Cloperastine Reduces IL-6 Expression via Akt/GSK3/Nrf2 Signaling in Monocytes/Macrophages and Ameliorates Symptoms in a Mouse Sepsis Model Induced by Lipopolysaccharide.

Cloperastine (CLP) is a drug with a central antitussive effect that is used to treat bronchitis. Therefore, we have attempted to examine the anti-inflammatory effects of CLP. CLP reduced the secretion of interleukin (IL)-6, a pro-inflammatory cytokine, from RAW264.7 monocyte/macrophage-linage cells treated with lipopolysaccharide (LPS). IL-6 is a biomarker of sepsis and has been suggested to exacerbate its symptoms. We found that the intraperitoneal administration of CLP reduced IL-6 levels in the lungs and also improved hypothermia in mice with LPS-induced sepsis. CLP ameliorated kidney pathologies such as congestion and increased the survival rate of mice administered with a lethal dose of LPS. To reveal the mechanisms underlying the anti-inflammatory function of CLP, we analysed the intracellular signaling in LPS-treated RAW264.7 cells. CLP induced the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3 (GSK3) and also increased the amount of nuclear factor erythroid-2-related factor 2 (Nrf2) in RAW264.7 cells with/without LPS. Wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), reduced the upregulated phosphorylation levels of Akt and GSK3 and the increased amount of Nrf2. It also halted the reduction of IL-6 secretion caused by CLP. These results suggest that CLP has an anti-inflammatory function via Akt/GSK3/Nrf2 signaling and could be a candidate drug for the treatment of inflammatory diseases, including sepsis.

Expression patterns of interleukin-6 and microRNA-146A during orthodontic relapse in a rat model.

In this study, we established an experimental rat model to simulate orthodontic tooth movement relapse and a human periodontal ligament stem cell (PDLScs) model. Our aim was to explore the relationship between microRNA-146a (miR-146a) expression in periodontal tissue, the inflammatory factor interleukin-6 (IL-6), and orthodontic relapse subsequent to mechanical intervention. In the animal experiment, a total of 30 healthy male Wistar rats were randomly allocated to either the control group (n=6) or the model group (n=24). In the model group, the orthodontic appliance was removed 14 days after force application. Gingival crevicular fluid (GcF) and periodontal tissue samples were collected at intervals of days 0, 7, 14, and 21 following removal of the orthodontic appliance to assess alterations in miR-146a and IL-6 expressions. In the in vitro cell culture study, human premolar tooth tissue was isolated 24 hours following the addition of the transfection reagent to harvest PDLScs. Reverse transcription quantitative polymerase chain reaction was employed to evaluate the expression levels of the miRNA-146a gene, while Western blot analysis was utilized to assess the production of the IL-6 protein. As a result in comparison to the control group, the protein expression of IL-6 notably escalated to its peak value in the model-day 7 group (p<0.05). Subsequently, although experiencing a slight decline, the IL-6 expression in the model-day 14 group remained significantly elevated compared to control group (p<0.05). In the model-day 21 group, the protein expression of IL-6 approached that of the control group, with no significant difference observed (p>0.05). conversely, in relation to the control group, the gene expression of miR-146a drastically decreased to its lowest point in the model-day 7 group (p<0.05). While exhibiting a slight increase, the miR-146a expression in the model-day 14 group remained significantly diminished compared to control group (p<0.05). Following the identification of human periodontal ligament cells (hPDLcs) through immunofluorescence in the in vitro study, a subsequent experiment was conducted to specifically inhibit miR-146a expression. In comparison to the control group, the protein expression of IL-6 demonstrated a significant increase in the anti-miRNA oligodeoxyribonucleotide (AMO) group, where miR-146a expression was effectively suppressed (p<0.05). Throughout the process of orthodontic tooth movement relapse in rats, there was a notable reduction in the gene expression of miR-146a, accompanied by a significant increase in the expression of IL-6.

The role of salivary vitamin D and interleukin-6 on non-scarring Alopecia.

