Integrated Approaches Revealed the Therapeutic Mechanisms of Zuojin Pill Against Gastric Mucosa Injury in a Rat Model with Chronic Atrophic Gastritis.

Background: The Zuojin Pill (ZJP) is widely used for treating chronic atrophic gastritis (CAG) in clinical practice, effectively ameliorating symptoms such as vomiting, pain, and abdominal distension in patients. However, the underlying mechanisms of ZJP in treating CAG has not been fully elucidated. Purpose: This study aimed to clarify the characteristic function of ZJP in the treatment of CAG and its potential mechanism. Methods: The CAG model was established by alternant administrations of ammonia solution and sodium deoxycholate, as well as an irregular diet. Therapeutic effects of ZJP on body weight, serum biochemical indexes and general condition were analyzed. HE staining and AB-PAS staining were analyzed to characterize the mucosal injury and the thickness of gastric mucosa. Furthermore, network pharmacology and molecular docking were used to predict the regulatory mechanism and main active components of ZJP in CAG treatment. RT-PCR, immunohistochemistry, immunofluorescence and Western blotting were used to measure the expression levels of apoptosis-related proteins, gastric mucosal barrier-associated proteins and PI3K/Akt signaling pathway proteins. Results: The results demonstrated that ZJP significantly improved the general state of CAG rats, alleviated weight loss and gastric histological damage and reduced the serum biochemical indicators. Network pharmacology and molecular docking found that ZJP in treating CAG by inhibiting inflammation, suppressing apoptosis, and protecting the gastric mucosal barrier via the PI3K/Akt signaling pathway. Further experiments confirmed that ZJP obviously modulated the expression of key proteins involved in gastric mucosal cell apoptosis, such as Bax, Bad, Apaf-1, cleaved-caspase-3, cleaved-caspase-9, Cytochrome C, Bcl-2, and Bcl-xl. Moreover, ZJP significantly reversed the protein expression of Occludin, ZO-1, Claudin-4 and E-cadherin. Conclusion: Our study revealed that ZJP treats CAG by inhibiting the PI3K/Akt signaling pathway. This research provided a scientific basis for the rational use of ZJP in clinical practice.

PI3K/AKT Signaling Pathway Mediated Autophagy in Oral Carcinoma - A Comprehensive Review.

Oral cancer is the most heterogeneous cancer at clinical and histological levels. PI3K/AKT/mTOR pathway was identified as one of the most commonly modulated signals in oral cancer, which regulates major cellular and metabolic activity of the cell. Thus, various proteins of PI3K/AKT/mTOR pathway were used as therapeutic targets for oral cancer, to design more specific drugs with less off-target toxicity. This review sheds light on the regulation of PI3K/AKT/mTOR, and its role in controlling autophagy and associated apoptosis during the progression and metastasis of oral squamous type of malignancy (OSCC). In addition, we reviewed in detail the upstream activators and the downstream effectors of PI3K/AKT/mTOR signaling as potential therapeutic targets for oral cancer treatment.

Crocin ameliorates neuroinflammation and cognitive impairment in mice with Alzheimer's disease by activating PI3K/AKT pathway.

BACKGROUND: Crocin has a good prospect in the treatment of Alzheimer's disease (AD), but the mechanisms underlying its neuroprotective effects remain elusive. This study aimed to investigate the neuroprotective effects of Crocin and its underlying mechanisms in AD. METHODS: AD mice were set up by injecting Aß25-35 solution into the hippocampus. Then, the AD mice were injected intraperitoneally with 40 mg/kg/day of Crocin for 14 days. Following the completion of Crocin treatment, an open-field test, Y-maze test and Morris water maze test were conducted to evaluate the impact of Crocin on spatial learning and memory deficiency in mice. The effects of Crocin on hippocampal neuron injury, proinflammatory cytokine expressions (IL-1ß, IL-6, and TNF-α), and PI3K/AKT signaling-related protein expressions were measured using hematoxylin and eosin staining, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) experiments, respectively. RESULTS: Crocin attenuated Aß25-35-induced spatial learning and memory deficiency and hippocampal neuron injury. Furthermore, the Western blot and qRT-PCR results showed that Crocin effectively suppressed inflammation and activated the PI3K/AKT pathway in Aß25-35-induced mice. CONCLUSION: Crocin restrained neuroinflammation via the activation of the PI3K/AKT pathway, thereby ameliorating the cognitive dysfunction of AD mice.

ALDH1A1 confers resistance to RAF/MEK inhibitors in melanoma cells by maintaining stemness phenotype and activating PI3K/AKT signaling.

