Activation Of P38 MAPK Pathway Research Articles

GATA4: Regulation of expression and functions in goat granulosa cells

GATA4 plays a pivotal role in the reproductive processes of mammals. However, the research on GATA4 in goat ovary is limited. This study aimed to study the expression and function of GATA4 in goat ovary. Utilizing real-time PCR and western blot analysis, we studied the expression and regulatory mechanisms of GATA4 in goat ovary and granulosa cells (GCs). We found that GATA4 was expressed in all follicle types in the goat ovary, with significantly higher levels in GCs of larger follicles (> 3 mm) compared to those in smaller follicles (< 3 mm). Additionally, we demonstrated that human chorionic gonadotrophin (hCG) induced GATA4 mRNA expression via the activation of PKA, MEK, p38 MAPK, PKC, and PI3K pathways in vitro. Our study also showed that hCG suppressed the levels of miR-200b and miR-429, which in turn directly target GATA4, thereby modulating the basal and hCG-induced expression of GATA4. Functionally, we examined the effect of siRNA-mediated GATA4 knockdown on cell proliferation and hormone secretion in goat GCs. Our results revealed that knockdown of GATA4, miR-200b, and miR-429 suppressed cell proliferation. Moreover, knockdown of GATA4 decreased estradiol and progesterone production by inhibiting the promoter activities of CYP11A1, CYP19A1, HSD3B, and StAR. Collectively, our findings suggest a critical involvement of GATA4 in regulating goat GC survival and steroidogenesis.

Dexmedetomidine alleviates blood-brain barrier disruption in rats after cerebral ischemia-reperfusion by suppressing JNK and p38 MAPK signaling.

Dexmedetomidine displays multiple mechanisms of neuroprotection in ameliorating ischemic brain injury. In this study, we explored the beneficial effects of dexmedetomidine on blood-brain barrier (BBB) integrity and neuroinflammation in cerebral ischemia/reperfusion injury. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1.5 h and reperfusion for 24 h to establish a rat model of cerebral ischemia/reperfusion injury. Dexmedetomidine (9 􀁐g/kg) was administered to rats 30 min after MCAO through intravenous injection, and SB203580 (a p38 MAPK inhibitor, 200 􀁐g/kg) was injected intraperitoneally 30 min before MCAO. Brain damages were evaluated by 2,3,5-triphenyltetrazolium chloride staining, hematoxylin-eosin staining, Nissl staining, and brain water content assessment. BBB permeability was examined by Evans blue staining. Expression levels of claudin-5, zonula occludens-1, occludin, and matrix metalloproteinase-9 (MMP-9) as well as M1/M2 phenotypes-associated markers were assessed using immunofluorescence, RT-qPCR, Western blotting, and gelatin zymography. Enzyme-linked immunosorbent assay was used to examine inflammatory cytokine levels. We found that dexmedetomidine or SB203580 attenuated infarct volume, brain edema, BBB permeability, and neuroinflammation, and promoted M2 microglial polarization after cerebral ischemia/reperfusion injury. Increased MMP-9 activity by ischemia/reperfusion injury was inhibited by dexmedetomidine or SB203580. Dexmedetomidine inhibited the activation of the ERK, JNK, and p38 MAPK pathways. Moreover, activation of JNK or p38 MAPK reversed the protective effects of dexmedetomidine against ischemic brain injury. Overall, dexmedetomidine ameliorated brain injury by alleviating BBB permeability and promoting M2 polarization in experimental cerebral ischemia/reperfusion injury model by inhibiting the activation of JNK and p38 MAPK pathways.

Hypoxia-Induced Myocardial Hypertrophy Companies with Apoptosis Enhancement and p38-MAPK Pathway Activation.

