling) assays were used to verify that cadmium induced apoptosis and immune responses in bovine mammary epithelial cells (BMECs) and in mouse mammary gland. Isolated BMECs cultured with or without cadmium were collected to screen miRNA (microRNA) using high-throughput sequencing. There were 42 differentially-expressed miRNAs among which 27 were upregulated and 15 downregulated including bta-miR-133a, bta-miR-23b-5p, bta-miR-29e, bta-miR-365–5p, bta-miR-615, bta-miR-7, bta-miR-11975, bta-miR-127, and bta-miR-411a. Among those, miR-133a (which can specifically target TGFB2 (Recombinant Transforming Growth Factor Beta 2) was the most significantly downregulated with a fold-change of 5.27 in BMECs cultured with cadmium. Application of the double luciferase reporter system, western blotting, and qRT-PCR (Quantitative Real-time PCR) revealed that circ08409 can directly bind to miR-133a. Experiments demonstrated that circRNA-08409 could adsorb bta-miR-133a. Both circ08409 and TGFB2 significantly increased apoptosis and altered expression level of a series of inflammatory factors in BMECs. In contrast, miR-133a decreased significantly apoptosis and inflammation in the cells. Compared with cultures receiving only cadmium, the miR-133a+cadmium cultures exhibited significant reductions in the occurrence of late apoptosis. Overall, results indicated that circ08409 could relieve the inhibitory effect of miR-133a on TGFB2 expression by combining with miR-133a and subsequently modulating cell proliferation, apoptosis and inflammation. Overall, the data suggested that the circ08409/miR-133a/TGFB2 axis might play a role in mediating the effect of cadmium on BMECs. As such, data provide novel insights into controlling hazards that cadmium could induce in the mammary gland.

Sevoflurane induces neuronal apoptosis via enhancing DNMT3L expression and promoting methylation of PSD95 promoter in postoperative cognitive dysfunction

Postoperative cognitive dysfunction (POCD) is a central nervous system (CNS) complication and occurs quite frequently after anesthesia, especially in elderly patients. DNA (cytosine-5)-methyltransferase 3-like (DNMT3L) is an enzymatically inactive regulatory factor that can modulate gene expression via methylation. Few studies specifically focused on the role of DNMT3L in sevoflurane-induced POCD. The cognitive dysfunction was determined by Morris water maze assay. Hippocampal neuron apoptosis was measured by TdT-mediated dUTP nick-end labeling (TUNEL) assay. Neuron proliferation and apoptosis were measured by cell counting kit-8 (CCK-8) and flow cytometry assay. The expression levels of DNMT3L and postsynaptic density protein-95 (PSD95) were measured by qRT-PCR and western blot. The sevoflurane-induced POCD aging rat model was established for in vivo study. Suppression of DNA methylation by 5-Aza-2′-deoxycytidine (5-AZA; a widely used methylation inhibitor) reversed sevoflurane-induced cognitive dysfunction and hippocampal neuron apoptosis in rats. Moreover, sevoflurane enhanced DNMT3L expression and induced methylation of PSD95 promoter in vivo. In vitro, sevoflurane induced neuronal apoptosis and DNMT3L expression to promote PSD95 methylation. Finally, rescue experiments indicated that sevoflurane induced neuron apoptosis by increasing DNMT3L expression and promoting methylation of PDS95 in POCD. Sevoflurane induced neuronal apoptosis by increasing DNMT3L expression and promoting methylation of PDS95 in vivo and in vitro, providing a novel therapeutic strategy for POCD.

CircCCND1 Regulates Oxidative Stress and FGF9 to Enhance Chemoresistance of Non-Small Cell Lung Cancer via Sponging miR-187-3p.