BACKGROUND: Alopecia, or hair loss, is an emerging global disease. Its etiopathogenesis includes nutritional deficiencies, oxidative stress, and deficiency of physiological factors. Around 2% of the general population has the probability of developing alopecia at any one period. Vitamin D and interleukin-6 (IL-6) have a major role in alopecia. The present goal of research is to investigate the role of vitamin D and IL-6 in the saliva of patients with non-scarring alopecia. METHODOLOGY: The study involved 51 cases of non-scarring alopecia and 50 healthy controls with an age range between 18 and 40 years. A detailed history and clinical examination were done. Salivary vitamin D and IL-6 were determined to compare within the groups. RESULTS: The average vitamin D level in cases (104.64 ± 46.95 pmol/L) was significantly lower as compared to controls (223 ± 12.03 pmol/L) (p < 0.001). Whereas the average amount of IL-6 was significantly higher (170.54 ± 63.68 ng/L) than the control group (56.38 ± 46.52 ng/L) (p < 0.001). No correlation of vitamin D level with IL-6 was detected in study subjects. CONCLUSION: Vitamin D significantly influences the development of non-scarring alopecia. Patients with non-scarring alopecia had low amount of vitamin D indicate its role in etiology of hair loss. IL-6 may cause a collapse of the hair bulb, having a significant part in the pathogenesis of alopecia indicating chronic inflammatory or autoimmune condition. This research will aid in diagnosing scalp disease using salivary biomarkers and improve the treatment of alopecia.

Effect of Endurance Exercise Training on Gut Microbiota and ER Stress.

Regular exercise as part of one's lifestyle is well-recognized for its beneficial effect on several diseases such as cardiovascular disease and obesity; however, many questions remain unanswered regarding the effects of exercise on the gut environment. This study aimed to investigate the impact of long-term endurance exercise on modulating inflammation and endoplasmic reticulum (ER) stress. Fifteen-week-old male Sprague-Dawley (SD) rats were subjected to six months of endurance treadmill training, while age-matched controls remained sedentary. Results showed that IL-6 mRNA levels in colon tissues were significantly higher in the exercise group compared to the sedentary group. Exercise activated a significant ER stress-induced survival pathway by increasing BiP and phosphorylation of eIF2α (p-eIF2α) expressions in the liver and colon, while decreasing CHOP in the liver. Gene expressions of MUC2, Occludin, and Claudin-2 were increased in the colon of the exercise group, indicating enhanced intestinal integrity. Furthermore, the data showed a positive correlation between microbiota α-diversity and BiP (r = 0.464~0.677, p < 0.05). Populations of Desulfovibrio C21 c20 were significantly greater in the exercise group than the sedentary group. Additionally, predicted functions of the gut microbial community in terms of enzymes and pathways supported the enhancement of fatty-acid-related processes by exercise. These findings suggest that prolonged endurance exercise can affect the colon environment, which is likely related to changes in inflammation, ER stress, mucin layers and tight junctions, associated with modifications in the gut microbiome.

Neuroprotective effect of Naochuxue prescription on intracerebral hemorrhage: inhibition of autophagy downregulating high mobility group box-1.

OBJECTIVE: To determine the molecular mechanisms underlying the neuroprotective effects of Naochuxue prescription (,NCXP) in rats with intracerebral hemorrhage (ICH). METHODS: Sprague-Dawley rats were injected with collagenase to generate ICH models, which were then randomly divided into six groups, including control, sham, model, and three intervention groups. The intervention groups received different doses of NCXP (0.13, 0.26, and 0.52 g/kg) daily for 10 d. High-performance liquid chromatography (HPLC) was used to analyze the chemical characteristics of NCXP. The neurobehavioral outcomes of the rats were evaluated using neurological deficit scores (Zea Longa 5) and the corner turn test. Pathomorphological changes in perihematomal tissues after ICH were observed using hematoxylin and eosin staining. Immunohistochemistry (IHC) was used to detect the inflammation expression of interleukin 6 (IL-6) and toll-like receptor 4 (TLR4). High mobility group box-1 (HMGB1), Beclin1, microtubule-associated protein 1 light chain 3 beta (LC3), and sequestosome 1 (p62) were detected using real-time quantitative polymerase chain reaction and Western blotting in perihematomal tissues. RESULTS: HPLC showed that the NCXP had good stability. Rats with ICH had severe neurological function deficits compared to the control group. IHC results showed that NCXP significantly downregulated the expression of the inflammatory proteins IL-6 and TLR4. ICH rats treated with NCXP showed less neurological injury than the model group, accompanied by a significantly decreased expression of HMGB1, Beclin1, and LC3 and an increased expression of p62. CONCLUSIONS: The neuroprotective effect of NCXP alleviated inflammation and autophagy possibly by downregulating HMGB1 expression. However, further research on the signaling pathways is required to verify this hypothesis.

Qingfei Zhisou oral liquid alleviates fever-induced inflammation by regulating arachidonic acid and lysophospholipids metabolism and inhibiting hypothalamus transient receptor potential ion channels expression.