Li, Xiaoxu, Zhijun Pu, Gang Xu, Yidong Yang, Yu Cui, Xiaoying Zhou, Chenyuan Wang, Zhifeng Zhong, Simin Zhou, Jun Yin, Fabo Shan, Chengzhong Yang, Li Jiao, Dewei Chen, and Jian Huang. Hypoxia-induced myocardial hypertrophy companies with apoptosis enhancement and p38-MAPK pathway activation. High Alt Med Biol. 00:00-00, 2024. Background: Right ventricular function and remodeling are closely associated with symptom severity and patient survival in hypoxic pulmonary hypertension. However, the detailed molecular mechanisms underlying hypoxia-induced myocardial hypertrophy remain unclear. Methods: In Sprague-Dawley rats, hemodynamics were assessed under both normoxia and hypobaric hypoxia at intervals of 7 (H7), 14 (H14), and 28 (H28) days. Morphological changes in myocardial tissue were examined using hematoxylin and eosin (HE) staining, while myocardial hypertrophy was evaluated with wheat germ agglutinin (WGA) staining. Apoptosis was determined through TUNEL assays. To further understand the mechanism of myocardial hypertrophy, RNA sequencing was conducted, with findings validated via Western blot analysis. Results: The study demonstrated increased hypoxic pulmonary hypertension and improved right ventricular diastolic and systolic function in the rat models. Significant elevations in pulmonary arterial systolic pressure (PASP), mean pulmonary arterial pressure (mPAP), right ventricular mean pressure (RVMP), and the absolute value of +dp/dtmax were observed in the H14 and H28 groups compared with controls. In addition, right ventricular systolic pressure (RVSP), -dp/dtmax, and the mean dp/dt during isovolumetric relaxation period were notably higher in the H28 group. Heart rate increased in the H14 group, whereas the time constant of right ventricular isovolumic relaxation (tau) was reduced in both H14 and H28 groups. Both the right heart hypertrophy index and the heart weight/body weight ratio (HW/BW) were elevated in the H14 and H28 groups. Myocardial cell cross-sectional area also increased, as shown by HE and WGA staining. Western blot results revealed upregulated HIF-1α levels and enhanced HIF-2α expression in the H7 group. In addition, phosphorylation of p38 and c-fos was augmented in the H28 group. The H28 group showed elevated levels of Cytochrome C (Cyto C), whereas the H14 and H28 groups exhibited increased levels of Cleaved Caspase-3 and the Bax/Bcl-2 ratio. TUNEL analysis revealed a rise in apoptosis with the extension of hypoxia duration in the right ventricle. Conclusions: The study established a link between apoptosis and p38-MAPK pathway activation in hypoxia-induced myocardial hypertrophy, suggesting their significant roles in this pathological process.

Exogenous Hydrogen Sulfide Prevents Necroptosis by Inhibiting p38MAPK Pathway Activation in JEG-3 Trophoblast Cells: A Role in Preeclampsia

Objectives: Necroptosis, a form of programmed cell death, can occur in the placenta of patients with preeclampsia (PE). Hydrogen sulfide (H2S) can inhibit necroptosis of human umbilical vein endothelial cells under the high glucose-induced injury. Whether H2S can protect trophoblasts against necroptosis underlying PE has not been elucidated. This study aimed to explore the protective role of H2S in trophoblast cells against necroptosis underlying PE. Design: This is an in vitro experimental study. Participants: A total of 10 pregnant women with severe PE and 10 matched control normotensive pregnant women were included. The placenta tissues were extracted from participators. The human JEG-3 trophoblasts were commercially available. Methods: The expression and localization of necrotic proteins were assayed in human placenta samples, and the effect of necrotic cell death on the proliferation and apoptosis of human JEG-3 trophoblasts was evaluated. The component expressions of inflammatory cytokine and p38MAPK signaling pathway were measured in samples pretreated with or without NaHS (H2S donor) and SB203580 (p38 inhibitor). Results: RIPA1, RIPA3, and p-p38 levels were significantly higher in PE placental tissue, whereas cystathionine β-synthase expression was decreased. In JEG-3 trophoblasts, necroptosis increased apoptotic cell numbers, suppressed cell proliferation, increased inflammatory cytokine expression, and increased p38MAPK activation, which can be prevented by NaHS. Limitations: In the present study, we did not provide sufficient evidence that necroptosis was a part of the pathogenesis of PE. Conclusions: We proposed the putative role of necroptosis in early-onset PE, reflected by the blockage of caspase-8/3 and increased expression of RIPA1 and RIPA3 in PE placenta tissues. Furthermore, we demonstrated that exogenous H2S protected cytotrophoblasts against ceramide-induced necroptosis via the p38MAPK pathway.

Macrophage-derived exosomes rescue the TNF-ɑ-suppressed osteo-/cementogenic differentiation of hPDLCs.

