p-JNK Protein Research Articles

Study on mechanisms of acupuncture underlying improvement of CUMS-induced depression in rats based on tandem mass spectrometry proteomics technique

To investigate the possible mechanism of "regulating qi and relieving depression" acupuncture underlying improvement of chronic unpredictable mild stress (CUMS)-induced depression in rats by using Tandem Mass Tags(TMT) quantitative proteomics technique. Thirty-six male SD rats were randomly divided into control, model and acupuncture groups, with 12 rats in each group. The depression model was induced by CUMS stress for 21 days. After the depression model was successfully established, the rats in the acupuncture group received manual acupuncture stimulation at "Baihui" (GV20) and "Yintang" (GV24+) for 20 min, once daily for 21 days. Open field test, sugar water preference test and forced swimming test (FST) were used to evaluate the behavioral changes. TMT quantitative proteomics was used to obtain differential proteins in the hippocampus tissue and related signaling pathways enrichment was analyzed, followed by verifying differential protein pathways by using Western blot and immunofluorescence methods. Behavior tests showed that on the 21st and 42nd days, the horizontal crossing times, walking distance and percentage of sugar water consumption were significantly decreased (P<0.05), while the immobility time of FST was obviously increased (P<0.05) in the model group relevant to the control group. After acupuncture intervention, the horizontal crossing times, walking distance and percentage of sugar water consumption were significantly increased (P<0.05), and the immobility time was apparently decreased (P<0.05) in the acupuncture group relevant to the model group. The TMT quantitative proteomics of hippocampus tissue displayed that of the 71 differential proteins (model group vs control group), 32 was down-regulated and 39 up-regulated in the model group; and among the above 71 differential proteins, there were 20 differential proteins between acupuncture group and model group, 15 down-regulated and 5 up-regulated in the acupuncture group (vs the model group). The expression of Mapk8ipl was up-regulated in the model group (vs the control group) and down-regulated in the acupuncture group (vs the model group). GO and KEGG enrichment analysis showed that these acupuncture-related differential proteins mainly involve the regulation of blood coagulation system, MAPK signaling pathway, etc. We selected the MAPK/JNK signaling pathway related to depression for verification. Western blot showed that the expression levels of c-JUN and phosphorylated c-JUN terminal kinase (p-JNK) proteins in the hippocampus were up-regulated in the model group relevant to the control group (P<0.05); while the expression levels of c-JUN and p-JNK proteins in the hippocampus were down-regulated in the acupuncture group relevant to the model group (P<0.05). The results of immunofluorescence showed that the mean fluorescence intensity of c-JUN and p-JNK in hippocampal CA1, CA3 and DG regions was increased in the model group relevant to the control group (P<0.05), while the mean fluorescence intensity of c-JUN and p-JNK in hippocampal CA1, CA3 and DG regions was obviously lower in the acupuncture group than in the model group (P<0.05). Acupuncture for "regulating qi and relieving depression" can significantly improve depression-like behavior in CUMS-induced depression model rats, which involves multiple targets and multiple pathways, including MAPK/JNK signaling.

Tubson-2 decoction ameliorates rheumatoid arthritis complicated with osteoporosis in CIA rats involving isochlorogenic acid A regulating IL-17/MAPK pathway

BackgroundOsteoporosis (OP) is considered as one of the major comorbidities of rheumatoid arthritis (RA), and is responsible for fragility fracture. However, there is currently no effective treatment for RA complicated with OP. Tubson-2 decoction (TBD), a Mongolian medicine also known as Erwei Duzhong Decoction, has been shown to exert a preventive effect on post-menopausal osteoporosis (PMOP). The preventive effects of TBD on RA-induced OP, as well as the bioactive compound responsible and the underlying mechanisms, remain to be elucidated. ObjectiveTo explore the effects of TBD on RA-induced OP in vivo, and to elucidate the mechanism of isochlorogenic acid A (ICA), the effective component of TBD, in vitro. MethodsTo evaluate the anti-arthritic and anti-osteoporotic effects of TBD, we conducted H&E straining and safranine O/fast green, TEM, immunohistochemistry (IHC), bone histomorphometry, micro-CT imaging, and biomechanical testing in collagen induced arthritis (CIA) rats. The active ingredient in TBD was identified using network pharmacology and molecular docking. The identification was supported by in vivo IHC assay, and further confirmed using qRT-PCR, Western blot, and SEM analysis in TNF-α-treated MH7A cells and/or in LPS-exposed RAW264.7 cells. ResultsOral administration of TBD attenuated the severity of arthritis and osteopenia as well as poor bone quality, in CIA rats. Additionally, TBD and the positive control, tripterygium glycosides (TG), exhibited similar effects in reducing inflammation in both the synovium and ankle joint. They also were both effective in improving bone loss, microarchitecture, and overall bone quality. TBD reduced the expression of MMP13, IL-17, and p-JNK protein in the synovium of CIA rats. ICA, which was screened, suppressed TNF-α or LPS-triggered inflammatory responses via down-regulating IL-17 signaling, involving in MMP13, IL-1β, IL-23, and IL-17, and the MAPK pathway including p-ERK, p-JNK, and p-P38, both in MH7A cells and in RAW264.7 cells. Furthermore, ICA prevented osteoclasts from differentiating and bone resoprtion in a dose-dependent manner in vitro. ConclusionThis study provides the first evidence that TBD exerts intervening effects on RA-induced OP, possibly through the downregulation of the IL-17/MAPK signaling pathway by ICA. The findings of our study provides valuable insights for further research in this area.

