P65 Protein Expression Research Articles

Interleukin-22 Alleviates Caerulein-Induced Acute Pancreatitis by Activating AKT/mTOR Pathway

BackgroundAcute pancreatitis (AP) is one of the most common acute abdominal disorders; due to the lack of specific treatment, the treatment of acute pancreatitis, especially serious acute pancreatitis (SAP), is difficult and challenging. We will observe the changes of Interleukin -22 levels in acute pancreatitis animal models, and explore the mechanism of Interleukin -22 in acute pancreatitis.ObjectiveThis study aims to assess the potential protective effect of Interleukin -22 on caerulein-induced acute pancreatitis and to explore its mechanism.MethodsBlood levels of amylase and lipase and Interleukin -22 were assessed in mice with acute pancreatitis. In animal model and cell model of caerulein-induced acute pancreatitis, the mRNA levels of P62 and Beclin-1 were determined using PCR, and the protein expression of P62, LC3-II, mTOR, AKT, p-mTOR, and p-AKT were evaluated through Western blot analysis.ResultsInterleukin -22 administration reduced blood amylase and lipase levels and mitigated tissue damage in acute pancreatitis mice model. Interleukin -22 inhibited the relative mRNA levels of P62 and Beclin-1, and the Interleukin -22 group showed a decreased protein expression of LC3-II and P62 and the phosphorylation of the AKT/mTOR pathway. Furthermore, we obtained similar results in the cell model of acute pancreatitis.ConclusionThis study suggests that Interleukin -22 administration could alleviate pancreatic damage in caerulein-induced acute pancreatitis. This effect may result from the activation of the AKT/mTOR pathway, leading to the inhibition of autophagy. Consequently, Interleukin -22 shows potential as a treatment.

Codonopsis pilosula water extract delays D-galactose-induced aging of the brain in mice by activating autophagy and regulating metabolism

Ethnopharmacological relevanceCodonopsis pilosula (C. pilosula), also called “Dangshen” in Chinese, is derived from the roots of Codonopsis pilosula (Franch.) Nannf. (C. pilosula), Codonopsis pilosula var. Modesta (Nannf.) L.D.Shen (C. pilosula var. modesta) or Codonopsis pilosula subsp. Tangshen (Oliv.) D.Y.Hong (C. pilosula subsp. tangshen), is a well-known traditional Chinese medicine. It has been regularly used for anti-aging, strengthening the spleen and tonifying the lungs, regulating blood sugar, lowering blood pressure, strengthening the body's immune system, etc. However, the mechanism, by which, C. pilosula exerts its therapeutic effects on brain aging remains unclear. Aim of the studyThis study aimed to investigate the underlying mechanisms of the protective effects of C. pilosula water extract (CPWE) on the hippocampal tissue of D-galactose-induced aging mice. Materials and methodsIn this research, plant taxonomy has been confirmed in the “The Plant List” database (www.theplantlist.org). First, an aging mouse model was established through the intraperitoneal injections of D-galactose solution, and low-, medium-, and high-dose CPWE were administered to mice by gavage for 42 days. Then, the learning and memory abilities of the mice were examined using the Morris water maze tests and step-down test. Hematoxylin and eosin staining was performed to visualize histopathological damage in the hippocampus. A transmission electron microscope was used to observe the ultrastructure of hippocampal neurons. Immunohistochemical staining was performed to examine the expression of glial fibrillary acidic protein (GFAP), the marker protein of astrocyte activation, and autophagy-related proteins, including microtubule-associated protein light chain 3 (LC3) and sequestosome 1 (SQSTM1)/p62, in the hippocampal tissues of mice. Moreover, targeted metabolomic analysis was performed to assess the changes in polar metabolites and short-chain fatty acids in the hippocampus. ResultsFirst, CPWE alleviated cognitive impairment and ameliorated hippocampal tissue damage in aging mice. Furthermore, CPWE markedly alleviated mitochondrial damage, restored the number of autophagosomes, and activated autophagy in the hippocampal tissue of aging mice by increasing the expression of LC3 protein and reducing the expression of p62 protein. Meanwhile, the expression levels of the brain injury marker protein GFAP decreased. Moreover, quantitative targeted metabolomic analysis revealed that CPWE intervention reversed the abnormal levels of L-asparagine, L-glutamic acid, L-glutamine, serotonin hydrochloride, succinic acid, and acetic acid in the hippocampal tissue of aging mice. CPWE also significantly regulated pathways associated with D-glutamine and D-glutamate metabolism, nitrogen metabolism, arginine biosynthesis, alanine, aspartate, and glutamate metabolisms, and aminoacyl-tRNA biosynthesis. ConclusionsCPWE could improve cognitive and pathological conditions induced by D-galactose in aging mice by activating autophagy and regulating metabolism, thereby slowing down brain aging.