OBJECTIVE: To explore how Qingfei Zhisou oral liquid (, QFZS) adjusts body temperature bias and the interaction of inflammatory factors levels and metabolomic differences. METHODS: Dry yeast was subcutaneously injected at 10 mL/kg to establish the pyrexia model. We randomly divided 60 Sprague-Dawley rats into five groups: control, model, positive, low dose of QFZS and high dose of QFZS. Inflammatory proteins were evaluated by Western blotting and immunohistochemistry. For the examination of the endogenous metabolites, enzyme linked immunosorbent assay and ultra-high-performance liquid chromatography high-resolution mass spectrometry were employed. RESULTS: QFZS significantly reduced rats' body temperature within 6 h after dry yeast injection and reduced the secretion of the arginine vasopressin, cyclic adenosine monophosphate, prostaglandin E-2, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß in serum. Meanwhile, we identified 41 metabolites between the model and QFZS groups, including arachidonic acid and lysophospholipids. QFZS restored normal arachidonic acid levels. Based on the differential metabolite enrichment analysis, QFZS's anti-inflammatory and anti-pyrexia effects might be related to the inflammatory pathway regulated by transient receptor potential. Additionally, QFZS treatment reduced transient receptor potential melastatin 2 ion channel expression and affected TNF-α, heat shock protein 70, and cyclooxygenase-2 expression in the hypothalamus. CONCLUSION: QFZS exerts its regulatory effects on fever by regulating the metabolism of lysophospholipids and arachidonic acid and the regulation of inflammation via transient receptor potential ion channels channels.

Regulatory T cells modulate monocyte functions in immunocompetent antiretroviral therapy naive HIV-1 infected people.

We previously demonstrated that the overall number of regulatory T (Treg) cells decrease proportionately with helper CD4+ T cells and their frequencies increase in antiretroviral therapy (ART)-naive human immunodeficiency virus type-1 (HIV-1) infected individuals. The question now is whether the discrepancies in Treg cell numbers and frequencies are synonymous to an impairment of their functions. To address this, we purified Treg cells and assessed their ability to modulate autologous monocytes functions. We observed that Treg cells were able to down modulate autologous monocytes activation as well as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) production during stimulation with polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (poly-ICLC). This activity of Treg cells has been shown to be influenced by immunocompetence including but not limited to helper CD4+ T cell counts, in individuals with HIV-1 infection. Compared to immunosuppressed participants (CD4 < 500 cells/µL), immunocompetent participants (CD4 ≥ 500 cells/µL) showed significantly higher levels of transforming growth factor beta (TGF-ß) and IL-10 (p < 0.001 and p < 0.05, respectively), key cytokines used by Treg cells to exert their immunosuppressive functions. Our findings suggest the contribution of both TGF-ß and IL-10 in the suppressive activity of Treg cells.

Acetylcholine receptor-ß inhibition by interleukin-6 in skeletal muscles contributes to modulating neuromuscular junction during aging.

BACKGROUND: Aging-related strength decline contributes to physiological deterioration and is a good predictor of poor prognosis. However, the mechanisms underlying neuromuscular junction disorders affecting contraction in aging are not well described. We hypothesized that the autocrine effect of interleukin (IL)-6 secreted by skeletal muscle inhibits acetylcholine receptor (AChR) expression, potentially causing aging-related strength decline. Therefore, we investigated IL-6 and AChR ß-subunit (AChR-ß) expression in the muscles and sera of aging C57BL/6J mice and verified the effect of IL-6 on AChR-ß expression. METHODS: Animal experiments, in vitro studies, bioinformatics, gene manipulation, dual luciferase reporter gene assays, and chromatin immunoprecipitation experiments were used to explore the role of the transcription cofactor peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α) and its interacting transcription factors in the IL-6-mediated regulation of AChR-ß expression. RESULTS: IL-6 expression gradually increased during aging, inhibiting AChR-ß expression, which was reversed by tocilizumab. Both tocilizumab and the PGC1α agonist reversed the inhibiting effect of IL-6 expression on AChR-ß. Compared to inhibition of signal transducer and activator of transcription 3, extracellular signal-regulated kinases 1/2 (ERK1/2) inhibition suppressed the effects of IL-6 on AChR-ß and PGC1α. In aging mouse muscles and myotubes, myocyte enhancer factor 2 C (MEF2C) was recruited by PGC1α, which directly binds to the AChR-ß promoter to regulate its expression. CONCLUSIONS: This study verifies AChR-ß regulation by the IL-6/IL-6R-ERK1/2-PGC1α/MEF2C pathway. Hence, evaluating muscle secretion, myokines, and AChRs at an earlier stage to determine pathological progression is important. Moreover, developing intervention strategies for monitoring, maintaining, and improving muscle structure and function is necessary.