LncRNA MEG3 regulates ASK1/JNK axis-mediated apoptosis and autophagy via sponging miR-23a in granulosa cells of yak tertiary follicles

Apoptosis and autophagy in granulosa cells (GCs) are highly related to follicular development and atresia. It has also been reported that they are related to LncRNA MEG3, miR-23a and apoptosis signal-regulating kinase 1 (ASK-1). However, their relationship to follicular development and the extent to which follicle stimulating hormone (FSH) or luteinizing hormone (LH) can regulate this process remain unknown. Here, we found that ASK1 and JNK were expressed in the GCs of gonadotropin-dependent follicles, and those levels were significantly higher (p < 0.05) in yak Tertiary follicles compared to that of Secondary follicles and Graafian follicles. Then, the effect of LncRNA MEG3 / miR-23a on apoptosis and autophagy via ASK1/JNK (c-Jun N-terminal kinase) in yak GCs was studied. Overexpressing LncRNA MEG3 reduced miR-23a levels and p-967 protein expression, but enhanced ASK1 and JNK mRNA levels as well as t-ASK1, p-845, t-JNK, and p-JNK proteins levels. And Up-regulation of LncRNA MEG3 promoted apoptosis while attenuating autophagy. The targeting relationship between miR-23a and the binding sites of LncRNA MEG3 and ASK1 was also confirmed with the dual luciferase reporter assay. And, the relationship between LncRNA MEG3 and miR-23a was observed as a negative feedback regulation, and changes in LncRNA MEG3 and miR-23a levels can alter the expression of ASK1/JNK axis in yaks GCs. In addition, FSH (10 μg/mL) or LH (100 μg/mL) ability to reverse the effects of LncRNA MEG3 on miR-23a levels and ASK1/JNK axis-mediated apoptosis and autophagy was verified in yak GCs. This is significantly beneficial for decreasing abnormal follicular atresia for yaks tertiary follicles.

Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on the expression of apoptosis-related factors in rats with premature ovarian insufficiency based on oxidative stress

To explore the possible mechanism of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on premature ovarian insufficiency (POI) from the perspective of oxidative stress. Sixty female SD rats were randomly divided into a blank group, a model group, a sham acupuncture group, a medication group, and an acupuncture group, 12 rats in each group. Except the blank group, the rats in the remaining groups were intraperitoneally injected with cyclophosphamide to establish the POI model. After the model was successfully established, the rats in the acupuncture group were treated with acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28), with a depth of about 12 mm, and the needle was retained for 30 min; the acupuncture was given once a day, for a total of 4 weeks. The rats in the sham acupuncture group were treated with blunt-head needle to tap the skin surface of "Zhibian" (BL 54), without penetrating the skin, once a day for 4 weeks. The rats in the medication group were treated with estradiol valerate by gastric gavage for 4 weeks. After the intervention, the level of reactive oxygen species (ROS) in the ovarian tissue was detected by fluorescence probe; the expression of c-Jun N-terminal kinase (JNK), forkhead box O1 (FoxO1), tumor suppressor gene protein 53 (p53) and p53 up-regulated modulator of apoptosis (Puma) mRNA and protein in ovarian tissue were detected by real-time fluorescence quantitative PCR and Western blot. Compared with the blank group, the level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the model group were increased (P<0.01). Compared with the model group, the level of ROS and the expression of p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the sham acupuncture group were slightly reduced, but the difference was not statistically significant (P>0.05). The level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the acupuncture group and the medication group were reduced (P<0.01). Acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) could improve the level of oxidative stress, down-regulate the expression of apoptosis-related factors JNK, FoxO1, p53 and Puma induced by oxidative stress, and inhibit the premature failure of ovarian reserve function caused by apoptosis of ovarian granulosa cells in POI rats.

Dimethyl fumarate abrogates striatal endoplasmic reticulum stress in experimentally induced late-stage Huntington's disease: Focus on the IRE1α/JNK and PERK/CHOP trajectories.

Introduction: Dimethyl fumarate (DMF) is FDA-approved for use in patients with relapsing multiple sclerosis, and it processes neuroprotection in several experimental settings; however, its impact on combating Huntington's disease (HD) remains elusive. This study aimed to explore the role of DMF post-treatment on HD mediated endoplasmic reticulum (ER) stress response in a selective striatal degeneration HD model. Methods: Rats, exposed to 3-nitropropionic acid, were either left untreated or post-treated with DMF for 14days. Results and Discussion: DMF reduced locomotion deficits in both the open field and beam walk paradigms, boosted the striatal dopamine (DA) content, improved its architecture at the microscopic level, and hindered astrogliosis. Mechanistically, DMF limited the activation of two of the ER stress arms in the striatum by reducing p-IRE1α, p-JNK, and p-PERK protein expressions besides the CHOP/GADD153 content. Downstream from both ER stress arms' suppression, DMF inhibited the intrinsic apoptotic pathway, as shown by the decrease in Bax and active caspase-3 while raising Bcl-2. DMF also decreased oxidative stress markers indicated by a decline in both reactive oxygen species and malondialdehyde while boosting glutathione. Meanwhile, it enhanced p-AKT to activate /phosphorylate mTOR and stimulate the CREB/BDNF/TrkB trajectory, which, in a positive feedforward loop, activates AKT again. DMF also downregulated the expression of miRNA-634, which negatively regulates AKT, to foster survival kinase activation. Conclusion: This study features a focal novel point on the DMF therapeutic ability to reduce HD motor manifestations via its ability to enhance DA and suppress the IRE1α/JNK and PERK/CHOP/GADD153 hubs to inhibit the mitochondrial apoptotic pathway through activating the AKT/mTOR and BDNF/TrkB/AKT/CREB signaling pathways and abating miRNA-634 and oxidative stress.