Mechanism of Fushen Granules treat peritoneal dialysis-related peritonitis by regulating TLR4/NF-κB pathway:based on network pharmacology and animal experiments

Network pharmacology was employed to probe into the mechanism of Fushen Granules in treating peritoneal dialysis-rela-ted peritonitis(PDRP) in rats. The main active components of Fushen Granules were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and their targets were predicted. PDRP-related targets were retrieved from DisGeNET and other databases. The common targets shared by the drug and the disease were identified by the online tool, and protein-protein interaction(PPI) network of the common targets. The obtained 276 common targets were imported into DAVID for GO function enrichment and KEGG pathway enrichment. The main signaling pathway of Fushen Granules in the treatment of PDRP was predicted as Toll-like receptor 4(TLR4)/nuclear factor(NF)-κB. The rat model of uremia was induced by 5/6 nephrectomy. From two weeks after operation, the rat model of peritoneal dialysis(PD) was established by intraperitoneal injection of 20 mL dialysate with 1.25% glucose every day. The sham operation group and model group received 2 mL normal saline by gavage every day. The rats in Fushen Gra-nules groups were administrated with 2 mL solutions of low-(0.54 g·kg~(-1)), medium-(1.08 g·kg~(-1)) and high-dose(2.16 g·kg~(-1)) Fushen Granules every day. The bifico group received 2 mL(113.4 mg·kg~(-1)) of bifico solution every day. At the end of the 8th week, the levels of serum creatinine(Scr) and blood urea nitrogen(BUN) in each group were measured. The serum levels of hypersensitive C reactive protein(hs-CRP), tumor necrosis factor(TNF)-α, and interleukin(IL)-6 were measured, and the pathological changes in the colon tissue were observed by hematoxylin-eosin(HE) staining. The serum levels of lipopolysaccharide(LPS) and lipopolysaccharide-binding protein(LBP) of rats were measured, and the expression levels of LBP, TLR4, NF-κB p65, inhibitor of κB kinase α(IκBα), TNF-α, and IL-1β in the colon tissue were determined. Compared with sham operation group, the model group had abnormal structure of all layers of colon tissue, sparse and shorter intestinal villi, visible edema in mucosal layer, wider gap, obvious local inflammatory cell infiltration, significantly decreased body weight(P<0.01), and significantly increased kidney function index(Scr, BUN) content(P<0.01). Serum levels of inflammatory cytokines(hs-CRP, TNF-α, IL-6), LPS and LBP were significantly increased(P<0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1β were significantly increased(P<0.01), and protein expressions of IκBα were significantly decreased(P<0.01). Compared with model group, intestinal villi damage in colonic tissue of rats in low-, medium-and high-dose Fushen Granules groups and bifico group were alleviated to different degrees, edema in submucosa was alleviated, space was narrowed, and inflammatory cell infiltration in lamina propria was reduced. The contents of renal function index(Scr, BUN) and serum inflammatory factors(hs-CRP, TNF-α, IL-6) were significantly decreased(P<0.05 or P<0.01) in medium-and high-dose Fushen Granules groups and bifico group(P<0.05 or P<0.01). Serum LPS and LBP contents in Fushen Granules group and bifico group were significantly decreased(P<0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1β in Fushen Granules group were significantly decreased(P<0.05 or P<0.01), and protein expressions of IκBα were significantly increased(P<0.01). The expression of LBP protein in bifico group was significantly decreased(P<0.01). The results suggest that Fushen Granules can protect the residual renal function of PD rats, reduce the inflammatory response, and protect the colon tissue. Based on network pharmacology, TLR4/NF-κB pathway may be the main signaling pathway of Fushen granule in the treatment of PDRP. The results showed that Fushen Granules could improve intestinal inflammation and protect intestinal barrier to prevent PDRP by regulating the expression of key factors in TLR4/NF-κB pathway in colon of PD rats.

Effect of beta-cypermethrin on the reproductive capacity of female mice in advanced age

The aim of the present study was to investigate whether exposure to pesticides beta-cypermethrin (β-CYP) harms the reproductive capacity of advanced-age female mice. The results evidenced that peri-implantation β-CYP exposure significantly reduced the number of fetuses per advanced-age female in the first litter, and the number and weight of implantation sites. The levels of decidualization markers were significantly reduced in β-CYP-administered advanced-age mice. Lower expression of Pcna, Cdk6, Foxo1, Ki67, and p62 protein and mRNA was found in the decidua of β-CYP-treated advanced-age mice. The levels of Bax, cleaved caspase-3, Lc3a/b, Atg, mTOR, and p-mTOR protein, and the ratio of p-mTOR/mTOR protein expression were clearly downregulated by peri-implantation β-CYP exposure. These results indicated that peri-implantation β-CYP exposure may elevate the decline in reproductive capacity of early pregnant mice in advanced age.

