Phosphorylation Levels Of P38 MAPK Research Articles

Novel trifluoromethyl ketone derivatives as oral cPLA2/COX-2 dual inhibitors for resolution of inflammation in rheumatoid arthritis

Thirty-five trifluoromethyl hydrazones and seventeen trifluoromethyl oxime esters were designed and synthesized via molecular hybridization. All the target compounds were initially screened for in vitro anti-inflammatory activity by assessing their inhibitory effect on NO release in LPS-stimulated RAW264.7 cells, and the optimal compound was finally identified as 2-(3-Methoxyphenyl)-N'-((6Z,9Z,12Z,15Z)-1,1,1-trifluorohenicosa-6,9,12,15-tetraen-2-ylidene)acetohydrazide (F26, IC50 = 4.55 ± 0.92 μM) with no cytotoxicity. Moreover, F26 potently reduced the production of PGE2 in LPS-stimulated RAW264.7 cells compared to indomethacin. The interaction of F26 with COX-2 and cPLA2 was directly verified by the CETSA technique. F26 was found to modulate the phosphorylation levels of p38 MAPK and NF-κB p65, as well as the protein expression of IκB, cPLA2, COX-2, and iNOS in LPS-stimulated rat peritoneal macrophages. Additionally, F26 was observed to prevent the nuclear translocation of NF-κB p65 in LPS-stimulated rat peritoneal macrophages by immunofluorescence localization. Therefore, the aforementioned in vitro experiments demonstrated that F26 blocked the p38 MAPK and NF-κB pathways by binding to COX-2 and cPLA2. In the adjuvant-induced arthritis model, F26 demonstrated a significant effect in preventing arthritis symptoms and inflammatory status in rats, exerting an immunomodulatory role by regulating the homeostasis between Th17 and Treg through inhibition of the p38 MAPK/cPLA2/COX-2/PGE2 and NF-κB pathways. Encouragingly, F26 caused less acute ulcerogenicity in rats at a dose of 50 mg/kg compared to indomethacin. Overall, F26 is a promising candidate worthy of further investigation for treating inflammation and associated pain with lesser gastrointestinal irritation, as well as other symptoms in which cPLA2 and COX-2 are implicated in the pathophysiology.

Demethylzeylasteral (T-96) Alleviates Allergic Asthma via Inhibiting MAPK/ERK and NF-κB Pathway

Introduction: Demethylzeylasteral (T-96), a new extract of Tripterygium wilfordii Hook F, exerted immunomodulatory properties in autoimmune diseases, but its effect on airway inflammatory diseases remains unclear. Our study aims to explore the protective effect and underlying mechanism of T-96 in allergic asthma. Methods: The OVA-induced asthmatic mice were administered by gavage with T-96 (0.1 mg/10 g, 0.3 mg/10 g, or 0.6 mg/10 g) 1 h before each challenge. The airway hyperresponsiveness was assessed, pathological changes were evaluated by HE and PAS staining, and expressions of Th2 cytokines were determined by PCR and ELISA. The activation of MAPK/ERK and NF-κB pathway was assessed by western blot. Results: T-96 significantly relieved airway hyperresponsiveness in asthmatic mice, evidenced by reduced airway resistance (Raw) and increased lung compliance dynamic compliance (Cdyn). Also, enhanced inflammatory infiltration and mucus hypersecretion were ameliorated in lungs of asthmatic mice following increasing doses of T-96 treatment, accompanied by decreased eosinophils in bronchoalveolar lavage fluid (BALF), IgE and OVA-specific IgE levels in serum, and downregulated IL-5 and IL-13 expressions in BALF and lung tissues as well. Notably, phosphorylation levels of p38 MAPK, ERK, and p65 NF-κB were obviously increased in asthmatic mice compared with the control group, which were then abrogated upon T-96 treatment. Conclusion: This study first revealed that T-96 alleviated allergic airway inflammation and airway hyperresponsiveness via inhibiting MAPK/ERK and NF-κB pathway. Thus, T-96 could potentially act as a new anti-inflammatory agent in allergic asthma.