Apelin-36 alleviates LPS-induced trophoblast cell injury by inhibiting GRP78/ASK1/JNK signaling

Pre-eclampsia (PE) is a type of hypertensive disorder of pregnancy that poses a serious threat to the health of both mother and fetus. Inhibition of the inflammatory environment on trophoblast cells is of great significance to improve PE. Apelin-36 is an endogenous active peptide with strong anti-inflammatory activity. Therefore, this study aims to investigate the effects of Apelin-36 on lipopolysaccharide (LPS)-induced trophoblast cells and its potential mechanism. The levels of inflammatory factors (TNF-α, IL-8, IL-6 and MCP-1) were detected by reverse transcription-quantitative PCR (RT-qPCR). The proliferation, apoptosis, migration and invasion capacities in trophoblast cells were detected by CCK-8, TUNEL staining, wound healing and Transwell assays, respectively. GRP78 was overexpressed by cell transfection. Western blot was applied for the identification of protein levels. Apelin concentration-dependently decreased the expression of inflammatory cytokines and p-p65 protein level in trophoblast cells induced by LPS. Apelin treatment reduced LPS-induced apoptosis and improved the proliferation, invasion and migration capacities of LPS-mediated trophoblast cells. Additionally, Apelin down-regulated GRP78, p-ASK1 and p-JNK protein levels. The inhibition on LPS-induced trophoblast cell apoptosis and the promotion on invasion and migration by Apelin-36 was counteracted by GRP78 overexpression. To sum up, Apelin-36 could alleviate LPS-induced cell inflammation and apoptosis and improve the invasion and migration of trophoblasts by inhibiting the GRP78/ASK1/JNK signaling.

Effect of palmitoylethanolamide on degeneration of a human-derived retinal pigment epithelial cell induced by all-trans retinal.

To study the effect of palmitoylethanolamide (PEA) on apoptosis of retinal pigment epithelial (RPE) cells induced by all-trans retinal (atRAL) and to explore the possible molecular mechanism. CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) was used to detect the effect of PEA on human-derived retinal epithelial cells (ARPE-19) viability induced by atRAL. A Leica DMi8 inverted microscope was used to observe cell morphology. Reactive oxygen species (ROS) production was evaluated with 2',7'-dichlorodihydrof-luorescein diacetate (H2DCFDA) staining and fluorescence microscopy. Expression of c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), c-Jun, phosphorylated c-Jun (p-c-Jun), Bak, cleaved caspase-3, C/EBP homologous protein (CHOP), and binding (Bip) protein levels were tested by Western blot. Abca4 -/- Rdh8 -/- mice, mouse models of atRAL clearance defects which displays some symbolic characteristics of dry age-related macular degeneration (AMD) and Stargardt disease (STGD1). In the animal models, PEA was injected intraperitoneally. The full-field electroretinogram was used to detect visual function under scotopic conditions traced from mice. Optical coherence tomography showed reconstitution or thickening of the retinal pigment epithelium layer. Effect of PEA on fundus injury induced by light in Abca4-/-Rdh8-/- mice was observed by fundus photography. PEA ameliorated ARPE-19 cells apoptosis and inhibited ROS (including mitochondrial ROS) production induced by atRAL. PEA improved the retinal functional, prohibited both RPE and photoreceptor from death, ameliorates light-induced fundus impairment in Abca4 -/- Rdh8 -/- mice. In vitro and in vivo, PEA inhibited JNK, p-JNK, c-Jun, p-c-Jun, Bak, cleaved caspase-3, CHOP, and Bip protein levels induced by all-trans retinal in ARPE-19 cells. PEA has effect on treating RPE cells apoptosis in retinopathy caused by atRAL accumulation. PEA is a potential treatment strategy for dry AMD and STGD1. The molecular mechanism is affecting the ROS-JNK-CHOP signaling pathway partly.

Immunopotentiation effects of apigenin on NK cell proliferation and killing pancreatic cancer cells.