Inflammatory stimuli compromise the differentiation potency of human periodontal ligament cells (hPDLCs). Macrophage-derived exosomes (M-Exo) play a role in several aspects of cellular activity. This study investigated how M-Exo contributes to the osteo-/cementogenic differentiation of hPDLCs under inflammation and the mechanism involved. M-Exo was identified by transmission electron microscopy, western blotting (WB), and dynamic light scattering. The internalization of M-Exo by hPDLCs was observed. After M-Exo treatment, the osteo-/cementogenic markers were detected by RT-qPCR and WB, and alkaline phosphatase (ALP) activity by ALP staining. Tumor necrosis factor alpha (TNF-ɑ) was applied to simulate inflammation. The rescue effect of M-Exo on TNF-ɑ-suppressed differentiation was validated. The p38 MAPK pathway activity was tested and a specific inhibitor was applied to explore the mechanism. M-Exo was successfully isolated, identified and internalized by hPDLCs. M-Exo enhanced the osteo-/cementogenic differentiation of hPDLCs, as indicated by upregulated osteo-/cementogenic markers and elevated ALP activity. Moreover, TNF-ɑ inhibited the differentiation capabilities of hPDLCs, on which M-Exo showed a rescue effect. M-Exo activated the p38 MAPK pathway and SB203580 attenuated its promotion effect. This study showed that M-Exo ameliorated the TNF-ɑ-suppressed osteo-/cementogenic differentiation of hPDLCs partly through the p38 MAPK pathway.

Manganese dioxide nanoparticles provoke inflammatory damage in BV2 microglial cells via increasing reactive oxygen species to activate the p38 MAPK pathway.

With the widespread use of manganese dioxide nanoparticles (nano MnO2), health hazards have also emerged. The inflammatory damage of brain tissues could result from nano MnO2, in which the underlying mechanism is still unclear. During this study, we aimed to investigate the role of ROS-mediated p38 MAPK pathway in nano MnO2-induced inflammatory response in BV2 microglial cells. The inflammatory injury model was established by treating BV2 cells with 2.5, 5.0, and 10.0μg/mL nano MnO2 suspensions for 12 h. Then, the reactive oxygen species (ROS) scavenger (20nM N-acetylcysteine, NAC) and the p38 MAPK pathway inhibitor (10μM SB203580) were used to clarify the role of ROS and the p38 MAPK pathway in nano MnO2-induced inflammatory lesions in BV2 cells. The results indicated that nano MnO2 enhanced the expression of pro-inflammatory cytokines IL-1β and TNF-α, elevated intracellular ROS levels and activated the p38 MAPK pathway in BV2 cells. Controlling intracellular ROS levels with NAC inhibited p38 MAPK pathway activation and attenuated the inflammatory response induced by nano MnO2. Furthermore, inhibition of the p38 MAPK pathway with SB203580 led to a decrease in the production of inflammatory factors (IL-1β and TNF-α) in BV2 cells. In summary, nano MnO2 can induce inflammatory damage by increasing intracellular ROS levels and further activating the p38 MAPK pathway in BV2 microglial cells.

Irisflorentin promotes bacterial phagocytosis and inhibits inflammatory responses in macrophages during bacterial infection

Bacterial infection remains a big concern in the patients of ICU, which is the main cause of life-threatening organ dysfunction, or even sepsis. The poor control of bacterial infection caused by antibiotic resistance, etc. or the overwhelming immune response are the most important patho genic factors in intensive care unit (ICU) patients. As main pathogens, antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), impose serious challenges during sepsis and require alternative therapeutic options. Irisflorentin (IFL) is one of the major bioactive compounds isolated from the roots of Belamcanda chinensis (Shegan). In this study, IFL could suppress inflammatory response induced by MRSA or a synthetic mimic of bacterial lipoprotein (Pam3CSK4). IFL treatment enhanced the ability of macrophages to phagocytose bacteria likely through up-regulating the expression of phagocytic receptors SR-A1 and FcγR2a. Furthermore, IFL inhibited Pam3CSK4-induced production of pro-inflammatory cytokines, including IL-6 and TNF-α in Raw 264.7 cells, mouse primary macrophages or dendritic cells. IFL treatment also inhibited heat-killed MRSA-induced secretion of IL-6 and TNF-α in mouse bone marrow-derived macrophages. Moreover, IFL attenuated M1 polarization of macrophages as indicated by the down-regulated expression of its polarization markers CD86 and iNOS. Mechanistically, IFL markedly decreased the Pam3CSK4-induced activation of ERK, JNK or p38 MAPK pathways in macrophages. Taken together, IFL may serve as a promising compound for the therapy of bacterial infection, particularly those caused by antibiotic-resistant bacteria, such as MRSA.