Circular RNAs have been shown to regulate cancer tumorigenesis and drug resistance. Recently, circCCND1 is reported to promote laryngeal squamous cell carcinoma; however, whether circCCND1 is implicated in non-small cell lung cancer (NSCLC) remains unclear. In this research, The Cancer Genome Atlas data of lung adenocarcinoma were analyzed to show gene expression and overall survival. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide assay and cell colony formation assay were utilized to measure cell viability and proliferation of A549 and HCC827. Apoptosis was detected by TdT-mediated dUTP Nick-End Labeling assay. Besides, reverse transcription-quantitative PCR was used to examine gene expression. We observed that circCCND1 was significantly upregulated in lung cancer cells and patients. circCCND1 knockdown attenuated cell proliferation and induced apoptosis under cisplatin treatment. Mechanistically, circCCND1 interacted with miR-187-3p to regulate reactive oxygen species and FGF9 in NSCLC cells. Finally, miR-187-3p was demonstrated to rescue circCCND1 knockdown-modulated chemoresistance of NSCLC cells. In this study, our conclusions facilitate the understanding of NSCLC drug resistance to cisplatin.

Neutral Ceramidase Is Required for the Reproduction of Brown Planthopper, Nilaparvata lugens (Stål).

Ceramides are bioactive sphingolipids that have been implicated in insect development; however, their role in insect reproduction remains poorly understood. Here, we report the pivotal role of neutral ceramidase (NCER) in the female reproduction of the brown planthopper (BPH), Nilaparvata lugens (Stål), a significant pest in rice cultivation in Asia. LC-MS/MS demonstrated that, among different developmental stages of BPH, the levels of ceramides were highest in 1st instar nymphs and lowest in adults. The transcription of NCER was negatively correlated with the levels of ceramides at different developmental stages of BPH, in that the transcript levels of NCER were the highest, whereas ceramides levels were the lowest in BPH adults. Knocking down NCER through RNA interference (RNAi) increased the levels of ceramides in BPH females and ovaries, which resulted in a delay in oocyte maturation, a reduction in oviposition and egg hatching rate, as well as the production of vulnerable offspring. Transmission electron microscopy (TEM) analysis and TdT-mediated dUTP Nick-End Labeling (TUNEL) assays showed mitochondrial deficiency and apoptosis in NCER-deficient oocytes. Taken together, these results suggest that NCER plays a crucial role in female reproduction in BPH, likely by regulating the levels of ceramides.

Study on the mechanisms of the intestinal tight-junction destruction caused by Blastocystis hominis infection in rats

To explore the mechanism of the intestinal barrier damage caused by Blastocystis hominis infections in rats. Thirty SD rats were randomly divided into the control group, and the 1-, 3-, 6- and 9-week-infection groups, of 6 rats in each group. Rats in each infection group were orally infected with B. hominis trophozoites at a density of 2 × 108 parasites per rat, and the control group was given an equal volume of phosphate buffered saline solution. The 7-hour urine samples were collected 1, 3, 6 and 9 weeks post-infection for the measurement of the intestinal permeability. Then, rats were sacrificed using the cervical dislocation method, and the cecum specimens were collected for the detection of the intestinal epithelial cell permeability. The expression of tight junction-related Occludin and Claudin - 1 genes and apoptosis-related Bcl - 2 and Bax genes was quantified in cecum epithelial cells using the real-time fluorescent quantitative PCR (qPCR) assay, and cell apoptosis was detected in the rat cecum using the TdT-mediated dUTP nick-end labeling (TUNEL) assay. The median urinary lactolose to mannitol ratios were 0.29, 0.72, 0.44, 0.46 and 0.38 in the control group, and the 1-, 3-, 6- and 9-week-infection groups, respectively, and the difference was statistically significant (H = 12.09, P < 0.05). B. hominis invasion and epithelial injury were observed in intestinal epithelial cells of rats infected with B. hominis, and transmission electron microscopy displayed the destruction of tight junctions between intestinal epithelial cells. The relative expression of Occludin, Claudin-1, Bcl-2 and Bax genes was 1.04, 0.62, 0.71, 0.68 and 0.96; 1.03, 0.61, 0.63, 0.76 and 0.86; 1.08, 0.70, 0.75, 0.74 and 1.03; and 1.00, 1.57, 1.33, 1.35 and 1.10 in the control group and the 1-, 3-, 6- and 9-week-infection groups, respectively, and all differences were statistically significant (F = 2.86, 2.85, 3.37 and 4.45, all P values < 0.05). The median number of positive staining cells were 1.00, 13.00, 9.00, 3.50 and 1.00 in rat cecum specimens in the control group, and the 1-, 3-, 6- and 9-week-infection groups, respectively, and the difference was statistically significant (H = 22.95, P < 0.01). B. hominis infection may cause an increase in the rat intestinal permeability through triggering the apoptosis of intestinal epithelial cells to destroy the tight junction between intestinal epithelial cells, thereby destroying the intestinal barrier function.