Vitamin A regulates mitochondrial biogenesis and function through p38 MAPK-PGC-1α signaling pathway and alters the muscle fiber composition of sheep

BackgroundVitamin A (VA) and its metabolite, retinoic acid (RA), are of great interest for their wide range of physiological functions. However, the regulatory contribution of VA to mitochondrial and muscle fiber composition in sheep has not been reported.MethodLambs were injected with 0 (control) or 7,500 IU VA palmitate into the biceps femoris muscle on d 2 after birth. At the age of 3 and 32 weeks, longissimus dorsi (LD) muscle samples were obtained to explore the effect of VA on myofiber type composition. In vitro, we investigated the effects of RA on myofiber type composition and intrinsic mechanisms.ResultsThe proportion of type I myofiber was greatly increased in VA-treated sheep in LD muscle at harvest. VA greatly promoted mitochondrial biogenesis and function in LD muscle of sheep. Further exploration revealed that VA elevated PGC-1α mRNA and protein contents, and enhanced the level of p38 MAPK phosphorylation in LD muscle of sheep. In addition, the number of type I myofibers with RA treatment was significantly increased, and type IIx myofibers was significantly decreased in primary myoblasts. Consistent with in vivo experiment, RA significantly improved mitochondrial biogenesis and function in primary myoblasts of sheep. We then used si-PGC-1α to inhibit PGC-1α expression and found that si-PGC-1α significantly abrogated RA-induced the formation of type I myofibers, mitochondrial biogenesis, MitoTracker staining intensity, UQCRC1 and ATP5A1 expression, SDH activity, and enhanced the level of type IIx muscle fibers. These data suggested that RA improved mitochondrial biogenesis and function by promoting PGC-1α expression, and increased type I myofibers. In order to prove that the effect of RA on the level of PGC-1α is caused by p38 MAPK signaling, we inhibited the p38 MAPK signaling using a p38 MAPK inhibitor, which significantly reduced RA-induced PGC-1α and MyHC I levels.ConclusionVA promoted PGC-1α expression through the p38 MAPK signaling pathway, improved mitochondrial biogenesis, and altered the composition of muscle fiber type.Graphical

CCN3/NOV inhibition attenuates oxidative stress-induced apoptosis of mouse neural stem/progenitor cells by blocking the activation of p38 MAPK: An in vitro study

Neural stem/progenitor cells (NSPCs) hold immense promise in clinical applications, yet the harsh conditions resulting from central nervous system (CNS) injuries, particularly oxidative stress, lead to the demise of both native and transplanted NSPCs. Cellular communication network factor 3 (CCN3) exhibits a protective effect against oxidative stress in various cell types. This study investigates the impact of CCN3 on NSPCs apoptosis induced by oxidative stress. To establish models of primary cultured mouse NSPCs under oxidative stress, we exposed them to 50 μM H2O2 for 4 h. Remarkably, pre-exposing CCN3 exacerbated the H2O2-induced decline in cell viability in a concentration-dependent manner. However, employing gene-targeted siRNA to inhibit CCN3 protected NSPCs against H2O2-induced cell death. Conversely, CCN3 replenishment reversed this protective effect, as evidenced by TUNEL staining, the ratio of Cleaved-caspase-3 to Pro-caspase-3, and Bcl-2/Bax. Further investigations revealed that CCN3 pretreatment increased the phosphorylation level of p38 MAPK, while silencing CCN3 diminished p38 MAPK activation. Ultimately, the impact of changes in CCN3 protein expression on H2O2-induced apoptosis was nullified using anisomycin (a p38 activator) and SB 203580 (a p38 inhibitor). Our findings suggest that CCN3 inhibition prevents H2O2-induced cell death in cultured mouse NSPCs via the p38 pathway. These discoveries may contribute to the development of strategies aimed at enhancing the survival of both endogenous and transplanted NSPCs following CNS oxidative stress insults.

Scutellarin alleviates tensile stress-induced proliferation and migration of venous smooth muscle cells via mediating the p38 MAPK pathway