Syringic acid modulates methyl cellosolve-induced hepatic upregulation of the PI3K-Akt-mTOR pathway and inhibition of p53-Bax-caspase-3-dependent apoptosis in rats

BackgroundMethyl cellosolve (METC) is a widely used industrial solvent due to its amphipathic nature. It is found in household materials such as liquid soaps, dyes, inks, brake fluids, herbicides, etc. This makes exposure in humans incessant and indiscriminate. The PI3K-Akt-mTOR and the intrinsic apoptotic pathways are very important cellular activities. While the former participates in cell growth, proliferation, and survival, the latter coordinates the death of abnormally functional cells, thereby stopping their proliferation. Given that background, we studied the hepatoprotective effect of syringic acid (SA) on METC-induced hepatotoxicity by checking the involvement of the PI3K-Akt-mTOR and apoptotic pathways in male Wistar rats. MethodsThirty (30) rats divided into six groups were used. Rats in group 1 served as control, those in group 2 were administered METC for 30 consecutive days, and those in groups 3, 4, and 5 were treated with 25, 50, and 75 mg/kg body weight of SA respectively also for 30 consecutive days immediately after each day METC administrations, while rats in group 6 received 75 mg/kg body weight of SA only throughout. ResultsFollowing analyses, METC-induced activation of the PI3K-Akt pathway and inhibition of apoptosis were marked by a significant elevation in the liver gene expressions of PI3K, Akt, mTOR, PTEN, FoxO1, and TSC2 as well as a significant downregulation in the protein expressions of p53, caspase 3, Bax, and a significant upregulation in Bcl2 protein expression compared with control rats. SA treatment (25 mg/kg only) significantly downregulated the hepatic mTOR, FoxO1, and TSC2 gene expressions, while in addition to mTOR, FoxO1, and TSC2 genes, treatments with 50 and 75 mg/kg significantly downregulated the liver gene expressions of PI3K, Akt, and PTEN compared with METC only exposed rats. For protein expression, all the doses of SA treatment significantly downregulated the liver Bcl2 level (an antiapoptotic protein), while 75 mg/kg of SA only significantly upregulated the liver p53, Bax, and caspase 3 protein expressions (thereby ensuring the apoptosis of damaged cells) compared with METC-only exposed rats. ConclusionsIn conclusion, METC-induced liver activation of PI3K-Akt-mTOR signaling and inhibition of intrinsic apoptotic pathway was recorded, suggesting that one mechanism of METC toxicity may be through the advancement of cell survival and growth as well as inhibition of apoptosis. SA treatment (most especially by 75 mg/kg) modulated these two biochemical processes through the inhibition of participatory players in the rats.

Three Artemisia pollens trigger the onset of allergic rhinitis via TLR4/MyD88 signaling pathway.

The prevalence of allergic rhinitis is high, making it a relatively common chronic condition. Countless patients suffer from seasonal Allergic rhinitis (AR). The objective of this investigation is to examine the potential involvement of common pollen allergens in seasonal allergic rhinitis, and study the proposed mechanism of Toll-like receptor 4 (TLR4)/Myeloid differentiation primary response gene 88 (MyD88) signaling pathway in the induction of AR. A mouse AR model (sensitized group) was constructed with pollen extracts and ovalbumin(OVA) of Artemisia annua (An), Artemisia argyi (Ar) and Artemisia Sieversiana (Si), and thereafter, AR symptom score was performed. After successful modeling, mouse serum and nasal mucosa tissues were extracted for subsequent experiments. The expression levels of immunoglobulin E (IgE), Interleukin (IL)-4, IL-5, IL-13 and Tumor Necrosis Factor-α (TNF-α) in serum were detected using Enzyme-linked immunosorbent assay (ELISA); Hematoxylin-eosin (H&E) staining methods were used to observe the pathological changes of the nasal mucosal tissue; Utilizing immunohistochemistry (IHC) staining, the expression levels of TLR4, MyD88 and Nuclear factor kappa B (NF-κB) p65 in mouse nasal mucosa were quantified; The mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of sensitized mice were detected with Quantitative reverse transcription PCR (qRT-PCR) and Western Blot. Finally, the in vitro culture of Human nasal mucosal epithelial cells (HNEpC) cells was conducted, and cells were treated with 200µg/ml Artemisia annua pollen extractand OVA for 24h. Western Blot assay was used to detect the expression level of TLR4, MyD88 and NF-κB p65 proteins before and after HNEpC cells were treated with MyD88 inhibitor ST-2825. On the second day after AR stimulation, the mice showed obvious AR symptoms. H&E results showed that compared to the control group, the nasal mucosal tissue in the sensitized group was significantly more inflamed. Furthermore, ELISA assay showed increased expression levels of IgE, IL-4, IL-5, IL-13 and TNF-α in serum of mice induced by OVA and Artemisia annua pollen, Artemisia argyi pollen and Artemisia Sieversiana pollen than those of the control group. However, the expression level of IL-2 was lower than that of the control group (P < 0.05). Using Immunohistochemistry staining visually observed the expression levels of TLR4, MyD88 and NF-κB p65 in mouse nasal mucosa tissues and quantitatively analyzed. The expression levels of TLR4, MyD88 and NF-κB p65 in the sensitized group were higher than those in the control group, and the differences were statistically significant (P < 0.05). The results from qRT-PCR and Western Blot showed that the mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of the sensitized group were significantly higher than those in the control group (P < 0.05). Finally, HNEpC cells were cultured in vitro and analyzed using Western Blot. The expression levels of TLR4, MyD88 and NF-κB p65 in OVA and An groups were significantly increased (P < 0.05). After ST-2825 treatment, TLR4 protein expression was significantly increased (P < 0.05) and MyD88 and NF-κB p65 protein expression were significantly decreased (P < 0.05). To sum up, the occurrence and development of AR induced by OVA and pollen of Artemisia annua, Artemisia argyi and Artemisia Sieversiana were related to TLR4/MyD88 signal pathway.