Aerobic exercise improves VCI through circRIMS2/miR-186/BDNF-mediated neuronal apoptosis

BackgroundVascular cognitive impairment (VCI) is a common cognitive disorder caused by cerebrovascular disease, ranging from mild cognitive impairment to dementia. Studies have shown that aerobic exercise might alleviate the pathological development of VCI, and our previous study observed that aerobic exercise could alleviate VCI through NF-κB/miR-503/BDNF pathway. However, there are few studies on the mechanism. Therefore, it is of great significance to fill the gaps in the mechanism for the early diagnosis of VCI and the clinical prevention and treatment of vascular dementia.MethodsCircRNA microarray analysis and quantitative real-time PCR were used to detect the expression of circRNA regulating synaptic be exocytosis 2 (RIMS2) (circRIMS2). Cell apoptosis was determined by TdT-mediated dUTP nick-end labeling (TUNEL) assay. The dual-luciferase reporter assay was performed to verify the interaction between circRIMS2 and miR-186, as well as miR-186 and BDNF. RNA pull-down assay detected the binding between circRIMS2 and miR-186. A VCI mouse model was established by repeated ligation of bilateral common carotid arteries (2VO). The lentiviral interfering vector was injected into the VCI mice through the lateral ventricle. The mice in the aerobic exercise group performed 30 min (12 m/min) running for 5 days a week. A Morris water maze test was performed after 4 weeks.ResultsThe expression of circRIMS2 and BDNF in the serum of VCI patients was significantly reduced, miR-186 expression was increased, and the expression of circRIMS2 was increased in the 2VO group of mice undergoing aerobic exercise. The expression levels of circRIMS2 and BDNF in the oxygen and glucose deprivation-treated (OGD-treated) cells were decreased, the miR-186 expression and cell apoptosis were increased, while the effect was weakened after transfection with the lentiviral vector pLO-ciR-RIMS2. CircRIMS2 could bind to miR-186, and after interference with circRIMS2 in HT22 cells, the expression of miR-186 was increased. Besides, miR-186 could bind to BDNF, and BDNF expression was decreased because of the overexpression of miR-186 in HT22 cells. The expression level of BDNF in the pLO-ciR-RIMS2 group was increased, and apoptosis was decreased, but the miR-186 mimic weakened the effect of pLO-ciR-RIMS2. Aerobic exercise could shorten the average time that mice reached the platform in the Morris water maze, increase the expression level of circRIMS2 and BDNF, reduce miR-186 expression, and inhibit neuronal apoptosis. However, the interference with circRIMS2 weakened this effect.ConclusionThe expression of circRIMS2 was down-regulated in VCI and aerobic exercise reduced neuronal apoptosis, and circRIMS2 improved VCI through the circRIMS2/miR-186/BDNF axis.

TXNIP positively regulates the autophagy and apoptosis in the rat müller cell of diabetic retinopathy