Oral glutamine supplementation relieves muscle loss in immobilized rats, altering p38MAPK and FOXO3a signaling pathways

BackgroundSkeletal muscle synthesizes, stores, and releases body L-glutamine (GLN). Muscle atrophy due to disabling diseases triggers the activation of proteolytic and pro-apoptotic cell signaling, thus impairing the body's capacity to manage GLN content. This situation has a poor therapeutic prognosis. ObjectiveEvaluating if oral GLN supplementation can attenuate muscle wasting mediated by elevated plasma cortisol and activation of caspase-3, p38MAPK, and FOXO3a signaling pathways in soleus and gastrocnemius muscles of rats submitted to 14-day bilateral hindlimbs immobilization. MethodsAnimals were randomly distributed into six groups: non-immobilized rats (Control), control orally supplemented with GLN (1 g kg−1) in solution with L-alanine (ALA: 0.61 g kg−1; GLN+ALA), control orally supplemented with dipeptide L-alanyl-L-glutamine (DIP; 1.49 g kg−1), hindlimbs immobilized rats (IMOB), IMOB orally GLN+ALA supplemented (GLN+ALA-IMOB), and IMOB orally DIP supplemented (DIP-IMOB). Plasma and muscle GLN concentration, plasma cortisol level, muscle caspase-3 activity, muscle p38MAPK and FOXO3a protein content (total and phosphorylated forms), and muscle cross-sectional area (CSA) were measured. ResultsCompared to controls, IMOB rats presented: a) increased plasma cortisol levels; b) decreased plasma and muscle GLN concentration; c) increased muscle caspase-3 activity; d) increased total and phosphorylated p38MAPK protein content; e) increased FOXO3a and decreased phosphorylated FOXO3a protein content; f) reduced muscle weight and CSA befitting to atrophy. Oral supplementation with GLN+ALA and DIP was able to significantly attenuate these effects. ConclusionsThese findings attest that oral GLN supplementation in GLN+ALA solution or DIP forms attenuates rats' skeletal muscle mass wasting caused by disuse-mediated muscle atrophy.

Single-cell transcriptome analysis indicates fatty acid metabolism-mediated metastasis and immunosuppression in male breast cancer

Male breast cancer (MBC) is a rare but aggressive malignancy with cellular and immunological characteristics that remain unclear. Here, we perform transcriptomic analysis for 111,038 single cells from tumor tissues of six MBC and thirteen female breast cancer (FBC) patients. We find that that MBC has significantly lower infiltration of T cells relative to FBC. Metastasis-related programs are more active in cancer cells from MBC. The activated fatty acid metabolism involved with FASN is related to cancer cell metastasis and low immune infiltration of MBC. T cells in MBC show activation of p38 MAPK and lipid oxidation pathways, indicating a dysfunctional state. In contrast, T cells in FBC exhibit higher expression of cytotoxic markers and immune activation pathways mediated by immune-modulatory cytokines. Moreover, we identify the inhibitory interactions between cancer cells and T cells in MBC. Our study provides important information for understanding the tumor immunology and metabolism of MBC.

Interfering with Dusp2 alleviates high glucose-induced vascular endothelial cell dysfunction by promoting p38 MAPK pathway activation

BackgroundHyperglycemia-induced vascular endothelial cell dysfunction is a major factor contributing to diabetic lower extremity ischemia. We intend to investigate the role of Dusp2 in hyperglycemia-induced vascular endothelial cell dysfunction and related mechanisms. MethodsThe human umbilical vein endothelial cells (HUVECs) were treated with high glucose (HG) as the cell model. Streptozotocin injection was performed to induce diabetes and femoral artery ligation was to induce hind limb ischemia in mice. The levels of Dusp2, p-p38 MAPK, E2F4, and p38 MAPK were evaluated by Western blot or quantitative real-time PCR. The laser Doppler perfusion imaging was conducted to measure blood flow recovery. The cell counting kit-8, transwell, and tube formation assay were performed to evaluate cell proliferation, migration, and angiogenesis, respectively. CD31 immunohistochemical staining was carried out to detect the capillary density of gastrocnemius. The dual-luciferase reporter gene assay and Chromatin immunoprecipitation assay were executed to explore the interaction between E2F4 and Dusp2. ResultsDusp2 was highly expressed in HG-induced HUVECs and diabetic lower extremity ischemia model mice. Interference with Dusp2 promoted cell proliferation, migration, and angiogenesis, as well as alleviated mouse diabetic hindlimb ischemia. Dusp2 knockdown up-regulated p-p38 MAPK levels. We verified the binding between E2F4 and Dusp2. Overexpressing E2F4 suppressed Dusp2 levels and promoted cell proliferation, migration, and angiogenesis, co-overexpression of Dusp2 reversed the results. ConclusionsOverexpressing E2F4 promotes endothelial cell proliferation, migration, and angiogenesis by inhibiting Dusp2 expression and activating p38 MAPK to alleviate vascular endothelial cell dysfunction under HG stimulation.