Astrocytic DLL4-NOTCH1 signaling pathway promotes neuroinflammation via the IL-6-STAT3 axis.

Under neuroinflammatory conditions, astrocytes acquire a reactive phenotype that drives acute inflammatory injury as well as chronic neurodegeneration. We hypothesized that astrocytic Delta-like 4 (DLL4) may interact with its receptor NOTCH1 on neighboring astrocytes to regulate astrocyte reactivity via downstream juxtacrine signaling pathways. Here we investigated the role of astrocytic DLL4 on neurovascular unit homeostasis under neuroinflammatory conditions. We probed for downstream effectors of the DLL4-NOTCH1 axis and targeted these for therapy in two models of CNS inflammatory disease. We first demonstrated that astrocytic DLL4 is upregulated during neuroinflammation, both in mice and humans, driving astrocyte reactivity and subsequent blood-brain barrier permeability and inflammatory infiltration. We then showed that the DLL4-mediated NOTCH1 signaling in astrocytes directly drives IL-6 levels, induces STAT3 phosphorylation promoting upregulation of astrocyte reactivity markers, pro-permeability factor secretion and consequent blood-brain barrier destabilization. Finally we revealed that blocking DLL4 with antibodies improves experimental autoimmune encephalomyelitis symptoms in mice, identifying a potential novel therapeutic strategy for CNS autoimmune demyelinating disease. As a general conclusion, this study demonstrates that DLL4-NOTCH1 signaling is not only a key pathway in vascular development and angiogenesis, but also in the control of astrocyte reactivity during neuroinflammation.

Protein Kinase C and Matrix Metalloproteinases Expression Using Phorbol Myristate Acetate in Degenerative Intervertebral Disc Cells.

Background: Degeneration of nucleus pulposus (NP) cells involves multiple factors. The relationship between the canonical Wnt/ß-catenin signaling pathway and matrix metalloproteinases (MMPs) is important in cellular senescence. Protein kinase C (PKC), an intermediate of the non-canonical Wnt pathway stimulated by phorbol myristate acetate (PMA), possibly prevents NP cell senescence, although not yet demonstrated in human-based studies. This study aimed to investigate the effect of PMA stimulation on the non-canonical and canonical Wnt pathways and MMP expression in human NP cells to ascertain its inhibitory effects on the senescence of NP cells. Methods: Human disc tissues of Pfirrmann grades 1 and 2 were collected from patients during spinal surgery and subsequently cultured. Protein and ribonucleic acid (RNA) were isolated from NP cells treated with PMA (400 nM) for 24 hours. Expression of MMP1, MMP13, tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), transient receptor potential vanilloid 4 (TRPV4), interleukin-6 (IL-6), and ß-catenin were detected using western blot analysis. Messenger RNA (mRNA) expression of type II collagen and glycosaminoglycan (GAG) were analyzed using reverse transcription polymerase chain reaction. IL-6 and prostaglandin E2 (PGE2) levels were measured using enzyme-linked immunosorbent assay. Results: Expression of PKC-δ (intermediate of the non-canonical Wnt pathway) and ß-catenin (intermediate of the canonical Wnt pathway) was increased by PMA treatment. The mRNA levels of type II collagen and GAG increased; however, their protein levels were not altered. PMA treatment increased the expression of MMP1, TIMP1, ADAMTS5, IL-6, PGE2, and TRPV4; however, the expression of MMP13 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was unaltered. Conclusions: PMA activated PKC-δ, affecting the non-canonical Wnt pathway; however, its effect on ß-catenin in the canonical Wnt pathway was limited. ß-catenin activation through the TRPV4 channel led to increased expression of MMP1 and ADAMTS5 and that of IL-6 and PGE2 owing to NF-κB expression. Consequently, the degeneration of NP cells was not prevented.

Pro-inflammatory cytokines gene expression in liver and kidneys of rats exposed to a sub-lethal dose of Bitis arietans snake venom.