AimsDiabetic retinopathy (DR) can cause vision loss in patients with diabetes. The present study evaluated the expression of thioredoxin interacting protein (TXNIP) and investigated the role of TXNIP in autophagy and apoptosis of DR. Main methodsReverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to measure the expression level of the targets. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/cas9) method was applied for knockout of TXNIP. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay and flow cytometry were utilized to detect the apoptosis. Cell Counting Kit-8 (CCK-8) assay was used to evaluate the cell viability. EdU assay was carried out to measure the cell proliferation ability. Retinal immunohistochemistry, retinal frozen section immunofluorescence as well as the electroretinogram (ERG) recording were implemented to detect the function of the retina. Key findingsTXNIP was up-regulated under hyperglycemic condition both in vivo and in vitro. Overexpression of TXNIP activated the autophagy and apoptosis in the rat müller cell. Knockout of TXNIP reduced the autophagy and apoptosis in the rat müller cell under high glucose condition. TXNIP positively regulates autophagy via inhibition of the PI3K/AKT/mTOR signaling pathway. Knockdown of TXNIP improved the visual response to light stimulus of DR. SignificanceOur study unraveled for the first time that TXNIP positively regulates the autophagy in rat müller cell under high glucose condition by inhibiting the PI3K/AKT/mTOR signaling pathway, providing a novel understanding in the pathogenesis of DR and suggesting a potential new therapeutic target of DR.

Montelukast attenuates interleukin IL-1β-induced oxidative stress and apoptosis in chondrocytes by inhibiting CYSLTR1 (Cysteinyl Leukotriene Receptor 1) and activating KLF2 (Kruppel Like Factor 2)

ABSTRACT Montelukast is a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist widely used to suppress the inflammatory response in asthma and allergic rhinitis. This study aimed to investigate the potential impacts of montelukast on osteoarthritis (OA) progression. To determine the role of montelukast in OA, the expression of CysLTR1 was first examined by quantitative reverse transcription PCR (RT-qPCR) and western blot in IL-1β-induced ATDC5 cells treated with or without montelukast. Subsequently, the impacts of montelukast on cell viability and oxidative stress were measured by Cell-Counting-Kit-8 (CCK-8), commercial kits and western blot. Oxidative stress-related protein expressions were determined by western blot analysis in Il-1β-induced ATDC5 cells. Cell apoptosis and cartilage degradation were examined by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, western blot and RT-qPCR. KLF2 expression was measured in IL-1β-induced ATDC5 cells treated with montelukast. After interference with small interfering RNA (siRNA)-KLF2 in ATDC5 cells, the loss-of-function assays were also performed in same ways. CysLTR1 expression was elevated in IL-1β-induced ATDC5 cells but inhibited significantly by montelukast. Montelukast attenuated the oxidative stress and apoptosis, improved cell viability. Moreover, montelukast enhanced KLF2 expression. After transfected with siRNA-KLF2, montelukast attenuated cell injury, oxidative stress, apoptosis and cartilage degradation in IL-1β-induced ATDC5 cells by activating KLF2.In summary, this work elaborates the evidence that montelukast could attenuate oxidative stress and apoptosis in IL-1β-induced chondrocytes by inhibiting CysLTR1 and activating KLF2, which can guide the therapeutic strategies of montelukast for OA development in the future.

Resveratrol-modified mesoporous silica nanoparticle for tumor-targeted therapy of gastric cancer

ABSTRACT Resveratrol (Res) has been shown to exhibit anti-cancer properties in gastric cancer. However, its clinical application is limited by its poor pharmacokinetics, stability, and low solubility. Hence, this study aimed to explore and verify a better delivery system for gastric cancer therapy. Using transmission electron microscopy, Fourier transform infrared (FTIR) spectroscopy, and ultraviolet (UV) spectrometry, we observed the shape and encapsulation of resveratrol-modified mesoporous silica nanoparticles (MSN-Res) that were synthesized by chemical methods. To explore the anti-cancer effects of these MSN-Res in vivo and in vitro, we established AGS and HGC-27 tumor-bearing mouse models. Meanwhile, the proliferation of gastric cancer cells in vitro and in vivo was assessed by Cell Counting Kit-8, EdU, and Ki-67 immunohistochemical staining methods, while cellular apoptosis, and invasion and migration were detected by TdT-mediated dUTP nick end labeling (TUNEL) and Transwell assays, respectively. FTIR and UV results showed that we successfully synthesized and loaded drugs. Safety evaluation experiments showed that neither MSN-SH nor MSN-Res had toxic effects on the normal tissues of animals. Moreover, in vitro experiments revealed that MSN-Res significantly inhibited the proliferation, invasion, and migration of gastric cancer cells. Furthermore, TUNEL assay showed that MSN-Res promoted apoptosis in gastric cancer. These results were confirmed by the nude mouse tumorigenesis experiment. In conclusion, we demonstrated that MSN-Res showed better inhibitory effect on the development of gastric cancer than Res alone, indicating that MSN-Res could be a promising drug delivery system for gastric cancer treatment.