Apigenin is a kind of flavonoid with many beneficial biological effects. It not only has direct cytotoxicity to tumor cells, but also can boost the antitumor effect of immune cells by modulating immune system. The purpose of this study was to investigate the proliferation of NK cells treated with apigenin and its cytotoxicity to pancreatic cancer cells in vitro, and explore its potential molecular mechanism. In this study, the effect of apigenin on NK cell proliferation and killing pancreatic cancer cells were measured by CCK-8 assay. Perforin, granzyme B (Gran B), CD107a, and NKG2D expressions of NK cells induced with apigenin were detected by flow cytometry (FCM). The mRNA expression of Bcl-2, Bax and protein expression of Bcl-2, Bax, p-ERK, and p-JNK in NK cells were evaluated by qRT-PCR and western blotting analysis, respectively. The results showed that appropriate concentration of apigenin could significantly promote the proliferation of NK cells in vitro and enhance the killing activity of NK cells against pancreatic cancer cells. The expressions of surface antigen NKG2D and intracellular antigen perforin and Gran B of NK cells were upregulated after treating with apigenin. Bcl-2 mRNA expression was increased, while Bax mRNA expression was decreased. Similarly, the expression of Bcl-2, p-JNK, and p-ERK protein was upregulated, and the expression of Bax protein was downregulated. The molecular mechanism of the immunopotentiation effects of apigenin may be that it up-regulates Bcl-2 and down-regulates Bax expression at the gene and protein levels to facilitate NK cell proliferation, and up-regulates the expression of perforin, Gran B, and NKG2D through the activation of JNK and ERK pathways to enhance NK cell cytotoxicity.

Monocyte Chemoattractant Protein-1-Supplemented Plasma Enhances Adiponectin and Adipogenesis-Related Gene Expression.

Increasing adipogenesis has been explored to treat metabolic diseases and atherosclerosis through the release of adiponectin. The effects and mechanism of platelet-rich plasma treatment on fat graft survival and adipogenesis have not been clarified. Here, we aimed to study the effects of monocyte chemoattractant protein-1 (MCP-1)-supplemented plasma on adipogenesis-related gene expression and adiponectin levels. Stromal vascular fractions (SVFs) purified from the inguinal adipose tissue of obese and diabetic (Leprdb/db) mice were treated with plasma from control (Lepr+/+) mice supplemented with 10 or 50 ng of MCP-1. The expression of adiponectin and interleukin-33 (IL-33) mRNA in adipose tissue was increased in Leprdb/db mice, whereas control (Lepr+/+) plasma reduced expression of IL-33 mRNA as well as peroxisome proliferator-activated receptor gamma (PPARγ), pJNK, and pNF-κB protein, and increased the expression of IL-10 mRNA in SVFs of Leprdb/db mice. MCP-1-supplemented control plasma increased the expression of adiponectin, CCAAT-enhancer-binding protein α (C/EBPα), dipeptidyl peptidase 4 (DPP4), IL-33, and PDGFα mRNA and the expression of adiponectin protein as well as PPARγ of SVFs and the expression of PPARγ mRNA in adipose tissue macrophages (ATMs). Injection of MCP-1-supplemented plasma into adipose tissue of Leprdb/db mice increased the expression of IL-33 and Col3a1 mRNA in SVFs and IL-33, FABP4, PDGFα, PPARγ and PPARγ2 of ATMs, protein expression of adiponectin and PPARγ of SVFs, and plasma adiponectin levels, as well as DPP4 activity. In conclusion, our results demonstrate that control plasma decreases adipogenesis and increases IL-10, and decreases IL-33, pJNK, and pNF-κB in adipose tissue. MCP-1-supplemented plasma enhances adipogenesis-related gene expression in SVFs and adiponectin levels, which may be mediated through an increase of IL-33 and PPARγ. Thus, our findings suggest that MCP-1-supplemented plasma represents a novel therapy to stimulate local adipogenesis and systemic adiponectin levels.

Rosiglitazone Protects against Acetaminophen-Induced Acute Liver Injury by Inhibiting Multiple Endoplasmic Reticulum Stress Pathways.

Excessive acetaminophen (APAP) use can lead to acute liver injury (ALI) by inducing endoplasmic reticulum stress (ERS). We previously found that pretreatment with the peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand rosiglitazone (RSG) alleviated ALI in APAP-treated mice. To examine if RSG-mediated hepatoprotection is associated with ERS suppression. Forty-eight male CD-1 mice were randomly divided into control, RSG, APAP 4 h, APAP 24 h, RSG + APAP 4 h, and RSG + APAP 24 h groups. The RSG and RSG + APAP groups received RSG (20 mg/kg) by gavage 48, 24, and 1 h before intraperitoneal injection of 300 mg/kg APAP, while the APAP group received APAP alone and the control group received only normal saline. Animals were sacrificed immediately (RSG and control groups), 4 h (APAP 4 h and RSG + APAP 4 h), or 24 h (APAP 24 h and RSG + APAP 24 h) post-APAP injection. Liver tissues were collected for hematoxylin-eosin staining, TUNEL staining, and Western blotting for ERS-associated proteins. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were also measured. A second cohort received APAP or RSG + APAP as described and were monitored for survival over one week. At 4 and 24 h following APAP injection alone, serum ALT and AST levels were significantly elevated, and central lobular necrosis of the liver was observed. Necrosis area reached 21.7% at 4 h and 32.1% at 24 h post-APAP, while apoptotic fractions reached 25.6% and 32.4%. Further, 50% of mice in the survival analysis cohort died within one week post-APAP. At 4 h post-APAP, the ERS marker glucose-regulated protein-78 (GRP78) and ERS-associated proteins pJNK, GRP78, p-eIF2α, pPERK, and pIRE were all significantly upregulated. Pretreatment with RSG significantly reduced serum ALT and AST, liver necrosis area, apoptosis rate, and expression of ERS-associated proteins compared to APAP alone, while increasing survival to 80%. Rosiglitazone pretreatment can alleviate APAP-induced ALI by suppressing three branches of ERS signaling.