CHOP promotes coelomocyte apoptosis through p38-MAPK pathway in Vibrio splendidus-challenged sea cucumber Apostichopus japonicus.

CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) belongs to the C/EBP family of transcription factors that has been proven to regulate apoptosis in many vertebrate species. However, the functional role of CHOP in invertebrates is largely unknown. In this paper, the open reading frame of CHOP was cloned and characterized in the sea cucumber Apostichopus japonicus (AjCHOP). The deuced amino acid of AjCHOP shared a conserved RTP801_C domain from 63 to 171 aa. Phylogenetic analysis indicated that AjCHOP clustered with CHOPs from Lytechinus variegatus and Strongylocentrotus purpuratus. To confirm the immune function of AjCHOP, the time-course expression profiles of AjCHOP were investigated, and the findings revealed AjCHOP was significantly induced in coelomocytes at mRNA and protein levels after Vibro splendidus challenge. Furthermore, knockdown of AjCHOP in coelomocyes by siRNA transfection significantly decreased the apoptosis level induced by V. splendidus. Mechanically, AjCHOP-mediated apoptosis was dependent on the activation of p38-MAPK pathway but not JNK/ERK-MAPK. Overall, our results supported that V. splendidus triggers apoptosis among the coelomocytes, whereas AjCHOP mediates through the p38-MAPK pathway in A. japonicus.

L-borneol promotes neurovascular unit protection in the subacute phase of transient middle cerebral artery occlusion rats: p38-MAPK pathway activation, anti-inflammatory, and anti-apoptotic effect.

Our previous study showed l-borneol reduced cerebral infarction in the acute stage after cerebral ischemia, but there is little about the study of subacute phase. We herein investigated the cerebral protective effects of l-borneol on neurovascular units (NVU) in the subacute phase after transient middle cerebral artery occlusion (t-MCAO). The t-MCAO model was prepared by the line embolus method. Zea Longa, mNss, HE, and TTC staining were used to evaluate the effect of l-borneol. We evaluated the mechanisms of l-borneol on inflammation, p38 MAPK pathway, and apoptosis, etc. through various technologies. l-borneol 0.2, 0.1, 0.05 g·kg-1 could significantly reduce cerebral infarction rate, alleviate the pathological injury, and inhibit inflammation reaction. l-borneol could also significantly increase brain blood supply, Nissl bodies, and the expression of GFAP. Additionally, l-borneol activated the p38 MAPK signaling pathway, inhibited cell apoptosis, and maintained BBB integrity. l-borneol had a neuroprotective effect, which was related to activating the p38 MAPK signaling pathway, inhibiting inflammatory response and apoptosis, and improving cerebral blood supply to protect BBB and stabilize and remodel NVU. The study will provide a reference for the use of l-borneol in the treatment of ischemic stroke in the subacute phase.

Melatonin protects against NMDA-induced retinal ganglion cell injury by regulating the microglia-TNFα-RGC p38 MAPK pathway.

Glaucoma, one of the most common ocular neurodegenerative diseases worldwide, is characterized by retinal ganglion cell (RGC) loss. There is a large body of literature that describes the neuroprotective role of melatonin against neurodegenerative diseases by regulating neuroinflammation, although the exact mechanism through which melatonin acts on RGC is still uncertain. This study assessed the protective effects of melatonin using a NMDA-induced RGC injury model, and studied the possible mechanisms involved in this process. Melatonin promoted RGC survival, improved retinal function, and inhibited the apoptosis and necrosis of retinal cells. To understand the mechanism of the neuroprotective effects of melatonin on RGC, microglia and inflammation-related pathways were assessed after melatonin administration and microglia ablation. Melatonin promoted RGC survival by suppressing microglia-derived proinflammatory cytokines, in particular TNFα, which in turn inhibited the activation of p38 MAPK pathway. Inhibiting TNFα or manipulating p38 MAPK pathway protected damaged RGC. Our results suggest that melatonin protects against NMDA-induced RGC injury by inhibiting the microglial TNFα-RGC p38 MAPK pathway. It should be considered a candidate neuroprotective therapy against retinal neurodegenerative diseases.