Bitis arietans (Puff adder) is a poisonous snake and its bite causes pain, edema, blistering, tissue damage and neutrophilia. There are limited studies on inflammatory process involved in Bitis arietans envenomation. We therefore investigated the role of proinflammatory cytokines in Bitis arietans venom (BAV)-induced liver and kidney toxicities in rats. Adult male Sprague Dawley rats were treated with BAV (0.5 mg/kg) and were sacrificed after specific time intervals (2 h, 24 h, 1 week). Blood samples were collected for liver and renal function tests and tissues were collected for histopathology and gene expression analysis of IL-1ß, IL-6, and TNF-α in liver and kidneys. There was no significant difference in serum ALT activities among different treatment groups. Serum AST was significantly increased at 24 h following BAV injection. In both organs, injection of BAV resulted in mild inflammatory cell infiltration at 2 h post-dosing which normalized after 1 week. In liver, there was a significant increase in IL-1ß expression in BAV-treated rats at 2 and 24 h post-dosing that reduced after one week. Significant increases in IL-6 and TNF-α were observed at 24 h and 1 week after BAV exposure. In kidneys, there were significant increases in IL-1ß and TNF-α expression at 24 h that subsided after 1 week. In conclusion, a single sub-lethal dose of BAV caused an acute phase inflammation in liver and kidneys. It is most probable that a higher dose of BAV may result in greater and irreversible damage to these organs.

[IDI2-AS1 influences the development of acute myocardial infarction by regulating NR4A2 through microRNA-33b-5p].

OBJECTIVE: To explore the effect and correlation of long non-coding RNA (lncRNA) IDI2-AS1/microRNA-33b-5p (miR-33b-5p)/nuclear receptor-associated protein NR4A2 competitive endogenous RNA (ceRNA) regulatory network on acute myocardial infarction (AMI), and to verify whether IDI2-AS1 regulates NR4A2 through miR-33b-5p to affect the occurrence and development of myocardial infarction. METHODS: The miRNA and mRNA expression chips related to myocardial infarction were obtained from gene expression omnibus (GEO), and the differential expression was analyzed. The upstream regulatory mechanism of NR4A2 was predicted using TargetScan database. Thirty-two male C57/BL6 mice were divided into Sham group, AMI model group, miR-33b-5p mimic group [miR-33b-5p mimic lentivirus (5×107 TU) was injected locally into the heart tissue during ligation] and miR-33b-5p inhibitor group [miR-33b-5p inhibitor lentivirus (5×107 TU) was injected locally into the heart tissue during ligation] according to random number table method, with 8 mice per group. Left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were asseessed by echocardiography, left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) were calculated. After the last weighing, the anesthetized mice were sacrificed and the heart tissues were taken. Masson staining of the heart tissues was observed under light microscope, myocardial collagen volume fraction (CVF) and infarct size were calculated. Cardiomyocytes of SPF grade SD rats were collected. They were divided into normal control group (control group), ischemia-hypoxia model group, miR-33b-5p mimic transfection group (miR-33b-5p mimic transfection group before ischemia and hypoxia treatment) and miR-33b-5p inhibitor transfection group (miR-33b-5p inhibitor transfection group before ischemia and hypoxia treatment). The activity of caspase-3/7 in cardiomyocytes was measured. The levels of interleukins (IL-1ß, IL-6) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). The levels of malondialdehyde (MDA), superoxide dismutase (SOD), creatine kinase (CK), MB isoenzyme of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) were detected by colorimetry. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of apoptosis-related proteins Bax and Bcl-2, cytochrome C (Cyt C) and IDI2-AS1/miR-33b-5p/NR4A2 regulatory axis genes. RESULTS: The myocardial infarction microarray analysis showed that NR4A2 expression was significantly up-regulated in myocardial infarction, with predicted upstream regulatory mechanisms indicating its possible influence through the IDI2-AS1/miR-33b-5p/NR4A2 regulatory axis. Echocardiographic detection showed that compared with AMI model group and miR-33b-5p inhibitor group, LVEF and LVFS in the heart tissue of mice in miR-33b-5p mimic group were significantly increased, while the levels of LVEDD, LVESD, CK, CK-MB and LDH were significantly decreased, with statistical significance. Light microscope showed myocardial fibrosis and myocardial infarction in AMI model group and miR-33b-5p inhibitor group. In the miR-33b-5p mimic group, the degree of myocardial fibrosis was decreased and the myocardial infarction size was significantly reduced. Compared with AMI model group and miR-33b-5p inhibitor group, the levels of MDA, IL-1ß, IL-6, TNF-α and the expressions of Bax and Cyt C in the heart tissue of mice in miR-33b-5p mimic group were significantly decreased, while the levels of SOD and Bcl-2 expression were significantly increased, and the differences were statistically significant. The expressions of IDI2-AS1 and NR4A2 in the heart tissue of mice in miR-33b-5p mimic group were significantly lower than those in AMI model group and miR-33b-5p inhibitor group [IDI2-AS1 (2-ΔΔCt): 1.96±0.08 vs. 2.73±0.08, 3.10±0.05, NR4A2 (2-ΔΔCt): 2.36±0.07 vs. 3.16±0.08, 3.80±0.08, all P < 0.01]. The expression of miR-33b-5p was significantly higher than that of AMI model group and miR-33b-5p inhibitor group (2-ΔΔCt: 0.88±0.07 vs. 0.57±0.07, 0.23±0.01, both P < 0.01). The cell experiment results showed that the caspase-3/7 activity of rat neonatal cardiomyocytes in the miR-33b-5p mimic transfection group was significantly lower than that in the ischemia-hypoxia model group and the miR-33b-5p inhibitor transfection group, suggesting that miR-33b-5p can significantly reduce the apoptosis level of the ischemia-hypoxia model. The levels of peroxidation and inflammation indexes, important genes of apoptosis pathway and the expression of IDI2-AS1/miR-33b-5p/NR4A2 regulatory axis of rat neonatal cardiomyocytes in all groups were consistent with the above. CONCLUSIONS: IDI2-AS1 can regulate NR4A2 through miR-33b-5p, thus affecting the occurrence and development of AMI.