ObjectiveAbnormal proliferation and migration of biomechanical force-induced venous smooth muscle cells (VSMCs) is a major cause to limit the efficacy of coronary artery bypass grafting (CABG) for coronary heart disease (CHD). Scutellarin is the main active ingredient of Erigeron Breviscapus, and has broad-spectrum pharmacological effects. Therefore, the present study was proposed to investigate the effect of Scutellarin on VSMCs under tensile stress. MethodsAfter interfering with VSMCs at different tensile stresses, the optimal tensile stress was screened. In a tensile stress environment, 100 μM Scutellarin and Hesperetin (p38 MAPK pathway activator) was used to treatment with VSMCs. CCK-8, EDU, Wound healing, flow cytometry and western blotting assays were used to detect cell proliferation, migration, apoptosis, and the expression of apoptosis-related proteins (Caspase3, Bcl2 and Bax). ResultsTensile stress with 10% significantly enhanced the activity, wound-healing ratio, and EDU+ cells of VSMCs, and decreased their apoptosis ratio. Moreover, it upregulated Bcl2 expression, and downregulated cleaved-Caspase3 and Bax expression of VSMCs. Hence, 10% tensile stress was selected to creates a tensile stress environment for VSMCs. Interestingly, 100 μM Scutellarin alleviated the effect of 10% tensile stress on the phenotype of VSMCs. Notably, 10% tensile stress increased the phosphorylation level of p38 MAPK (Thr180 +Tyr182) in VSMCs, which was restricted by Scutellarin. Further, Hesperetin restored the effect of Scutellarin on the phenotype of VSMCs. ConclusionScutellarin alleviates tension stress-induced proliferation and migration of VSMCs via suppressing p38 MAPK pathway. Scutellarin may be used as an adjunctive strategy for future GABG treatment in CHD patients.

Dihydrotanshinone I reduces H9c2 cell damage by regulating AKT and MAPK signaling pathways.

Cardiovascular disease is the deadliest disease in the world. Previous studies have shown that Dihydrotanshinone I (DHT) can improve cardiac function after myocardial injury. This study aimed to observe the protective effect and mechanism of DHT on H9c2 cells by establishing an oxygen-glucose deprivation/reoxygenation (OGD/R) injury model. By constructing OGD/R injury simulation of H9c2 cells in a myocardial injury model, the proliferation of H9c2 cells treated with DHT concentrations of 0.1μmol/L were not affected at 24, 48, and 72h. DHT can significantly reduce the apoptosis of H9c2 cells caused by OGD/R. Compared with the OGD/R group, DHT treatment significantly reduced the level of MDA and increased the level of SOD in cells. DHT treatment of cells can significantly reduce the levels of ROS and Superoxide in mitochondria in H9c2 cells caused by OGD/R and H2O2. DHT significantly reduced the phosphorylation levels of P38MAPK and ERK in H9c2 cells induced by OGD/R, and significantly increased the phosphorylation levels of AKT in H9c2 cells. DHT can significantly reduce the oxidative stress damage of H9c2 cells caused by H2O2 and OGD/R, thereby reducing the apoptosis of H9c2 cells. And this may be related to regulating the phosphorylation levels of AKT, ERK, and P38MAPK.

Thyroid hormone induces restrictive cardiomyopathy in β1-adrenoceptor knockout mice.

The purpose of this study was to characterize the role of β1-AR signaling and its cross-talk between cardiac renin-angiotensin system and thyroid-hormone-induced cardiac hypertrophy. T3 was administered at 0.5 mg·kg-1·day-1 for 10 days in β1-KOT3 and WTT3 groups, while control groups received vehicle alone. Echocardiography and myocardial histology was performed; cardiac and serum ANGI/ANGII and ANP and cardiac levels of p-PKA, p-ERK1/2, p-p38-MAPK, p-AKT, p-4EBP1, and ACE were measured. WTT3 showed decreased IVSTd and increased LVEDD versus WTsal (p < 0.05). β1-KOT3 exhibited lower LVEDD and higher relative IVSTd versus β1-KOsal, the lowest levels of ejection fraction, and the highest levels of cardiomyocyte diameter (p < 0.05). Cardiac ANP levels decreased in WTT3 versus β1-KOT3 (p < 0.05). Cardiac ACE expression was increased in T3-treated groups (p < 0.05). Phosphorylated-p38 MAPK levels were higher in WTT3 versus WTsal or β1-KOT3, p-4EBP1 was elevated in β1-KO animals, and p-ERK1/2 was up-regulated in β1-KOT3. These findings suggest that β1-AR signaling is crucial for TiCH.