Knockout of S1PR3 attenuates acute lung injury in mice by inhibiting the MAPK pathway

To investigate whether knockout of S1PR3 improves lipopolysaccharide (LPS)-induced acute lung injury in mice by inhibiting mitogen activated protein kinases (MAPKs). Male C57BL/6J and S1PR3 knockout (S1PR3-/-) mice were both randomized into two groups (n=8) for intratracheal instillation of normal saline or LPS to induce acute lung injury. The expressions of S1PR3, IL-1β and IL-18 in the lung tissues were detected using RT-qPCR, lung tissue injury was observed with HE staining, and cell apoptosis was detected using flow cytometry. Western blotting was performed to detect the expression levels of caspase-1, GSDMD, p-JNK, p-ERK and p-p38 proteins. In the cell experiment, type II alveolar epithelial cells (MLE-12 cells) were treated with PBS, LPS, CYM5541 (a S1PR3 agonist), or CYM5541 + LPS, and the cell apoptosis and expression levels of MAPK signal pathway molecules were detected. The expression of S1PR3 was up-regulated and serum IL-1β and IL-18 levels were elevated significantly in the nontransgenic mice with acute lung injury (P < 0.001). By comparison, the elevation of IL-1β and IL-18 levels was obviously reduced in S1PR3 knockout mice with acute lung injury, which also showed significant improvement of pulmonary hemorrhage, inflammation and exudation, lowered wet-to-dry ratio of the lungs, and decreased cell apoptosis and expressions of cleaved caspase-1 and GSDMD (P < 0.05). In MLE-12 cells, treatment with the S1PR3 agonist significantly increased the expression of pyroptosis-associated proteins (P < 0.05). S1PR3 knockout strongly inhibited the activation of MAPKs family (JNK and ERK p38; P < 0.05), but their expressions were significantly increased following treatment with the S1PR3 agonist (P < 0.05). Inhibition of S1PR3 can improve LPSinduced acute lung injury in mice by inhibiting the activation of MAPK signaling.

Involvement of AMPKα and MAPK-ERK/-JNK Signals in Docetaxel-Induced Human Tongue Squamous Cell Carcinoma Cell Apoptosis.

Cancers of the oral cavity can develop in the anatomic area extending from the lip, gum, tongue, mouth, and to the palate. Histologically, about 85-90% of oral cavity cancers are of the type squamous cells carcinomas (SCCs). The incidence of oral tongue SCC is higher in the tongue than any other anatomic area of the oral cavity. Here, we investigated the therapeutic effects and molecular mechanisms of docetaxel, which is a paclitaxel antitumor agent, on the cell growth of a human tongue SCC-derived SAS cell line. The results showed that docetaxel (10-300 nM) induced cytotoxicity and caspase-3 activity in SAS cells. Moreover, docetaxel (100 nM) promoted the expression of apoptosis-related signaling molecules, including the cleavages of caspase-3, caspase-7, and poly (ADP-ribose) polymerase (PARP). In mitochondria, docetaxel (100 nM) decreased the mitochondrial membrane potential (MMP) and Bcl-2 mRNA and protein expression and increased cytosolic cytochrome c protein expression and Bax mRNA and protein expression. In terms of mitogen-activated protein kinase (MAPK) and adenosine monophosphate-activated protein kinase (AMPK) signaling, docetaxel increased the expression of phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-c-Jun N-terminal kinase (JNK), and p-AMPKα protein expression but not p-p38 protein expression. Moreover, the increase in caspase-3/-7 activity and Bax protein expression and decreased Bcl-2 protein expression and MMP depolarization observed in docetaxel-treated SAS cells could be reversed by treatment with either SP600125 (a JNK inhibitor), PD98059 (an MEK1/2 (mitogen-activated protein kinase kinase 1/2) inhibitor), or compound c (an AMPK inhibitor). The docetaxel-induced increases in p-JNK, p-ERK, and p-AMPKα protein expression could also be reversed by treatment with either SP600125, PD98059, or compound c. These results indicate that docetaxel induces human tongue SCC cell apoptosis via interdependent MAPK-JNK, MAPK-ERK1/2, and AMPKα signaling pathways. Our results show that docetaxel could possibly exert a potent pharmacological effect on human oral tongue SCC cell growth.

Low doses of fumonisin B1 exacerbate ochratoxin A-induced renal injury in mice and the protective roles of heat shock protein 70