Examining the Relationship Between Polystyrene Microplastics and Male Fertility: Insights From an In Vivo Study and In Vitro Sertoli Cell Culture.

BACKGROUND: While polystyrene microplastics (PS-MPs) are emerging as potentially significant health threats, linked to cancer and reproductive dysfunction, their precise effects on human health remain largely unknown. We aimed to investigate the underlying mechanisms promoting microplastic-induced damage in the reproductive system. METHODS: Thirty C57BL/6 male mice were randomly allocated into six equal-sized groups. Mice were exposed to fluorescent PS-MPs (5 µm, < 18%, green) at a dose of 1 and 3 mg/dL via oral gavage for 28 and 56 days, respectively (control, 0 mg/dL). The presence of antibodies and inflammatory and oxidative stress markers were evaluated using western blotting. Sperm analysis was also performed. Mouse testis Sertoli TM4 cells were divided into two groups: control (medium only) and PS-MPs (medium containing, 1,000 µg/mL) groups and cultured in vitro for 1, 24, 48, or 72 hours. The cells were cultured in a Ham's F12: Dulbecco's Modified Eagle Medium medium with 0.25% fetal bovine serum at 37°C with humidified atmosphere of 5% carbon dioxide in the air. Protein analyses for interleukin (IL)-6, IL-10, NADPH-oxidase (NOX)-2, NOX-4, hypoxia-inducible transcription factor (HIF)-2α, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-ß were performed using western blotting. RESULTS: The testes were evaluated after 28 and 56 days of exposure. Varying sizes of PS-MPs were detected in the testes (ranging from 5.870 to 7.768 µm). Significant differences in sperm concentration, motility, and the proportion of normal sperm were observed between the two groups. An increase in TGF-ß, HIF-2α, and NOX-4 levels was observed using western blot analysis. However, no dose-dependent correlations were observed between the two groups. In vitro evaluation of the PS-MPs group displayed PS-MP penetration of the lumen of Sertoli cells after 1 hour. Further PS-MP aggregation within Sertoli cells was observed at 24, 48, and 72 hours. A significant increase in inflammatory protein expressions (IL-10, TGF-ß, MCP-1, IL-6, TNF-α, and HIF-2α) was observed through western blotting, although oxidative agents did not show a significant increase. CONCLUSION: PS-MPs induced reproductive dysfunction in male mice provide new insights into PS-MPs-associated toxicity in mammals.

Effect of napabucasin and doxorubicin via the Jak2/Stat3 signaling pathway in suppressing the proliferation of neuroblastoma cells.

PURPOSE: Napabucasin (NP) is a natural compound that can suppress cancer cell proliferation and cell cycle by inhibition of the signal transducer and activator of transcription 3 (STAT3) gene. We examined the effects of NP on the proliferation and invasion of neuroblastoma cells (SH-SY5Y). METHODS: Human neuroblastoma SH-SY5Y cell line was used in this study. NP was administered to groups at the doses of 0.3-1 µM. Cell viability was analyzed by MTT assay. Real-time quantitative reverse transcription polymerase chain reaction and western blot analysis assessed the expressions of interleukin (IL)-6 dependent Jak2/Stat3 signaling pathway. The MTT cell viability method was applied to determine the antagonistic-synergistic effects and inhibitory concentration (IC50) doses of doxorubicin (DX) and NP. RESULTS: It was determined that 0.3-1 µM doses of NP killed the cells almost completely after 48 hours, and also that Jak2/Stat3 expressions decreased dose-dependently via IL-6. At the protein level, NP and DX were found to reduce Jak2 and Stat3 levels. CONCLUSIONS: NP showed that it suppresses the proliferation of neuroblastoma cells. Due to its inhibitory effect on Jak2 and Stat3, it can be used to prevent invasion of SH-SY5Y cells. NP, which can inactivate Jak2/Stat3, can be used as a treatment agent by combining with DX in proliferation pathway in neuroblastoma.