Short-Term Changes in Gut Microflora and Intestinal Epithelium in X-Ray Exposed Mice

Background: Gut microflora contributes to the nutritional metabolism of the host and to strengthen its immune system. However, if the intestinal barrier function of the living body is destroyed by radiation exposure, the intestinal bacteria harm the health of the host and cause sepsis. Therefore, this study aims to trace short-term radiation-induced changes in the mouse gut microflora-dominant bacterial genus, and analyze the degree of intestinal epithelial damage. Materials and Methods: Mice were irradiated with 0, 2, 4, 8 Gy X-rays, and the gut microflora and intestinal epithelial changes were analyzed 72 hours later. Five representative genera of Actinobacteria, Firmicutes, and Bacteroidetes were analyzed in fecal samples, and the intestine was pathologically analyzed by Hematoxylin-Eosin and Alcian blue staining. In addition, DNA fragmentation was evaluated by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. Results and Discussion: The small intestine showed shortened villi and reduced number of goblet cells upon 8 Gy irradiation. The large intestine epithelium showed no significant morphological changes, but the number of goblet cells were reduced in a radiation dose-dependent manner. Moreover, the small intestinal epithelium of 8 Gy-irradiated mice showed significant DNA damaged, whereas the large intestine epithelium was damaged in a dose-dependent manner. Overall, the large intestine epithelium showed less recovery potential upon radiation exposure than the small intestinal epithelium. Analysis of the intestinal flora revealed fluctuations in lactic acid bacteria excretion after irradiation regardless of the morphological changes of intestinal epithelium. Altogether, it became clear that radiation exposure could cause an immediate change of their excretion. Conclusion: This study revealed changes in the intestinal epithelium and intestinal microbiota that may pave the way for the identification of novel biomarkers of radiation-induced gastrointestinal disorders and develop new therapeutic strategies to treat patients with acute radiation syndrome.

Therapeutic effects of losartan on prostatic hyperplasia in spontaneously hypertensive rats

AimsWe investigated the therapeutic effects of losartan, an angiotensin II type 1 receptor blocker, on prostatic hyperplasia in spontaneously hypertensive rats (SHRs). Main methodsMale SHRs (age, 36 weeks) were perorally treated with losartan (3 or 10 mg·kg−1) or vehicle once daily for 18 weeks. Age-matched Wistar Kyoto rats (WKYs) were used as vehicle-treated controls (n = 8). The effects of losartan were evaluated by analyzing prostate weight, blood pressure, and prostatic blood flow. The tissue malondialdehyde (MDA), interleukin-6 (IL-6), and basic fibroblast growth factor (bFGF) levels were measured. Histological analysis for the ventral prostate involved hematoxylin and eosin staining and TdT-mediated dUTP nick-end labeling (TUNEL) assay. Key findingsCompared to the vehicle-treated WKYs, the vehicle-treated SHRs had significantly higher prostate weight, prostate weight/body weight ratio (PBR), blood pressure, glandular epithelial area, and tissue MDA, IL-6, and bFGF levels in the ventral prostate and lower prostatic blood flow. Treatment with losartan caused significant recovery of blood flow and decreased PBR and glandular epithelial area as well as tissue MDA, IL-6, and bFGF levels in the SHR ventral prostate without affecting blood pressure. High-dose losartan significantly decreased blood pressure and increased TUNEL-positive cells in the ventral prostate in SHRs. SignificanceChronic losartan treatment could ameliorate prostatic hyperplasia via recovery of reduced prostatic blood flow and induction of apoptosis in the ventral prostate in SHRs. Losartan might have therapeutic effects on not only hypertension but also prostatic hyperplasia in humans.