Myricetin improves pathological changes in 3×Tg-AD mice by regulating the mitochondria-NLRP3 inflammasome-microglia channel by targeting P38 MAPK signaling pathway

BackgroundAlzheimer's disease (AD) represents the common neurodegenerative disease featured by the manifestations of cognitive impairment and memory loss. AD could be alleviated with medication and improving quality of life. Clinical treatment of AD is mainly aimed at improving the cognitive function of patients. Donepezil, memantine and galantamine are commonly used drug. But they could only relieve AD, not cure it. Therefore, new treatment strategies focusing on AD pathogenesis are of great significance and value. Myricetin (Myr) is a natural flavonoid extracted from Myrica rubra. And it shows different bioactivities, such as anti-inflammation, antioxidation as well as central nervous system (CNS) activities. Nonetheless, its associated mechanism in treating AD remains unknown. PurposeHere we focused on investigating Myr's effect on treating AD and exploring if its protection on the nervous system activity was associated with specifically inhibiting P38 MAPK signaling pathway while regulating mitochondria-NLRP3 inflammasome-microglia. Study design and methodsThis work utilized triple transgenic mice (3 × Tg-AD) as AD models and Aβ25–35 was used to induce BV2 cells to build an in vitro AD model. Behavioristics, pathology and related inflammatory factors were examined. Molecular mechanisms are investigated by western-blot, immunofluorescence staining, CETSA, molecular docking, network pharmacology. ResultsAccording to our findings, Myr could remarkably improve memory loss, spatial learning ability, Aβ plaque deposition, neuronal and synaptic damage in 3 × Tg-AD mice through specifically inhibiting P38 MAPK pathway activation while restraining microglial hyperactivation. Furthermore, Myr promoted the transformation of microglial phenotype, restored the mitochondrial fission-fusion balance, facilitated mitochondrial biogenesis, and restrained NLRP3 inflammasome activation and neuroinflammation. For the in-vitro experiments, P38 agonist dehydrocorydaline (DHC) was utilized to confirm the key regulatory role of P38 MAPK signaling pathway on the mitochondria-NLRP3 inflammasome-microglia channel. ConclusionsOur results revealed the therapeutic efficacy of Myr in experimental AD, and implied that the associated mechanism is possibly associated with inhibiting tmitochondrial dysfunction, activating NLRP3 inflammasome, and neuroinflammation which was mediated by P38 MAPK pathway. Myr is the drug candidate in AD therapy via targeting P38 MAPK pathway.

Porphyromonas gingivalis-induced periodontitis could contribute to cognitive impairment in Sprague-Dawley rats via the P38 MAPK signaling pathway.

Periodontitis is one of the most common oral diseases and has been shown to be a risk factor for systemic diseases. Our aim was to investigate the relationship between periodontitis and cognitive impairment and to explore the role of the P38 MAPK signaling pathway in this process. We established a periodontitis model by ligating the first molars of SD rats with silk thread and injecting Porphyromonas gingivalis (P. gingivalis) or P. gingivalis plus the P38 MAPK inhibitor SB203580 at the same time for ten weeks. We assessed alveolar bone resorption and spatial learning and memory using microcomputed tomography and the Morris water maze test, respectively. We used transcriptome sequencing to explore the genetic differences between the groups. The gingival tissue, peripheral blood and hippocampal tissue were assessed for the cytokines TNF-α, IL-1β, IL-6, IL-8 and C reactive protein (CRP) with enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). We observed the presence of P. gingivalis in the hippocampus of rats by paraffin-fluorescence in situ hybridization (FISH). We determined the activation of microglia by immunofluorescence. Finally, Western blot analysis was employed to determine the expression of amyloid precursor protein (APP), β-site APP-cleaving enzyme 1 (BACE1) and P38MAPK pathway activation. We demonstrated that silk ligature-induced periodontitis plus injection of P. gingivalis into subgingival tissue could lead to memory and cognitive impairment. Transcriptome sequencing results suggested that there were neurodegenerative diseases in the P. gingivalis group, and the MWM test showed that periodontitis reduced the spatial learning and memory ability of mild cognitive impairment (MCI) model rats. We found high levels of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) and CRP in the gingiva, peripheral blood and hippocampus, and the expression of APP and BACE1 was upregulated, as was the P38 MAPK pathway activation. Activated microglia and the presence of P. gingivalis were also found in the hippocampus. P38 MAPK inhibitors mitigated all of these changes. Our findings strongly suggest that topical application of P. gingivalis increases the inflammatory burden in the peripheral and central nervous systems (CNS) and that neuroinflammation induced by activation of P38 MAPK leads to impaired learning and memory in SD rats. It can also modulate APP processing. Therefore, P38 MAPK may serve as a linking pathway between periodontitis and cognitive impairment.

The long noncoding RNA Meg3 mediates TLR4-induced inflammation in experimental obstructive nephropathy.