Nicotine promotes pathogenic bacterial growth and biofilm formation in peri-implant.

Introduction. Peri-implantitis is a plaque-associated disease that leads to implant loss and arises from bacterial biofilms on the surface of the implant. Smoking is a risk factor for peri-implantitis and impedes treatment effectiveness. Additionally, aryl hydrocarbon receptor (AHR), IL-6, and IL-22 levels are related to peri-implantitis.Aim. We aimed to investigate the effects of nicotine on inflammatory response, bacterial growth and biofilm formation.Hypothesis/Gap Statement. We hypothesized that nicotine promoted pathogenic bacterial growth and biofilm formation, thereby aggravating inflammation.Methodology. The expression of AHR, IL-6 and IL-22 was measured in peri-implant sulci fluid using quantitative PCR and Western blot analyses. The cementum was incubated with bacterial suspension including Porphyromonas gingivalis, Streptococcus sanguinis and Fusobacterium nucleatum and treated with 100, 200, 250 and 300 µg ml-1 nicotine, and then, the absorbance and number of colony-forming units were detected. Biofilm formation was evaluated using the tissue culture plate method and safranin O staining. Carbohydrates and proteins were measured by the phenol-sulfuric acid method and the bicinchoninic acid method, respectively.Results. The results indicated that smoking increased the levels of AHR, IL-6 and IL-22. Functionally, nicotine promoted the growth of P. gingivalis, S. sanguinis and F. nucleatum. Additionally, it promoted the biofilm formation of these bacteria and increased the contents of carbohydrates and proteins.Conclusion. Nicotine promoted bacterial growth and biofilm build-up, suggesting that smoking may aggravate the progression of peri-implantitis.

Investigation of the Effects of Curcumin and Gallic Acid in the Liver and Kidneys of Rat Ovarian Transplant Recipients.

OBJECTIVES: Recent studies have shown that oxidative stress, inflammation, and apoptosis play substantial roles in the pathogenesis of tissue damage and distant organ damage during ovarian transplantation. Because of their potential antioxidant and anti-inflammatory properties, investigating natural compounds like curcumin and gallic acid is crucial. This study investigated the effects of curcumin, gallic acid, and their combination on liver and kidney tissues in a rat model of ovarian transplantation. MATERIALS AND METHODS: For this study, we used 42 adult female Sprague-Dawley rats aged 11 to 12 weeks, which we randomly divided into 6 groups. After the transplant procedures, we performed histopathological analysis, biochemical examinations, and statistical analysis. RESULTS: Significant damage was shown in the liver and kidney tissues of the ovarian transplant only and transplant plus corn oil groups compared with the control group, as evidenced by histopathological evaluation, increased BAX/BCL-2 intensity indicating apoptotic activity, and elevated interleukin 6 levels. However, treatment with curcumin, gallic acid, or their combination attenuated these adverse effects, suggesting potential protective effects against transplant-induced injury. CONCLUSIONS: Our findings highlight the therapeutic potential of these compounds in mitigating tissue damage and inflammation associated with ovarian transplant.

Assessment of the Anti-cancer Effects of Camel Milk Exosomes (CMEXOs) on Murine Colorectal Cancer Cell Line (CT-26).

Today, camel milk consumption in the Middle East is trendy because it is believed that it reduces the risk of cancer. Recently, studies have discovered that most of milk's beneficial effects are because of its nanoparticles, especially exosomes. The objective of the present research was to investigate the anti-cancer effects of camel milk exosomes (CMEXOs) in the murine colorectal cancer cell line (CT-26). Our findings verified the existence of exosomes measuring approximately 114.1±3.4 nm in diameter. Through MTT and migration assays, we established that CMEXOs exhibit dose-dependent anti-proliferative and anti-migration effects on the CT-26 cell line. Furthermore, our study showed that treatment with CMEXOs led to a reduction in TNF-α and IL-6 gene expression in CT-26 cells. While additional in vivo studies are required, our data demonstrate that CMEXOs have anti-proliferative and anti-migration effects on CT-26, possibly by influencing crucial genes within the inflammation pathway.

Study on PINK1 Expression and Its Clinical Value in Diabetic Nephropathy.