Fumonisin B1 (FB1) and ochratoxin A (OTA) possess nephrotoxicity to animals and widely co-exist in food and feedstuffs. FB1 rarely, while OTA often, causes toxicosis in animals. Heat shock protein 70 (Hsp70) resists lung injury induced by pneumolysin, but whether Hsp70 could remission mycotoxins-induced renal injury is still unknown. The present study aims to explore the impacts of nontoxic doses of FB1 on OTA-induced nephrotoxicity and the protective roles of Hsp70. In the mycotoxins-challenge experiment, ICR mice were co-exposed to nontoxic doses of FB1 (0, 0.2, 0.5, 1.0 mg/kg bw, IP) and toxic dose of OTA (0.4 mg/kg bw, IP) for 16 d. The results showed that the levels of BUN, Cr, MDA in serum, the Cyto C in renal tubes or glomerulus, pro-apoptosis genes and p-JNK protein expression in kidney were significantly increased. Histopathological results revealed the glomerular swelling. The above all indexes were dose-dependent. In the protection experiment, the mice were pretreated with the eukaryotic plasmid of pEGFP-C3-Hsp70, these increasing parameters in the mycotoxins-challenge experiment were reversed. In vitro, after pK-15 cells were treated with 8 μM FB1 and 5 μM OTA for 48 h, the mitochondrial membrane potential was significantly reduced, mitochondrial ROS was remarkably increased, more Cyto C was leaked from mitochondria into cytoplasm, and pro-apoptosis genes were significantly up-regulated. After the Hsp70 level was up-regulated by pEGFP-C3-Hsp70 or ML346 in pK-15 cells, these above indexes were reversed. However, activation of JNK by anisomycin significantly suppressed the protective effects of Hsp70. Our results demonstrate that the nontoxic doses of FB1 exacerbate the toxic dose of OTA-induced renal injury, while Hsp70 alleviates renal injury by inhibiting the JNK/MAPK signaling pathway. Hsp70 up-regulation may be an efficient strategy for protecting against tissue damage and bio-function impairment induced by co-exposure to FB1 and OTA.

Salvianolic acid B inhibits autophagy and activation of hepatic stellate cells induced by TGF-β1 by downregulating the MAPK pathway.

In liver fibrosis, transforming growth factor-β1 (TGF-β1) can stimulate autophagy and activation of hepatic stellate cells (HSCs). Autophagy, playing a crucial role in HSCs activation, is related to liver fibrosis. Increasing evidence have suggested that antifibrosis effects of salvianolic acid B (Sal B) and their mechanisms of action, however, remain unclear. The aim of the article is to understand the role of Sal B in HSCs autophagy in liver fibrosis. Herein, we demonstrated that inducing TGF-β1 led to dramatic increase in autophagosome formation and autophagic flux in JS1 and LX2, which was mediated through the ERK, JNK, and p38 MAPK cascades. TGF-β1 significantly increased the protein of autophagy and liver fibrosis, including LC3BⅡ, ATG5, α-SMA, and Col.I; Sal B inhibits JS1 autophagy and activation by inhibiting the formation of autophagosomes and autophagic flux. Sal B significantly decreased the LC3BⅡ, ATG5, α-SMA, and Col.I protein expressions; pretreatment with autophagy inhibitors, chloroquine (CQ) and 3-methyladenine (3-MA) or silencing ATG7 further increase these reductions. However, pretreatment with autophagy agonist, rapamycin (Rapa), or overexpressed ATG5 attenuated this decrease. To further assess the importance of this mechanism, the antibody chip was used to detect the change of phosphorylation protein expression of the MAPK signaling pathway after treating JS1 with Sal B. Eleven differentially expressed proteins were verified. Sal B inhibits activation and autophagy of JS1 induced by TGF-β1 through downregulating the ERK, p38, and JNK signaling pathways, as demonstrated by downregulating p-ERK, p-JNK, and p-p38 MAPK protein expressions. In conclusion, Sal B inhibits autophagy and activation induced by TGF-β1 of HSCs possibly by downregulating the MAPK pathway.

Friedelin Attenuates Neuronal Dysfunction and Memory Impairment by Inhibition of the Activated JNK/NF-κB Signalling Pathway in Scopolamine-Induced Mice Model of Neurodegeneration.

Oxidative stress (OS) and c-Jun N-terminal kinase (JNK) are both key indicators implicated in neuro-inflammatory signalling pathways and their respective neurodegenerative diseases. Drugs targeting these factors can be considered as suitable candidates for treatment of neuronal dysfunction and memory impairment. The present study encompasses beneficial effects of a naturally occurring triterpenoid, friedelin, against scopolamine-induced oxidative stress and neurodegenerative pathologies in mice models. The treated animals were subjected to behavioural tests i.e., Y-maze and Morris water maze (MWM) for memory dysfunction. The underlying mechanism was determined via western blotting, antioxidant enzymes and lipid profile analyses. Molecular docking studies were carried out to predict the binding modes of friedelin in the binding pocket of p-JNK protein. The results reveal that scopolamine caused oxidative stress by (1) inhibiting catalase (CAT), peroxidase enzyme (POD), superoxide dismutase (SOD), and reduced glutathione enzyme (GSH); (2) the up-regulation of thiobarbituric acid reactive substances (TBARS) in mice brain; and (3) affecting the neuronal synapse (both pre- and post-synapse) followed by associated memory dysfunction. In contrast, friedelin administration not only abolished scopolamine-induced oxidative stress, glial cell activation, and neuro-inflammation but also inhibited p-JNK and NF-κB and their downstream signaling molecules. Moreover, friedelin administration improved neuronal synapse and reversed scopolamine-induced memory impairment accompanied by the inhibition of β-secretase enzyme (BACE-1) to halt amyloidogenic pathways of amyloid-β production. In summary, all of the results show that friedelin is a potent naturally isolated neuro-therapeutic agent to reverse scopolamine-induced neuropathology, which is characteristic of Alzheimer’s disease.