P38 MAPK involvement in the thermal stress response occurs via HSP27 and caspase3 in the large yellow croaker (Larimichthys crocea)

The p38 mitogen-activated protein kinase (p38 MAPK) is a multifunctional molecule that is involved in cellular response to various stressful stimuli. In the present study, the full-length cDNA sequence of p38 MAPK (Lcp38 MAPK) was identified from the large yellow croaker Larimichthys crocea, which encoded a polypeptide of 361 amino acid residues. The predicted Lcp38 MAPK protein contained a highly conserved Thr-Gly-Tyr (TGY) motif, a glutamate and aspartate (ED) site, a substrate binding site (Ala-Thr-Arg-Trp < ATRW>), and a serine/threonine kinase catalytic (S_TKc) domain characteristic of the MAPK family. The constitutive expression of Lcp38 MAPK was detected in most of the tissues examined with the strongest expression in intestine. Subcellular localization in LCK cells (kidney cell line from a L. crocea) revealed that Lcp38 MAPK existed in both the cytoplasm and cell nucleus. The expression of Lcp38 MAPK after temperature stress was tested in LCK cells. The results indicated that Lcp38 MAPK transcripts were significantly upregulated under both cold (10 °C) and heat stress (35 °C) (P < 0.05). Furthermore, the phosphorylation levels of p38 MAPK as well the transcriptional levels of heat shock protein 27 (HSP27) and caspase3 in LCK cells were significantly induced under thermal exposure (P < 0.05). However, the cold- and heat induced HSP27 and caspase3 expression was significantly suppressed by SB203580, a specific inhibitor of p38-MAPK (P < 0.05). These findings indicated that Lcp38 MAPK might be involved in the cellular stress response via HSP27 and caspase3 in large yellow croaker.

Regulation of microglia-mediated inflammation by host lncRNA Gm20559 upon flaviviral infection

BackgroundJapanese Encephalitis Virus (JEV) and West Nile Viruses (WNV) are neurotropic flaviviruses which cause neuronal death and exaggerated glial activation in the central nervous system. Role of host long non coding RNAs in shaping microglial inflammation upon flavivirus infections has been unexplored. This study attempted to decipher the role of lncRNA Gm20559 in regulating microglial inflammatory response in context of flaviviruses. MethodsAntisense oligonucleotide LNA Gapmers designed against lncRNA Gm20559 and non-specific site (negative control) were used for Gm20559 knockdown in JEV and WNV-infected N9 microglial cells. Upon establishing successful Gm20559 knockdown, expression of various proinflammatory cytokines, chemokines, interferon-stimulated genes (ISGs) and RIG-I were checked by qRT-PCR and cytometric bead array. Western Blotting was done to analyse the phosphorylation level of various inflammatory markers and viral non-structural protein expression. Plaque Assays were employed to quantify viral titres in microglial supernatant upon knocking down Gm20559. Effect of microglial supernatant on HT22 neuronal cells was assessed by checking expression of apoptotic protein and viral non-structural protein by Western Blotting. ResultsUpregulation in Gm20559 expression was observed in BALB/c pup brains, primary microglia as well as N9 microglia cell line upon both JEV and WNV infection. Knockdown of Gm20559 in JEV and WNV-infected N9 cell led to the reduction of major proinflammatory cytokines – IL-1β, IL-6, IP-10 and IFN-β. Inhibition of Gm20559 upon JEV infection in N9 microglia also led to downregulation of RIG-I and OAS-2, which was not the case in WNV-infected N9 microglia. Phosphorylation level of P38 MAPK was reduced in case of JEV-infected N9 microglia and not WNV-infected N9 microglia. Whereas phosphorylation of NF-κB pathway was unchanged upon Gm20559 knockdown in both JEV and WNV-infected N9 microglia. However, treating HT22 cells with JEV and WNV-infected microglial supernatant with and without Gm20559 could not trigger cell death or influence viral replication. ConclusionKnockdown studies on lncRNA Gm20559 suggests its pivotal role in maintaining the inflammatory milieu of microglia in flaviviral infection by modulating the expression of various pro-inflammatory cytokines. However, Gm20559-induced increased microglial proinflammatory response upon flavivirus infection fails to trigger neuronal death.

Osthole protects H9c2 cardiomyocytes against trastuzumab-induced damage by enhancing autophagy through the p38MAPK/mTOR signaling pathway

Trastuzumab (TRZ) is a novel targeted anti-tumor agent that significantly improve the survival of patients with human epidermal growth factor receptor (HER2) positive breast cancer. However, its clinical application is limited due to the side effects of cardiotoxicity. Osthole (OST), a coumarin derivative isolated from Cnidium monnieri (L.) Cusson, has previously demonstrated cardioprotective effects. The aim of this study was to observe the protective effect of OST on TRZ-induced cardiomyocytes damage and to explore its potential mechanism. The results showed that OST pretreatment could significantly inhibit TRZ-induced cardiomyocytes damage, markedly increase the ratio of LC3II/I and Beclin-1 protein expression, and reduce the protein expression of p62. OST pretreatment significantly attenuated oxidative stress and apoptosis induced by TRZ, as evidenced by reducing intracellular ROS level, the Bax/Bcl-2 ratio, and Caspase-3 protein expression. Additionally, OST markedly increased the phosphorylation level of p38MAPK and decreased the mTOR phosphorylation level. However, the effects of OST on enhancing autophagy, reducing oxidative stress, apoptosis, and the phosphorylation level of mTOR were reversed after the addition of 3-MA or SB203580. Molecular docking results indicated that OST exerted a good binding ability with the p38MAPK protein. Our findings suggested that OST could protect TRZ-induced cardiomyocytes damage by enhancing autophagy via the p38MAPK/mTOR signaling pathway.