Kidney inflammation contributes to the progression of chronic kidney disease (CKD). Modulation of Toll-like receptor 4 (TLR4) signaling is a potential therapeutic strategy for this pathology, but the regulatory mechanisms of TLR4 signaling in kidney tubular inflammation remains unclear. Here, we demonstrated that tubule-specific deletion of TLR4 in mice conferred protection against obstruction-induced kidney injury, with reduction in inflammatory cytokine production, macrophage infiltration and kidney fibrosis. Transcriptome analysis revealed a marked down-regulation of long noncoding RNA (lncRNA) Meg3 in the obstructed kidney from tubule-specific TLR4 knockout mice compared with wild-type control. Meg3 was also induced by lipopolysaccharide in tubular epithelial cells via a p53-dependent signaling pathway. Silencing of Meg3 suppressed LPS-induced cytokine production of CCL-2 and CXCL-2 and the activation of p38 MAPK pathway in vitro and ameliorated kidney fibrosis in mice with obstructive nephropathy. Together, these findings identify a proinflammatory role of lncRNA Meg3 in CKD and suggest a novel regulatory pathway in TLR4-driven inflammatory responses in tubular epithelial cells.

Low temperature exposure inhibits proliferation and induces apoptosis of bovine subcutaneous preadipocytes via p38 MAPK/JNK activation.

Cold stress can cause changes in cell growth states. Cell proliferation and apoptosis are important physiological processes to maintain normal growth and development of cells and tissues. We sought to identify the impact of low temperature exposure on bovine subcutaneous preadipocyte proliferation and apoptosis. We first cultured preadipocytes at different low temperatures (35°C, 33°C, and 31°C) for 12h, and then at 31°C for 24 and 48h. The results showed that compared with the 37°C group, exposure to 31°C significantly reduced cell viability and number as well as inhibited cell cycle progression in preadipocytes. Moreover, low temperature also significantly upregulated the apoptosis rate of preadipocytes. After low temperature treatment, the mRNA levels of Cyclin E, CDK2 and B-cell lymphoma 2 (Bcl-2) were decreased in preadipocytes, whereas that of p53, p21 and Bcl-2 associated x protein (Bax) were increased in preadipocytes. Concurrently, low temperature increased the proteins levels of p53, Bax and Cleaved Caspase3, and reduced the protein level of Bcl-2 in preadipocytes. Furthermore, the elevated phosphorylation levels of p38 mitogen-activated protein kinases (p38 MAPK) and c-jun NH2-terminal kinase (JNK) were detected in cold-treated preadipocytes. The influence of low temperature exposure on preadipocyte proliferation and apoptosis were obviously weakened after blocking the p38 MAPK and JNK signaling pathways. In conclusion, low temperature exposure could inhibit proliferation and cell cycle progression and induce apoptosis through activation of p38 MAPK and JNK signaling pathways in bovine subcutaneous preadipocytes.

Magnetofection of miR-21 promoted by electromagnetic field and iron oxide nanoparticles via the p38 MAPK pathway contributes to osteogenesis and angiogenesis for intervertebral fusion

BackgroundMagnetofection-mediated gene delivery shows great therapeutic potential through the regulation of the direction and degree of differentiation. Lumbar degenerative disc disease (DDD) is a serious global orthopaedic problem. However, even though intervertebral fusion is the gold standard for the treatment of DDD, its therapeutic effect is unsatisfactory. Here, we described a novel magnetofection system for delivering therapeutic miRNAs to promote osteogenesis and angiogenesis in patients with lumbar DDD.ResultsCo-stimulation with electromagnetic field (EMF) and iron oxide nanoparticles (IONPs) enhanced magnetofection efficiency significantly. Moreover, in vitro, magnetofection of miR-21 into bone marrow mesenchymal stem cells (BMSCs) and human umbilical endothelial cells (HUVECs) influenced their cellular behaviour and promoted osteogenesis and angiogenesis. Then, gene-edited seed cells were planted onto polycaprolactone (PCL) and hydroxyapatite (HA) scaffolds (PCL/HA scaffolds) and evolved into the ideal tissue-engineered bone to promote intervertebral fusion. Finally, our results showed that EMF and polyethyleneimine (PEI)@IONPs were enhancing transfection efficiency by activating the p38 MAPK pathway.ConclusionOur findings illustrate that a magnetofection system for delivering miR-21 into BMSCs and HUVECs promoted osteogenesis and angiogenesis in vitro and in vivo and that magnetofection transfection efficiency improved significantly under the co-stimulation of EMF and IONPs. Moreover, it relied on the activation of p38 MAPK pathway. This magnetofection system could be a promising therapeutic approach for various orthopaedic diseases.Graphical

Optimization of ultrasound-assisted extraction based on response surface methodology using HPLC-DAD for the analysis of red clover (Trifolium pretense L.) isoflavones and its anti-inflammatory activities on LPS-induced 3D4/2 cell.