Osteocalcin reduces fat accumulation and inflammatory reaction by inhibiting ROS-JNK signal pathway in chicken embryonic hepatocytes

Osteocalcin (OCN) has a function in preventing fatty liver hemorrhagic syndrome (FLHS) in poultry. The aim of this study was to investigate the effects of OCN on fat emulsion stimulated chicken embryonic hepatocytes and related signaling pathways. The primary chicken embryonic hepatocytes were isolated from the incubated 15-day (E15) pathogen free eggs and cultured with dulbecco's modified eagle medium (DMEM). After the hepatocyte density reached 80%, the cells were divided into 5 groups: control group (CONT), fat emulsion group (FE, 10% FE, v/v), FE with ucOCN at 1 ng/mL (FE-LOCN), 3 ng/mL (FE-MOCN), and 9 ng/mL (FE-HOCN). In addition, 2 mM N-Acetyl-L-cysteine (NAC) a reactive oxygen species (ROS) scavenger, and 5 μM SP600125, a Jun N-terminal kinase (JNK) inhibitor, were added separately in to the DMEM with 10% FE to test effects of FE on the function of ROS-JNK signal pathway. The number of hepatocytes, cell ultra-microstructure, viability, and apoptosis were detected after 48 h treatment, and the protein expressions and enzyme concentrations were detected after 72 h treatment. The results showed that, compared to the control group, FE increased the triglyceride (TG) concentration and lipid droplets (LDs) in chicken embryonic hepatocytes (P < 0.05), and induced hepatocytic edema with obviously mitochondrial swelling, membrane damage, and cristae rupture. FE also decreased ATP concentration, increased ROS concentrations and mitochondrial DNA (mtDNA) copy number, promoted inflammatory interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α) concentrations and hepatocytic apoptosis rate, and raised phospho-c-Jun N-terminal kinase (p-JNK) protein expressions. Compared to the FE group, ucOCN significantly increased hepatocyte viability, reduced hepatocytic TG concentrations and LDs numbers, and alleviated hepatocytic edema and mitochondrial swelling. Furthermore, ucOCN significantly decreased ROS concentrations, increased ATP concentrations, reduced IL-1, IL-6, TNF-α concentrations and hepatocytic apoptosis rate, and inhibited p-JNK protein expressions (P < 0.05). NAC had the similar functions of ucOCN reduced the ROS concentration and inhibited the TNF-α protein expression and p-JNK/JNK ration. Similarly, SP600125 reduced p-JNK/JNK protein expression, IL-1, IL-6, TNF-α, and TG concentrations without effects on ROS concentration and hepatocytic apoptosis. These results suggest that ucOCN alleviates FE-induced mitochondrial damage, cellular edema, and apoptosis of hepatocytes. These results reveal that the functions of ucOCN in reducing fat accumulation and inflammatory reaction in chicken embryonic hepatocytes are mostly via inhibiting the ROS-JNK signal pathway.

The Role of Microglia/Macrophages Activation and TLR4/NF-κB/MAPK Pathway in Distraction Spinal Cord Injury-Induced Inflammation.

Distraction spinal cord injuries (DSCIs) often occur as the neurological complication of distraction forces following the implantation of internal fixation devices during scoliosis correction surgery. However, the underlying mechanism behind these injuries remains unclear. The present study aimed to explore the activation of microglia and macrophages, as well as changes in TLR4-mediated NF-κB and MAPK pathway activity after DSCIs in Bama miniature pigs. Prior to surgical intervention, the pigs were randomly divided into three groups: the sham group, the complete distraction spinal cord injury (CDSCI) group, and the incomplete distraction spinal cord injury (IDSCI) group. After surgery, the Tarlov scale and individual limb motor scale (ILMS) were used to evaluate changes in the pigs’ behavior. All pigs were euthanized 7 days after surgery, and histopathological examinations of the spinal cord tissues were performed. Immunohistochemistry was used to detect Caspase-3 expression in the anterior horn of spinal gray matter tissues. Immunofluorescence staining was utilized to assess the M1/M2 phenotype changes in microglia/macrophages and NF-κB P65 expression in central DSCI lesions, while western blotting was performed to determine the expression of TLR4/NF-κB/MAPK pathway-related proteins. The results of the present study showed that the Tarlov and ILMS scores decreased significantly in the two DSCI groups compared with the sham group. Hematoxylin and eosin (HE) and Nissl staining revealed that the tissue structure and nerve fiber tracts in the distracted spinal cord tissues were destroyed. Both DSCI groups showed the number of survived neurons decreased and the Caspase-3 expression increased. The results of the immunofluorescence staining indicated that the CD16 and CD206 expression in the microglia/macrophages increased. Between the two DSCI groups, the CDSCI group showed increased CD16 and decreased CD206 expression levels. The intensity of the fluorescence of NF-κB P65 was found to be significantly enhanced in pigs with DSCIs. Moreover, western blot results revealed that the expression of TLR4, p-IκBα, NF-κB P65, p-JNK, p-ERK, and p-P38 proteins increased in spinal cord tissues following DSCI. The present study was based on a porcine DSCI model that closely mimicked clinical DSCIs while clarifying DSCI-associated neuroinflammation mechanisms, in turn providing evidence for identifying potential anti-inflammatory targets.