Anti-sepsis effect of Xiaochaihu decoction based on the TLR4/MyD88/NF-κB signalling pathway

The aims of this study were to explore the protective effect of Xiaochaihu decoction in mice with sepsis induced by intraperitoneal injection; to explore its anti-inflammatory effect on the TLR4-MyD88-NF-κB signalling pathway; and to explore the main material basis of the anti-inflammatory effect of Xiaochaihu decoction, with the aim of supplementing and expanding the associated research and providing a scientific foundation for the clinical use of the decoction. The effects of Xiaochaihu decoction on septic mice were analysed by measurements of white blood cells (WBC) and Platelets (PLT); Nitric Oxide (NO) level in serum; IL-6, IL-1β and TNF-α levels in serum; RT-PCR; Haematoxylin-Eosin (HE) immunohistochemistry; western blotting (WB). The results showed the excellent in vivo anti-inflammatory effects of Xiaochaihu decoction in LPS-induced septic mice, through down regulation of the gene and protein expression of TLR4, MYD88, TRAF6, IKK, IKBα and p65 and the subsequent reduction in the release of inflammatory mediators IL-6, IL-1β, TNF-α and NO. Moreover, significant anti-septic effect was observed from high and medium doses of Xiaochaihu decoction, but not from the low dose.

Inhibition of p38 MAPK/NF-κB p65 signaling pathway activity by rare ginsenosides ameliorates cyclophosphamide-induced premature ovarian failure and KGN cell injury

Ethnopharmacological relevancePanax ginseng C. A. Mey., one of the most used herbs in the world, shows effective treatment in reproductive injury. Recent studies have proven that the processed product, red ginseng, which is more active than ginseng itself. Therefore, it is speculated that its main functional component, rare ginsenosides (heat-transformed saponin, HTS), may be effective in treating premature ovarian failure (POF), but its efficacy has not yet been experimentally confirmed. Aim of the studyTo evaluate whether HTS could attenuate cyclophosphamide-induced inflammation and oxidative damage in POF model rats and the human granulosa-like KGN cell line and protect granulosa cell proliferation. Material and methodsHTS were isolated from ginsenosides and high performance liquid chromatography (HPLC) analysis was used to analyze the HTS components. Cyclophosphamide (CP) was used to establish a POF rat model and KGN cell injury model. Reactive oxygen species (ROS) and antioxidant enzyme production was determined using specific assays, while inflammatory cytokine secretion was measured by enzyme-linked immunosorbent assay (ELISA). The proliferative function of granulosa cells was assessed using high-content screening and immunohistochemistry to determine the Ki67 protein level. Protein expression in ovarian tissues and KGN cells was analyzed by Western blotting, quantitative real-time PCR (qRT-PCR) was used to determine the transcriptional changes in ovarian tissues and KGN cells. ResultsIn CP-treated POF model rats, HTS significantly decreased malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels, increased glutathione oxidase (GSH) levels, and upregulated Ki67 expression in ovarian granulosa cells. In addition, HTS significantly increased cell survival and Ki67 expression levels in CP-treated cells, and superoxide dismutase (SOD) levels were significantly increased. HTS significantly downregulated IL-6, TNF-α, and interleukin-1β (IL-1β) mRNA expression and significantly inhibited nuclear factor kappa-B p65 (NF-κB p65) and p38 mitogen activated protein kinase (p38 MAPK) phosphorylation in POF model rats and KGN cells. Moreover, NF-κB p65 and p38 MAPK levels were significantly increased in ovarian granulosa cells. p65 and p38 protein and gene expression was significantly downregulated. ConclusionHTS ameliorated CP-induced POF and human granulosa cell injury, possibly by inhibiting inflammation and oxidative damage mediated by the p38 MAPK/NF-κB p65 signaling pathway.

Mitochondrial calcium uniporter promotes mitophagy by regulating the PINK1/Parkin pathway in caerulein‑treated pancreatic ductal epithelial cells invitro.

The mitochondrial calcium uniporter (MCU) is a major protein for the uptake of mitochondrial calcium to regulate intracellular energy metabolism, including processes such as mitophagy. The present study investigated the effect of the MCU on mitophagy in pancreatic ductal epithelial cells (PDECs) in acute pancreatitis (AP) in vitro. The normal human PDECs (HPDE6-C7) were treated with caerulein (CAE) to induce AP-like changes, with or without ruthenium red to inhibit the MCU. The mitochondrial membrane potentials (MMPs) and mitochondrial Ca2+ levels were analyzed by fluorescence. The expression levels of MCU, LC3, p62, and translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20), putative kinase 1 (PINK1), and Parkin were measured by western blotting and immunofluorescence. Mitophagy was observed by confocal fluorescence microscopy and transmission electron microscopy. The results showed that CAE increased the MCU protein expression, mitochondrial Ca2+ levels, MMP depolarization and the protein expression of mitophagy markers including the LC3II/I ratio, PINK1, and Parkin. CAE decreased the protein expression of p62 and TOMM20, and promoted the formation of mitophagosomes in HPDE6-C7 cells. Notably, changes in these markers were reversed by inhibiting the MCU. In conclusion, an activated MCU may promote mitophagy by regulating the PINK1/Parkin pathway in PDECs in AP.