Renalase regulates renal tubular injury in diabetic nephropathy via the p38MAPK signaling pathway.

Diabetic nephropathy (DN) is an important complication of diabetes and the leading cause of end-stage renal disease globally. Renal tubular damage occurs to varying degrees in the early stages of DN prior to glomerular damage. Renalase (RNLS) is an amine oxidase, which is produced and secreted by the renal tubular epithelial cells. RNLS is reportedly closely related to renal tubular injury in acute and chronic kidney diseases. Herein, we aimed to evaluate the changes in tubular RNLS expression in DN and its correlation with DN-associated renal tubular injury. Conditional permanent renal tubular epithelial rat-cell line NRK-52E was transfected with pcDNA3-RNLS plasmid or administered recombinant rat RNLS protein and high glucose (HG) dose. A total of 22 adult Sprague-Dawley rats were randomly divided into the control (CON, n = 10) or diabetic nephrology (DN, n = 12) group. Random blood glucose levels of the rats were measured by sampling of the caudal vein weekly. After 8 weeks, the rat's body weight, 24-h urinary albumin concentration, and right kidney were evaluated. Our study suggested the decreased expression levels of RNLS in renal tissue and renal tubular epithelial cells in DN rats, accompanied by renal tubulointerstitial fibrosis, apoptosis of renal tubular epithelial cells, and activation of the p38MAPK signal pathway. Reversing the low RNLS expression can reduce the level of p38MAPK phosphorylation and delay renal tubular injury. Thus, the reduction of renal tubular RNLS expression in DN mediates tubulointerstitial fibrosis and cell apoptosis via the activation of the p38MAPK signal pathway. RNLS plays a key mediating role in DN-associated tubular injury via p38MAPK, which provides new therapeutic targets and a theoretical basis for early prevention and treatment of DN.

Positive Regulation of Acetate in Adipocyte Differentiation and Lipid Deposition in Obese Mice.

Acetate is associated with adipocyte differentiation and lipid deposition. To further develop this scientific point, obese mice on a high-fat diet were given an intragastric administration of acetate for 8 weeks and mouse adipose mesenchymal stem cells (mAMSCs) were treated with acetate for 24 h. The results showed that the body weight, food intake, Lee's index, adipose tissue coefficient, liver index, blood lipid levels, insulin resistance, pro-inflammatory factors levels and fatty lesions in liver and adipose tissue in obese mice treated with acetate increased markedly, while anti-inflammatory factors levels and liver function decreased significantly (p < 0.05). The mRNA expression levels of PPAR-γ, C/EBP-α, SREBP, AFABP, FAS, ACC-1, SCD-1, LPL, LEPR, GPR41 and GPR43 genes in adipose tissue and mAMSCs were significantly increased, while the mRNA expression levels of HSL, CPT-1, CPT-2, AMPK, AdipoR1 and AdipoR2 genes were significantly reduced (p < 0.05). Except for AMPK-α signaling pathway proteins, the phosphorylation levels of p38 MAPK, ERK1/2, JNK and mTOR were significantly increased (p < 0.05) and these changes were dose-dependent. The findings indicated that acetate played a positive role in regulating adipocyte differentiation and lipid deposition by activating MAPKs and mTOR signaling pathways (the expression up-regulation of genes such as PPAR-γ, C/EBP-α and SREBP-1, etc.) and inhibiting the AMPK signaling pathway (the expression down-regulation of genes such as HSL, CPT-1 and AMPK-α, etc.).