Umbilical cord-derived mesenchymal stem cell conditioned medium reverses neuronal oxidative injury by inhibition of TRPM2 activation and the JNK signaling pathway

BackgroundThe mechanism by which MSC-CM protects neuronal cells against ischemic injury remains to be elucidated. In this study, we aimed to clarify the protective effect of umbilical cord-derived mesenchymal stem cell conditioned medium (UC-MSC-CM) on neuronal oxidative injury and its potential mechanism.Methods and ResultsNeuronal oxidative damage was mimicked by H2O2 treatment of the HT22 cell line. The numbers of cleaved-Caspase-3-positive cells and protein expression of Caspase-9 induced by H2O2 treatment were decreased by UC-MSC-CM treatment. Furthermore, SOD protein expression was increased in the MSC-CM group compared with that in the H2O2 group. The H2O2-induced TRPM2-like currents in HT22 cells were attenuated by MSC-CM treatment. In addition, H2O2 treatment downregulated the expression of p-JNK protein in HT22 cells, and this the downward trend was reversed by incubation with MSC-CM.ConclusionsUC-MSC-CM protects neurons against oxidative injury, possibly by inhibiting activation of TRPM2 and the JNK signaling pathway.

Study on the Mechanism of Shenjing Guben Prescription Regulating PI3K and NRF2 Signaling Pathway in the Treatment of Immune Infertility.

Objective To explore the mechanism of Shenjing Guben prescription (SP) in the treatment of immune infertility by regulating PI3K-NRF2/p38 signal pathway. Methods 60 adult male SD rats were randomly divided into control group (NC group), ACN group, low concentration AP intervention group (low group), middle concentration SP intervention group (middle group), and high concentration SP intervention group (high group). 12 rats in each group were administered by gavage once a day, 6 days/w, and the rats were killed after 28 days. Bilateral testis and epididymis were removed and weighed and organ coefficients were calculated, and testicular histopathological sections were prepared to evaluate the changes of testicular tissue structure. The relative expression levels of PI3K, MKK7, JNK, p38 mRNA, and protein in testis were measured by QRT-PCR and western blot. Results (1) Compared with the control group, the proportion of grade A and B sperms in ACN group increased significantly, and the proportion of grade D sperm decreased significantly (P < 0.05). After SP intervention, compared with ACN group, there was no significant difference in the proportion of sperm at all levels in low, medium, and high SP intervention groups (P > 0.05). (2) Compared with the control group, the sperm VCL, VSL, VAP, and mad in ACN group increased significantly, and the BCF decreased significantly (P < 0.05). After SP intervention, compared with ACN group, there was no significant difference in sperm motility parameters among low, medium, and high SP intervention groups (P > 0.05). (3) Compared with the control group, the activities of AKP and SDH in testicular tissue of rats in ACN group decreased significantly (P < 0.05). After SP intervention, compared with ACN group, AKP activity increased significantly and LDH activity decreased significantly in low, medium, and high SP intervention groups (P < 0.05). (4) Compared with the control group, the expression levels of PI3K, p-PI3K, MKK7, p-MKK7, JNK, p-JNK, p38, and p-p38 proteins and the ratios of p-JNK/JNK and p-p38/p38 increased in the testis of ACN group (P < 0.05). After SP intervention, compared with ACN group, the protein expression levels of PI3K, p-PI3K, MKK7, p-MKK7, JNK, p-JNK, p38, and p-p38 in testicular tissue of SP intervention group decreased, and the ratio of p-JNK/JNK and p-p38/p38 decreased (P < 0.05). Conclusion SP can reduce the oxidative stress of testis induced by ACN and inhibit the activation of PI3K-NRF2/p38 signal pathway.

MicroRNA-181a-5p prevents the progression of esophageal squamous cell carcinoma in vivo and in vitro via the MEK1-mediated ERK-MMP signaling pathway.

MicroRNAs (miRNAs) have been revealed to play a crucial role in oncogenesis of esophageal squamous cell carcinoma (ESCC). However, the biological role of miR-181a-5p in ESCC is currently less explored. The current study was designed to assess whether miR-181a-5p affects ESCC progression and further investigate relevant underlying mechanisms. Based on the data of GSE161533, GSE17351, GSE75241 and GSE67269 downloaded from GEO database, MAP2K1 (MEK1) was revealed to be one overlapping gene of the top 300 DGEs. Additionally, using the predicting software, miR-181a-5p was projected as the presumed target miRNA. Immunohistochemical staining and RT-qPCR research revealed that miR-181a-5p expression was decreased in human tumor tissues relative to surrounding peri-cancerous tissues. In an in vivo experiment, miR-181a-5p mimics could inhibit tumor growth and metastasis of ESCC. Gene expression profiles in combination with gene ontology (GO) and KEGG pathway analysis revealed that MAP2K1 (MEK1) gene and ERK-MMP pathway were implicated in ESCC progression. MiR-181a-5p mimics inhibited the activity of p-ERK1/2, MMP2 and MMP9 in vivo, as shown by Western blotting and immunohistochemistry labeling. There were no variations in the expression of p-P38 and p-JNK proteins. Additionally, miR-181a-5p mimics lowered p-ERK1/2, MMP2 and MMP9 levels in ECA109 cells, which were restored by MEK1-OE lentivirus. MEK1-OE Lentivirus significantly reversed the function induced by miR-181a-5p mimics in ECA109 cells. Moreover, further investigation indicated that the capability of migration, invasion and proliferation was repressed by miR-181a-5p mimics in ECA109 cells. In short, repressed ERK-MMP pathway mediated by miR-181a-5p can inhibit cell migration, invasion and proliferation by targeting MAP2K1 (MEK1) in ESCC.