Protective effects of piperlongumine against adjuvant-induced arthritis in rats through modulating OPG/RANKL/NF-κB signaling pathway.

We examined the antirheumatoid effects of piperlongumine (PLM) on rat adjuvant-induced arthritis (AIA) and explored the underlying mechanisms involved. PLM (2.5, 5, and 10 mg/kg) was administered intraperitoneally to AIA rats to assess its effectiveness. Blood, thymus, spleen, ankle joint, and synovial tissue samples were gathered for subsequent analyses, like enzyme-linked immunosorbent assay, thymus/spleen index measurement, ankle joint pathological examination, immunohistochemistry assay, polymerase chain reaction, and western blot assay. Moreover, the involvement of osteoprotegerin (OPG)/receptor activators of nuclear factor κB ligand (RANKL)/nuclear factor-κB (NF-κB) signaling was investigated. PLM effectively relieved inflammation and joint destruction in AIA rats, as indicated by reductions in hind paw swelling, arthritis index, thymus/spleen index, ankle joint pathological damage, production of TNF-α, IL-1β, and IL-6 in both serum and synovium, and osteoclast formation. Also, PLM treatment raised OPG production, reduced RANKL expression, and elevated the OPG/RANKL ratio in synovial tissues. Furthermore, PLM prevented IκBα degradation and phosphorylation, resulting in a reduced expression of the nuclear NF-κB p65 protein in AIA rat synovial tissues. PLM demonstrated strong antiarthritic effects in rats with AIA by influencing the OPG/RANKL/NF-κB signaling pathway, highlighting its potential clinical relevance in treating rheumatoid arthritis.

IL1RAP Exacerbates Sepsis-Induced Pulmonary and Spleen Injury Through Regulating CD4+ T Lymphocyte Differentiation

ABSTRACT Background Complex pathophysiological the specific mechanism of sepsis on CD4+ T-cell responses is less well understood. IL1 receptor accessory protein (IL1RAP) was found to be involved in activating host immune responses. Method Cecum ligation and puncture (CLP) was utilized to build a mouse sepsis model. The experiment was randomly divided into four groups: Sham, CLP, CLP + shNC, and CLP + shIL1RAP group. Results qRT-PCR suggested mRNA levels of IL1RAP were decreased when IL1RAP was knocked down with the mRNA levels of IL-1β, NF-κB, and p38 decreased. Histopathology showed severe pathological damage with alveolar integrity lost, red blood cells in the alveoli, massive inflammatory cell infiltration, and the alveolar wall was thickening in the CLP group. The inflammatory cytokine levels of TNF-α, IL-1β, and IFN-γ were elevated in CLP mice by ELISA. The counts of CD4+ T cells were decreased in sepsis mice in peripheral blood, spleen, and BALF by flow cytometry. However, the above was blocked down when using shIL1RAP. Western blot suggested sh IL1RAP inhibited IL-1β, NF-κB, and p38 protein expressions. Conclusions We defined IL1RAP as a new target gene through NF-κB/MAPK pathways regulating CD4+ T lymphocyte differentiation mediated the progression of sepsis, which is potentially exploitable for immunotherapy.

Chlamydia trachomatis upregulates lncRNA CYTOR to mediate autophagy through miR-206/MAPK1 axis.

Chlamydia trachomatis infection can be regulated by autophagy-related genes. LncRNA CYTOR has been proven to be involved in autophagy. In this research, we investigated the role of CYTOR in autophagy induced by C. trachomatis and the potential mechanisms. After C. trachomatis infection, CYTOR and MAPK1 were up-regulated and miR-206 was down-regulated, meanwhile, the autophagy-related protein Beclin1 and LC3-Ⅱ/LC3-Ⅰ ratio were increased. Interference with CYTOR or overexpression with miR-206 downregulated the autophagy-related protein Beclin1 and the number of autophagic spots LC3, decreased the protein ratio of LC3-II/LC3-I, and upregulated the expression of P62 protein. The luciferase reporter assay confirmed that CYTOR acted as a sponge for miR-206 to target MAPK1. In addition, CYTOR promoted autophagy induced by C. trachomatis infection through the MAPK1/ERK signaling pathway activation. Taken together, we have identified a novel molecular mechanism that the CYTOR/miR-206/MAPK1 axis was involved in the regulation of autophagy in C. trachomatis infection. This work provides an experimental basis for elucidating the pathogenesis of C. trachomatis for the treatment, prevention and control of related infectious diseases.