Synthesis and neuroprotective effects of new genipin derivatives against glutamate-induced oxidative damage

Glutamate-induced oxidative stress is well-known to play a crucial role in the development of neurodegenerative diseases, such as stroke. Genipin, a natural iridoid compound, has demonstrated potential neuroprotective properties but is unstable in physiological conditions. The present study aimed to develop new derivatives of genipin that exhibit improved stability and activity for the treatment of stroke. Nineteen new derivatives were thus designed and synthesized. Their neuroprotective effect against glutamate-induced injury was evaluated in HT22 cells. Among the newly synthesized derivatives, 3e demonstrated significantly greater neuroprotection and improved stability compared to genipin. Specifically, 0.01 μM of 3e was found to effectively attenuate glutamate-induced oxidative damage by inhibiting ROS over-accumulation, reducing MDA content, and restoring the endogenous antioxidative system. Further investigation revealed that 3e inhibited oxidative stress by downregulating the phosphorylation levels of p38 MAPK and activating Nrf2 and HO-1 proteins. These results suggested that 3e has the potential to serve as a promising candidate for the treatment of stroke by protecting against glutamate-induced oxidative stress.

Network pharmacology and transcriptomics to determine Danggui Yifei Decoction mechanism of action for the treatment of chronic lung injury

Ethnopharmacological relevanceSeveral children with pneumonia (especially severe cases) have symptoms of cough and expectoration during the recovery stage after standard symptomatic treatment, which eventually results in chronic lung injury. Danggui yifei Decoction (DGYFD), a traditional Chinese formula, has shown clinical promise for the treatment of chronic lung injury during the recovery stage of pneumonia, however, its mechanism of action is yet to be deciphered. Aim of this studyTo investigate the therapeutic mechanism of DGYFD for the treatment of chronic lung injury by integrating network pharmacology and transcriptomics. Materials and methodsBALB/c mice were used to establish the chronic lung injury mouse model by intratracheal instillation of lipopolysaccharide (LPS). Pathological analysis of lung tissue, lung injury histological score, lung index, protein levels in bronchoalveolar lavage fluid (BALF), immunohistochemical staining, blood rheology, inflammatory cytokines, and oxidative stress levels were used to evaluate the pharmacological effects of DGYFD. Chemical components of DGYFD were identified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Integrated network pharmacology together with transcriptomics was used to predict potential biological targets. Western blot analysis was used to verify the results. ResultsIn this study, we demonstrated that DGYFD could improve lung injury pathological changes, decreases lung index, down-regulate NO and IL-6 levels, and regulate blood rheology. In addition, DGYFD was able to reduce the protein levels in BALF, up-regulate the expression levels of occludin and ZO-1, improve the ultrastructure of lung tissues, and reverse the imbalance of AT I and AT II cells to repair the alveolar-capillary permeability barrier. Twenty-nine active ingredients of DGYFD and 389 potential targets were identified by UPLC-MS/MS and network pharmacology, and 64 differentially expressed genes (DEGs) were identified using transcriptomics. GO and KEGG analysis revealed that the MAPK pathway may be the molecular target. Further, we found that DGYFD inhibits phosphorylation levels of p38 MAPK and JNK in chronic lung injury mouse models. ConclusionsDGYFD could regulate the imbalance between the excessive release of inflammatory cytokines and oxidative stress, repair the alveolar-capillary permeability barrier and improve the pathological changes during chronic lung injury by regulating the MAPK signaling pathway.

Rickettsial pathogen inhibits tick cell death through tryptophan metabolite mediated activation of p38 MAP kinase

Anaplasma phagocytophilum modulates various cell signaling pathways in mammalian cells for its survival. In this study, we report that A. phagocytophilum modulates tick tryptophan pathway to activate arthropod p38 MAP kinase for the survival of both this bacterium and its vector host. Increased level of tryptophan metabolite, xanthurenic acid (XA), was evident in A. phagocytophilum-infected ticks and tick cells. Lower levels of cell death markers and increased levels of total and phosphorylated p38 MAPK was noted in A. phagocytophilum-infected ticks and tick cells. Treatment with XA increased phosphorylated p38 MAPK levels and reduced cell death in A. phagocytophilum-infected tick cells. Furthermore, treatment with p38 MAPK inhibitor affected bacterial replication, decreased phosphorylated p38 MAPK levels and increased tick cell death. However, XA reversed these effects. Taken together, we provide evidence that rickettsial pathogen modulates arthropod tryptophan and p38 MAPK pathways to inhibit cell death for its survival in ticks.