INTRODUCTION: To explore PTEN-induced putative kinase 1 (PINK1) expression and its clinical value in diabetic nephropathy. METHODS: Ninety patients with diabetic nephropathy were recruited and divided into metformin hydrochloride monotherapy group, telmisartan monotherapy group and combination therapy (metformin and telmisartan) group. Renal function indices and PINK1 expression, inflammatory factors, reactive oxygen species (ROS), mitochondrial membrane potential (MMP) levels were detected. The correlation between PINK1 and inflammatory factors, renal function indicators including estimated glomerular filtration rate (eGFR), urinary albumin-to-creatinine ratio (UACR) and serum creatinine (SCr)] were analyzed by Pearson correlation. RESULTS: Following treatments, the combination therapy group exhibited increased PINK1 expression levels and decreased ROS levels compared to the groups receiving metformin hydrochloride or telmisartan monotherapy. The combination therapy group showed significant improvements in renal function indices and inflammatory markers. Additionally, the MMP ratio in the combination therapy group was higher compared to the two monotherapy groups. Furthermore, PINK1 was negatively correlated with UACR, SCr, tumor necrosis factor (TNF-α) and interleukin-6 (IL-6), while positively correlated with eGFR and interleukin-2 (IL-2). CONCLUSION: PINK1 exhibits low expression levels in patients with diabetic nephropathy and its expression is strongly associated with the inhibition of disease progression, thereby offering significant clinical diagnostic value. Additionally, it may serve as a potential biological marker for clinical diagnosis and treatment of diabetic nephropathy.

In Vitro Profiling of the Antiviral Peptide TAT-I24.

The synthetic peptide TAT-I24 (GRKKRRQRRRPPQCLAFYACFC) exerts antiviral activity against several double-stranded (ds) DNA viruses, including herpes simplex viruses, cytomegalovirus, some adenoviruses, vaccinia virus and SV40 polyomavirus. In the present study, in vitro profiling of this peptide was performed with the aim of characterizing and improving its properties for further development. As TAT-I24 contains three free cysteine residues, a potential disadvantageous feature, peptide variants with replacements or deletions of specific residues were generated and tested in various cell systems and by biochemical analyses. Some cysteine replacements had no impact on the antiviral activity, such as the deletion of cysteine 14, which also showed improved biochemical properties, while the cyclization of cysteines 14 and 20 had the most detrimental effect on antiviral activity. At concentrations below 20 µM, TAT-I24 and selected variants did not induce hemolysis in red blood cells (RBCs) nor modulated lipopolysaccharide (LPS)-induced release of cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), in human peripheral blood mononuclear cells (PBMCs). These data indicate that TAT-I24 or its peptide variants are not expected to cause unwanted effects on blood cells.

Impact of Mygalin on Inflammatory Response Induced by Toll-like Receptor 2 Agonists and IFN-γ Activation.

Several natural products are being studied to identify new bioactive molecules with therapeutic potential for infections, immune modulation, and other pathologies. TLRs are a family of receptors that play a crucial role in the immune system, constituting the first line of immune defense. They recognize specific products derived from microorganisms that activate multiple pathways and transcription factors in target cells, which are vital for producing immune mediators. Mygalin is a synthetic acylpolyamine derived from hemocytes of the spider Acanthoscurria gomesiana. This molecule negatively regulates macrophage response to LPS stimulation by interacting with MD2 in the TLR4/MD2 complex. Here, we investigated the activity of Mygalin mediated by TLR2 agonists in cells treated with Pam3CSK4 (TLR2/1), Pam2CSK4, Zymosan (TLR2/6), and IFN-γ. Our data showed that Mygalin significantly inhibited stimulation with agonists and IFN-γ, reducing NO and IL-6 synthesis, regardless of the stimulation. There was also a significant reduction in the phosphorylation of proteins NF-κB p65 and STAT-1 in cells treated with Pam3CSK4. Molecular docking assays determined the molecular structure of Mygalin and agonists Pam3CSK4, Pam2CSK4, and Zymosan, as well as their interaction and free energy with the heterodimeric complexes TLR2/1 and TLR2/6. Mygalin interacted with the TLR1 and TLR2 dimer pathway through direct interaction with the agonists, and the ligand-binding domain was similar in both complexes. However, the binding of Mygalin was different from that of the agonists, since the interaction energy with the receptors was lower than with the agonists for their receptors. In conclusion, this study showed the great potential of Mygalin as a potent natural inhibitor of TLR2/1 and TLR2/6 and a suppressor of the inflammatory response induced by TLR2 agonists, in part due to its ability to interact with the heterodimeric complexes.