Trifolium pratense L. has anti-inflammatory, antioxidant, cardiovascular disease prevention, and estrogen-like effects. The existing method for the assay of effective components is commonly based on a spectrophotometer, which could not meet the requirement of quality control. Furthermore, although there have been many studies on the anti-inflammation effect of red clover, a few have been reported on the regulatory effect of red clover isoflavones (RCI) on lipopolysaccharide (LPS)-induced inflammatory response in porcine alveolar macrophages (3D4/2 cells), and its mechanism of action is still unclear. The main components of RCI including daidzein, genistein, and biochanin A were accurately quantified by high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) after optimizing the extraction process through response surface methodology. The anti-inflammatory potential of RCI was carried out by detecting the level of inflammatory cytokines and mRNA expression of related genes. Furthermore, its anti-inflammatory mechanism was explored by investigating two signaling pathways (NF-κB and MAPK). The optimal extraction conditions of RCI were as follows: the concentration of ethanol is 86% and the solid-liquid ratio is 1:29, with the herb particle size of 40 mesh sieve. Under the optimal conditions, the total extraction of target components of RCI was 2,641.469 μg/g. The RCI could significantly suppress the production and expression of many pro-inflammatory cytokines. The results of the Western blot revealed that RCI dramatically reduced the expression of p65, p-p65, IκB-α, p38, and p-p38. These results are associated with the suppression of the signal pathway of p38 MAPK, and on the contrary, activating the NF-κB pathway. Collectively, our data demonstrated that RCI reversed the transcription of inflammatory factors and inhibited the expression of p65, p-p65, IκB-α, and p38, indicating that RCI had excellent anti-inflammatory properties through disturbing the activation of p38 MAPK and NF-κB pathways. The extraction conditions of RCI were optimized by HPLC-DAD combined with response surface methodology, which will contribute to the quality control of RCI. RCI had anti-inflammatory effects on the LPS-induced 3D4/2 cells. Its mechanism is to control the activation of NF-κB and p38 MAPK pathways, thereby reducing the expression of inflammatory-related genes and suppressing the release of cytokines.

Endoplasmic reticulum stress mediates nickel chloride-induced epithelial‑mesenchymal transition and migration of human lung cancer A549 cells through Smad2/3 and p38 MAPK activation

BackgroundThe endoplasmic reticulum (ER) is a cellular membrane-bound organelle whereby proteins are synthesized, folded and glycosylated. Due to intrinsic (e.g., genetic) and extrinsic (e.g., environmental stressors) perturbations, ER proteostasis can be deregulated within cells which triggers unfolded protein response (UPR) as an adaptive stress response that may impact the migration and invasion properties of cancer cells. However, the mechanisms underlying the nickel compounds on lung cancer cell migration and invasion remain uncertain. ObjectiveWe aimed to study whether Nickel chloride (NiCl2) induces ER stress in lung cancer cells, and whether ER stress is involved in modulating epithelial-mesenchymal transition (EMT) and migration by Smads and MAPKs pathways activation following NiCl2 treatment. MethodsA549 cells were treated with NiCl2 to determine the cell viability using MTT assay. The wound healing assay was used to evaluate cell migration ability. ER ultrastructure was observed by transmission electron microscopy. Western blotting assay was performed to evaluate the protein levels of BIP, PERK, IRE-1α, XBP-1 s, and ATF6 for ER stress and UPR, E-cadherin and Vimentin for EMT, p-Smad2/3, p-ERK, p-JNK, and p-P38 for activation of Smads and MAPKs signaling pathways. ResultsThe expression levels of BIP, PERK, IRE-1α, XBP-1 s, and ATF6 were significantly increased following treatment with NiCl2 in time- and dose-effect relationship. The ER stress inhibitor 4-PBA downregulated the expression levels of the above five proteins, and reversed the decrease in E-cadherin protein level and the increase in vimentin protein expression and cell migration abilities caused by NiCl2. Furthermore, 4-PBA significantly reduced nickel chloride-induced Smad2/3 and p38 MAPK pathway activation, while not affected ERK and JNK MAPK pathways. ConclusionNiCl2 triggers ER stress and UPR in A549 cells. Moreover, 4-PBA alleviates NiCl2-induced EMT and migration ability of A549 cells possibly through the Smad2/3 and p38 MAPK pathways activation, rather than ERK and JNK MAPK pathways.