Knockdown of PKMYT1 is associated with autophagy inhibition and apoptosis induction and suppresses tumor progression in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a cancerous tumor that ranks as the third leading cause of cancer death across the globe. Protein kinase membrane-associated tyrosine/threonine kinase 1 (PKMYT1) is overexpressed in many cancer types, including HCC, but the potential mechanism and biological function of PKMYT1 are not fully understood. Materials and methodsThe expression level of PKMYT1 was detected in human HCC tissues and adjacent tissues. We then established HCC cell lines with PKMYT1 knockdown and observed proliferation, migration, autophagy, apoptosis in cell lines and tumor growth in a nude mouse model. To investigate the underlying mechanism by which PKMYT1 regulates autophagy and apoptosis, RNA sequencing was performed in HCC-LM3 cells with and without PKMYT1 knockdown. ResultsHere, we observed that human HCC tissues had higher expression of PKMYT1 than adjacent tissues. Overexpression of PKMYT1 was closely associated with poor prognosis in HCC patients. PKMYT1 knockdown inhibited the proliferative potential and migration of HCC cell lines. We also found that downregulation of PKMYT1 inhibited autophagy and induced apoptosis. RNA sequencing analysis showed that the MAPK and PI3K-AKT pathways, which have been reported to affect autophagy and apoptosis, may be regulated after PKMYT1 knockdown by KEGG pathway enrichment analysis. Furthermore, we identified that knockdown of PKMYT1 attenuated the phosphorylation levels of p38 MAPK, ERK and PI3K/Akt/mTOR, which might mediate autophagy inhibition and apoptosis induction via these signaling pathways to inhibit the development of HCC. ConclusionOur study suggests that PKMYT1 functions as an oncogene and may be a new target for HCC treatment.

Effects of Dietary Vegetable Oils Replacing Fish Oil on Fatty Acid Composition, Lipid Metabolism and Inflammatory Response in Adipose Tissue of Large Yellow Croaker (Larimichthys crocea)

The present study was conducted to investigate the effects of dietary vegetable oils (VOs) replacing fish oil (FO) on fatty acid composition, lipid metabolism and inflammatory response in adipose tissue (AT) of large yellow croaker (Larimichthys crocea). The initial body weight of a large yellow croaker was 10.07 ± 0.13 g. Three iso-nitrogenous and iso-lipidic diets were formulated by replacing FO with 0% (the control group), 100% soybean oil (SO) and 100% linseed oil (LO). Results showed that the contents of C18:2n-6 and C18:3n-3 were significantly increased in AT of fish fed the SO and LO diets compared with the FO diet, respectively. The proportion of n-6 polyunsaturated fatty acid (PUFA) was increased in SO and LO diets, while the proportions of saturated fatty acid and n-3 LC-PUFA were decreased. Moreover, dietary SO and LO significantly induced excess fat accumulation of AT by increasing the triglyceride content and the hypertrophy of adipocytes. Dietary SO and LO significantly increased lipogenesis-related gene expressions (dagt2, fabp10, srebp1, cebpα and pparγ), while decreasing the gene expression of lpl. Meanwhile, dietary SO increased the expression of genes related to fatty acid β-oxidation (cpt1 and aco), while LO showed no differences. Furthermore, dietary SO and LO increased the pro-inflammatory gene expressions and decreased the anti-inflammatory gene il10 expression. The phosphorylation levels of p38 MAPK and NF-κB were significantly upregulated by dietary SO and LO. In addition, there was a significant increase in macrophage infiltration and M1 polarization in AT of fish fed SO and LO diets. In conclusion, the present study revealed that dietary SO and LO replacing FO affected fatty acid composition and induced lipid dysmetabolism and inflammatory response in the adipose tissue of large yellow croaker.

MiR-328-3p Affects Axial Length Via Multiple Routes and Anti-miR-328-3p Possesses a Potential to Control Myopia Progression.