Jaceosidin induces apoptosis and inhibits migration in AGS gastric cancer cells by regulating ROS-mediated signaling pathways

ABSTRACT Jaceosidin (JAC) is a natural flavonoid with anti-oxidant and other pharmacological activities; however, its anti-cancer mechanism remains unclear. We investigated the mechanism of action of JAC in gastric cancer cells. Cytotoxicity and apoptosis assays showed that JAC effectively killed multiple gastric cancer cells and induced apoptosis in human gastric adenocarcinoma AGS cells via the mitochondrial pathway. Network pharmacological analysis suggested that its activity was linked to reactive oxygen species (ROS), AKT, and MAPK signaling pathways. Furthermore, JAC accumulated ROS to up-regulate p-JNK, p-p38, and IκB-α protein expressions and down-regulate the p-ERK, p-STAT3, and NF-κB protein expressions. Cell cycle assay results showed that JAC accumulated ROS to up-regulate p21 and p27 protein expressions and down-regulate p-AKT, CDK2, CDK4, CDK6, Cyclin D1, and Cyclin E protein expressions to induce G0/G1 phase arrest. Cell migration assay results showed JAC accumulated ROS to down-regulate Wnt-3a, p-GSK-3β, N-cadherin, and β-catenin protein expressions and up-regulate E-cadherin protein expression to inhibit migration. Furthermore, N-acetyl cysteine pre-treatment prevented the change of these protein expressions. In summary, JAC induced apoptosis and G0/G1 phase arrest and inhibited migration through ROS-mediated signaling pathways in AGS cells.

TRPM2 knockdown attenuates myocardial apoptosis and promotes autophagy in HFD/STZ-induced diabetic mice via regulating the MEK/ERK and mTORC1 signaling pathway

Diabetic cardiomyopathy (DCM) is a major complication of diabetes. Transient receptor potential melastatin 2 (TRPM2) activity increases in diabetic oxidative stress state, and it is involved in myocardial damage and repair. We explore the protective effect of TRPM2 knockdown on the progression of DCM. A type 2 diabetes animal model was established in C57BL/6N mice by long-term high-fat diet (HFD) feeding combined with a single injection of 100-mg/kg streptozotocin (STZ). Genetic knockdown of TRPM2 in heart was accomplished by the intravenous injection via the tail vein of adeno-associated virus type 9 carrying TRPM2 shRNA. Neonatal rat ventricular myocytes was exposed to 45 mM of high-glucose (HG) stimulation for 72 h in vitro to mimic the in vivo conditions. Western blot, real-time quantitative PCR (RT-qPCR), immunohistochemistry and fluorescence, electron, CCK-8, and flow cytometry were used to evaluate the phenotype of cardiac inflammation, fibrosis, apoptosis, and autophagy. Mice with HFD/STZ-induced diabetes exhibited systolic and diastolic dysfunction, as demonstrated by increased myocardial apoptosis and autophagy inhibition in the heart. Compared to control group, the protein expression of TRPM2, bax, cleaved caspase-3, and P62 was significantly elevated, and the protein expression of bcl-2 and LC3-II was significantly decreased in the myocardial tissues of the HFD/STZ-induced diabetes group. Knockdown of TRPM2 significantly reversed the HFD/STZ-induced myocardial apoptosis and autophagy inhibition. TRPM2 silencing attenuated HG-induced apoptosis and autophagy inhibition in primary cardiomyocytes via regulating the MEK/ERK mTORC1 signaling pathway. TRPM2 knockdown attenuates hyperglycemia-induced myocardial apoptosis and promotes autophagy in HFD/STZ-induced diabetic mice or HG-stimulated cardiomyocytes via regulating the MEK/ERK and mTORC1 signaling pathway.

Exercise ameliorates lipid droplet metabolism disorder by the PLIN2–LIPA axis-mediated lipophagy in mouse model of non-alcoholic fatty liver disease

Excessive hepatic lipid droplets (LDs) accumulation-induced lipid metabolism disorder contributes to the development of non-alcoholic fatty liver disease (NAFLD). Exercise is a promising therapeutic strategy for NAFLD. However, the mechanism by which exercise ameliorates NAFLD through regulating the catabolism of hepatic LDs remains unclear. In the present study, we investigated the effect of perilipin2 (PLIN2)–lysosomal acid lipase (LIPA) axis mediating exercise-triggered lipophagy in a high-fat diet (HFD)-induced NAFLD mouse model. Our results showed that exercise could reduce HFD-induced hepatic LDs accumulation and change the expression of lipolysis-related enzymes. Moreover, exercise upregulated the expression of microtubule associated protein 1 light chain 3 (LC3) and autophagy-related proteins, and downregulated sequestosome 1 (P62) expression and promoted autophagosomes formation. Interestingly, exercise downregulated PLIN2 expression, upregulated LIPA expression, and increased the activity of hepatic LIPA and serum levels of LIPA in the NAFLD mouse model. Further mechanistic studies demonstrated that adenosine monophosphate-activated protein kinase (AMPK) activator-5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr) treatment significantly increased mRNA levels and protein expression of LIPA and LC3II and decreased levels of PLIN2 and P62 in palmitic acid (PA)-treated HepG2 cells. PLIN2 silencing and LIPA overexpression notably increased the mRNA level and protein expression of LC3II and decreased the mRNA level and protein expression of p62, respectively. In summary, our findings reveal novel insights into the effect of exercise on improving lipid droplet metabolism disorder in NAFLD. Enhancing the PLIN2–LIPA axis-mediated lipophagy may be one of the key mechanisms involved in NAFLD alleviation by exercise.