We previously reported miR-328-3p as a novel risk factor for myopia through a genetic association study of the PAX6 gene. In the present study, we first explored the effects of miR-328-3p on other myopia-related genes, and then tested whether anti-miR-328-3p may be used for myopia control. The luciferase report assay and transient transfection were used to confirm miR-328-3p target genes. The chromatin immunoprecipitation (ChIP) assay was used to investigate retinoic acid receptor on the miR-328-3p promoter. Mice and pigmented rabbits were induced to have myopia by the form deprivation method, and then anti-miR-328-3p oligonucleotide was topically instilled to the myopic eyes. The axial length was measured to assess the therapeutic effect of anti-miR-328-3p. A toxicity study using much higher doses was conducted to assess the safety and ocular irritation of anti-miR-328-3p. The report assay and transfection of miR-328-3p mimic confirmed that miR-328-3p dose-dependently decreased both mRNA and protein expression of fibromodulin (FMOD) and collagen1A1 (COL1A1). We subsequently showed that FMOD promoted TGF-β1 expression, and overexpression of FMOD increased the phosphorylation levels of p38-MAPK and JNK. The ChIP study showed that retinoic acid binds to miR-328-3p promoter and up-regulates miR-328-3p expression. In myopic animal studies, anti-miR-328-3p was as effective as 1% atropine and had a dose-dependent effect on suppressing axial elongation. In the toxicity study, anti-miR-328-3p did not cause any unwanted effects in the eyes or other organs. Micro (mi)R-328-3p affects myopia development via multiple routes. anti-miR-328-3p possesses a potential as a novel therapy for myopia control.

Conioselinum tenuissimum Root Extract Modulates Macrophage Activation via the Calcium–STAT3 Pathway

Despite the development of many antibiotics, excessive inflammation caused by endotoxins is still a subject of interest to biomedical researchers. The hyper-inflammatory response of macrophages activated by endotoxins is an important topic in the development of natural product-based anti-inflammatory drugs. Conioselinum tenuissimum, a perennial herb of the family Apiaceae, contains levistolide A, demethylsuberosin, and fraxetin. One of the synonyms of Conioselinum tenuissimum is Angelica tenuissima. The objective of this study was to determine the effects of Conioselinumtenuissimum root water extract (AT) on the hyper-inflammatory responses of macrophages activated by endotoxin (lipopolysaccharide; LPS) and the mechanisms involved in such effects. Levels of cytokines, nitric oxide (NO), hydrogen peroxide, and cytosolic calcium in LPS-activated RAW 264.7 murine macrophages were evaluated by the multiplex cytokine assay (MCA), Griess reagent assay (GRA), dihydrorhodamine 123 assay (DHR), and Fluo-4 calcium assay (FCA), respectively. Additionally, real-time PCR and the flow cytometry assay (FLA) was performed to determine the effects of AT on LPS-activated RAW 264.7. Data from MCA, GRA, DHR, and FCA revealed that AT lowered levels of IL-6, MCP-1, TNF-α, G-CSF, GM-CSF, VEGF, M-CSF, LIF, LIX, MIP-1α, MIP-1β, MIP-2, RANTES, IP-10, NO, hydrogen peroxide, and calcium in LPS-activated RAW 264.7. Real-time PCR results revealed that AT significantly lowered mRNA expression levels of inflammatory genes such as Chop, Nos2, c-Jun, Stat1, Stat3, c-Fos, Camk2a, Ptgs2, Fas, and Jak2. FLA showed that AT significantly reduced phosphorylation levels of P38 MAPK and STAT3 in LPS-activated RAW 264.7. These results indicate that AT can exert anti-inflammatory effects in LPS-activated macrophages via the calcium–STAT3 pathway.

CPKCγ Inhibits Caspase-9-Initiated Neuronal Apoptosis in an Ischemia Reperfusion Model In Vitro Through p38 MAPK-p90RSK-Bad Pathway.

Strokes are one of the leading causes of death and disability in the world. Previously we have found that conventional protein kinase Cγ (cPKCγ) plays neuroprotective role in ischemic strokes. Further, we found that cPKCγ knockdown increased the level of cleaved (cl)-Caspase-3. However, the precise mechanisms underlying cPKCγ-mediated neuronal death remain unclear. To this end, a model incorporating 1h oxygen-glucose deprivation/24h reoxygenation (1h OGD/24h R) was established in cortical neurons. We found that cPKCγ knockdown remarkably increased neuronal death after OGD. We also found that cPKCγ knockdown increased the level of cl-Caspase-3 through the upstream initiators Capsases-9 (not Caspase-8/12) in OGD-treated neurons. Overexpression of cPKCγ could decrease neuronal death and cl-Caspase-3 and -9 levels. Moreover, cPKCγ knockdown further reduced the phosphorylation levels of p38 MAPK, p90RSK, and Bad. In addition, the protein levels of Bcl-2 and Bcl-xl were decreased after cPKCγ knockdown, whereas that of Bax was increased. In conclusion, our results suggest that cPKCγ partly alleviates ischemic injury through activating the p38 MAPK-p90RSK-Bad pathway and inhibiting Caspase-9 initiated apoptosis. This may have potential as a therapeutic target for ischemic stroke.