Coated sodium butyrate ameliorates high-energy and low-protein diet induced hepatic dysfunction via modulating mitochondrial dynamics, autophagy and apoptosis in laying hens

BackgroundFatty liver hemorrhagic syndrome (FLHS), a fatty liver disease in laying hens, poses a grave threat to the layer industry, stemming from its ability to trigger an alarming plummet in egg production and usher in acute mortality among laying hens. Increasing evidence suggests that the onset and progression of fatty liver was closely related to mitochondria dysfunction. Sodium butyrate was demonstrated to modulate hepatic lipid metabolism, alleviate oxidative stress and improve mitochondrial dysfunction in vitro and mice models. Nevertheless, there is limited existing research on coated sodium butyrate (CSB) to prevent FLHS in laying hens, and whether and how CSB exerts the anti-FLHS effect still needs to be explored. In this experiment, the FLHS model was induced by administering a high-energy low-protein (HELP) diet in laying hens. The objective was to investigate the effects of CSB on alleviating FLHS with a focus on the role of CSB in modulating mitochondrial function.MethodsA total of 288 healthy 28-week-old Huafeng laying hens were arbitrarily allocated into 4 groups with 6 replicates each, namely, the CON group (normal diet), HELP group (HELP diet), CH500 group (500 mg/kg CSB added to HELP diet) and CH750 group (750 mg/kg CSB added to HELP diet). The duration of the trial encompassed a period of 10 weeks.ResultsThe result revealed that CSB ameliorated the HELP-induced FLHS by improving hepatic steatosis and pathological damage, reducing the gene levels of fatty acid synthesis, and promoting the mRNA levels of key enzymes of fatty acid catabolism. CSB reduced oxidative stress induced by the HELP diet, upregulated the activity of GSH-Px and SOD, and decreased the content of MDA and ROS. CSB also mitigated the HELP diet-induced inflammatory response by blocking TNF-α, IL-1β, and F4/80. In addition, dietary CSB supplementation attenuated HELP-induced activation of the mitochondrial unfolded protein response (UPRmt), mitochondrial damage, and decline of ATPase activity. HELP diet decreased the autophagosome formation, and downregulated LC3B but upregulated p62 protein expression, which CSB administration reversed. CSB reduced HELP-induced apoptosis, as indicated by decreases in the Bax/Bcl-2, Caspase-9, Caspase-3, and Cyt C expression levels.ConclusionsDietary CSB could ameliorate HELP diet-induced hepatic dysfunction via modulating mitochondrial dynamics, autophagy, and apoptosis in laying hens. Consequently, CSB, as a feed additive, exhibited the capacity to prevent FLHS by modulating autophagy and lipid metabolism.Graphical

Inhibition of p62-Keap1-Nrf2 Pathway Activation by Realgar Promotes the Inhibition of Esophageal Cancer Cell Proliferation, Migration, and Ferroptosis.

Realgar, a Chinese herbal decoction, has been used to treat various types of tumors with positive outcomes; however, there is a lack of convincing evidence on its use for the treatment of esophageal cancer (EC). In this study, the role of the p62-Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in the regulation of EC cell proliferation, migration, and ferroptosis in response to realgar was assessed. Different concentrations of realgar (0, 10, 20, 40, 60, 80, and 100 μmol/L) were applied to the EC cell lines Eca109 and KYSE150. The inhibition rate and half-inhibitory concentration (IC50) were determined using the Cell Counting Kit-8 (CCK-8) method. Subsequently, the cells were treated with realgar (1/2IC50, IC50, 2IC50). Cell migration was measured using the scratch assay, and cell invasion was measured using the transwell assay. The mRNA expression of p62, Keap1, and Nrf2 was measured by quantitative real-time polymerase chain reaction (qRT-PCR), and the protein expression of p62, Keap1, Nrf2, matrix metalloproteinase (MMP)-2, MMP-9, E-cadherin, Slug, N-cadherin, and vimentin was measured by Western blot. The control, 2IC50, shRNA-NC, shRNA-p62, 2IC50 + shRNA-NC, 2IC50 + shRNA-p62, shRNA-Keap1, 2IC50 + shRNA-Keap1, and 2IC50 + shRNA-p62 + shRNA-Keap1 groups were defined. The CCK-8 method was used to measure the cell inhibition rate, and the clone formation assay was used to measure the clone formation ability. Moreover, the scratch assay was used to detect the cell migration ability, and the transwell assay was used to detect the cell invasion ability. Transmission electron microscopy was used to observe the mitochondrial morphology, Prussian blue staining was used to observe the intracellular iron particle distribution, and flow cytometry was used to detect changes in intracellular reactive oxygen species. In addition, qRT-PCR was performed to detect p62, Keap1, Nrf2, and glutathione peroxidase 4 (GPX4) mRNA expression, and Western blot was performed to detect p62, Keap1, Nrf2, E-cadherin, Slug, N-cadherin, and GPX4 protein expression. Realgar inhibited Eca109 and KYSE150 cell proliferation in a time- and concentrationdependent manner. It also significantly inhibited the migration and invasion of Eca109 and KYSE150 cells and affected the mRNA and protein expression of p62, Keap1, and Nrf2. In response to realgar, low p62 expression inhibited the proliferation, migration, and invasion of Eca109 and KYSE150 cells, as well as ferroptosis induction. The findings demonstrate that inhibiting the p62-Keap1-Nrf2 signaling pathway promotes the inhibitory effects of realgar on EC cells.

Inhibitory Effect of Metformin and Arsenic Trioxide on KG1a Cell Proliferation

To investigate the effect of metformin and arsenic trioxide on KG1a cells proliferation of acute myeloid leukemia and its possible mechanism. CCK-8 method was used to detect the killing effect of metformin, arsenic trioxide and combined application on KG1a cells. Annexin V-FITC/PI Dual Stain Flow Cytometry was used to detect the effect of combined application on apoptosis of KG1a cells. Western blot was used to detect the expression of intracellular apoptosis-,autophagy-related protein. Metformin and arsenic trioxide alone or in combination could inhibit the proliferation of KG1a cells and induce apoptosis of KG1a cells, and the proliferation inhibition rate and apoptosis rate in the combined drug group were higher than those in the drug group alone(P <0.05). The combination of drugs induced upregulation of Caspase 8 protein and P62 protein expression and was higher than that in the drug group alone(P <0.05). Metformin can synergize with arsenic trioxide to kill KG1a cells, and its mechanism of action may be related to inducing apoptosis and enhancing autophagy.

Taohong Siwu decoction ameliorates atherosclerosis in rats possibly through toll-like receptor 4/myeloid differentiation primary response protein 88/nuclear factor-κB signal pathway.

To investigate the effect of Taohong Siwu decoction (, TSD) on atherosclerosis in rats as well as investigate the underlying mechanism based on molecular docking. Sixty healthy male Sprague-Dawley rats were randomly divided into 6 groups with 10 rats in each group: control group, model group, atorvastatin group (AT, 2.0 mg/kg), and TSD groups (20, 10, 5 g/kg) after 7 d of acclimation. The model of atherosclerosis was successfully established except the control group by high fat diet (HFD) and vitamin D2. Biochemical analyzers were used to detect the levels of triglyceride (TG), total cholestero (TC), low density lipoprotein-cholesterol (LDL-C) and high density lipid-cholesterol (HDL-C) in blood lipid. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were determined by enzyme-linked immunosorbent assay. Sudan IV staining and Hematoxylin and eosin staining (HE staining) were performed to observe the pathological changes in aortic tissue. Molecular docking technology was used to predict the best matching between the main components of TSD and the target proteins. The expression of target proteins was further detected by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot analysis. The results showed that TSD restricted atherosclerosis development and decreased the inflammatory cytokines in plasma. Molecular docking results predicted that the main components of TSD showed a strong binding ability with toll-like receptor (TLR4), myeloid differentiation primary response protein 88 (MyD88), and nuclear factor kappa-B (NF-κB). The results of qRT-PCR and Western blot analysis showed that the mRNA and protein expressions of TLR4, MyD88 and NF-κB p65 in the aorta were reduced in atorvastatin group and TSD group. TSD can ameliorate atherosclerosis in rats, and the underlying mechanism is supposed be related to the suppression of inflammatory response by regulating TLR4/MyD88/NF-κB signal pathway.

Exploring the Action Mechanism and Validation of the Key Pathways of Dendrobium officinale Throat-clearing Formula for the Treatment of Chronic Pharyngitis Based on Network Pharmacology.

This study investigated the molecular action mechanism of a compound herb, also known as the Dendrobium officinale throat-clearing formula (QYF), by using network pharmacology and animal experimental validation methods to treat chronic pharyngitis (CP). The active ingredients and disease targets of QYF were determined by searching the Batman-TCM and GeneCards databases. Subsequently, the drug-active ingredient-target and protein-protein interaction networks were constructed, and the core targets were obtained through network topology. The Metascape database was screened, and the core targets were enriched with Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. In total, 1403 and 241 potential targets for drugs and diseases, respectively, and 81 intersecting targets were yielded. The core targets included TNF, IL-6, and IL-1β, and the core pathways included PI3K-Akt. The QYF treatment group exhibited effectively improved general signs, enhanced anti-inflammatory ability in vitro, reduced serum and tissue expressions of TNF- α, IL-6, and IL-1β inflammatory factors, and decreased blood LPS levels and Myd88, TLR4, PI3K, Akt, and NF-κB p65 protein expression in the tissues. QYF could inhibit LPS production, which regulated the expression of the TLR4/PI3K/Akt/NF-κB signaling pathway to suppress the expression of the related inflammatory factors (i.e., TNF-α, IL-6, and IL-1β), thereby alleviating the CP process.