The mitogen-activated protein kinase (MAPK/ERK) pathway is pivotal in controlling the proliferation and survival of melanoma cells. Several mutations, including those in BRAF, exhibit an oncogenic effect leading to increased cellular proliferation. As a result, the combination therapy of a MEK inhibitor with a BRAF inhibitor demonstrated higher efficacy and lower toxicity than BRAF inhibitor alone. This combination has become the preferred standard of care for tumors driven by BRAF mutations. Aldehyde dehydrogenase 1A1 (ALDH1A1) is a known marker of stemness involved in drug resistance in several type of tumors, including melanoma. This study demonstrates that melanoma cells overexpressing ALDH1A1 displayed resistance to vemurafenib and trametinib through the activation of PI3K/AKT signaling instead of MAPK axis. Inhibition of PI3K/AKT signaling partially rescued sensitivity to the drugs. Consistently, pharmacological inhibition of ALDH1A1 activity downregulated the activation of AKT and partially recovered responsiveness to vemurafenib and trametinib. We propose ALDH1A1 as a new potential target for treating melanoma resistant to MAPK/ERK inhibitors.

Duloxetine protected indomethacin-induced gastric mucosal injury by increasing serotonin-dependent RANTES expression and activating PI3K-AKT-VEGF pathway.

Antidepressant duloxetine has been shown protective effect on indomethacin-induced gastric ulcer, which was escorted by inflammation in the gastric mucosa. Cytokines are the principal mediators of inflammation. Thus, by screening the differential expression of cytokines in the gastric mucosa using cytokine array at 3 h after indomethacin exposure, when the gastric ulcer began to format, we found that indomethacin increased cytokines which promoted inflammation responses, whereas duloxetine decreased pro-inflammatory cytokines increased by indomethacin and increased RANTES expression. RANTES was consistently increased by pretreated with both 5 mg/kg and 20 mg/kg duloxetine at 3 h and 6 h after indomethacin exposure in male rats. Selective blockade of RANTES-CCR5 axis by a functional antagonist Met-RANTES or a CCR5 antagonist maraviroc suppressed the protection of duloxetine. Considering the pharmacologic action of duloxetine on reuptake of monoamine neurotransmitters, we examined the serotonin (5-HT), norepinephrine and dopamine contents in the blood and discovered 20 mg/kg duloxetine increased 5-HT levels in platelet-poor plasma, while treatment with 5-HT promoted expression of RANTES in the gastric mucosa and alleviated the indomethacin-induced gastric injury. Furthermore, duloxetine activated PI3K-AKT-VEGF signaling pathway, which was regulated by RANTES-CCR5, and selective inhibitor of VEGF receptor axitinib blocked the prophylactic effect of duloxetine. Furthermore, duloxetine also protected gastric mucosa from indomethacin in female rats, and RANTES was increased by duloxetine after 6 h after indomethacin exposure too. Together, our results identified the role of cytokines, particularly RANTES, and the underlying mechanisms in gastroprotective effect of duloxetine against indomethacin, which advanced our understanding in inflammatory modulation by monoamine-based antidepressants.

Mitigating cognitive deficits with teriflunomide: unraveling PI3K-modulated behavioral outcomes in mice.

BACKGROUND: Alzheimer's disease is a leading neurological disorder that gradually impairs memory and cognitive abilities, ultimately leading to the inability to perform even basic daily tasks. Teriflunomide is known to preserve neuronal activity and protect mitochondria in the brain slices exposed to oxidative stress. The current research was undertaken to investigate the teriflunomide's cognitive rescuing abilities against scopolamine-induced comorbid cognitive impairment and its influence on phosphatidylinositol-3-kinase (PI3K) inhibition-mediated behavior alteration in mice. METHODS: Swiss albino mice were divided into 7 groups; vehicle control, scopolamine, donepezil + scopolamine, teriflunomide (10 mg/kg) + scopolamine; teriflunomide (20 mg/kg) + scopolamine, LY294002 and LY294002 + teriflunomide (20 mg/kg). Mice underwent a nine-day protocol, receiving scopolamine injections (2 mg/kg) for the final three days to induce cognitive impairment. Donepezil, teriflunomide, and LY294002 treatments were given continuously for 9 days. MWM, Y-maze, OFT and rota-rod tests were conducted on days 7 and 9. On the last day, blood samples were collected for serum TNF-α analysis, after which the mice were sacrificed, and brain samples were harvested for oxidative stress analysis. RESULTS: Scopolamine administration for three consecutive days increased the time required to reach the platform in the MWM test, whereas, reduced the percentage of spontaneous alternations in the Y-maze, number of square crossing in OFT and retention time in the rota-rod test. In biochemical analysis, scopolamine downregulated the brain GSH level, whereas it upregulated the brain TBARS and serum TNF-α levels. Teriflunomide treatment effectively mitigated all the behavioral and biochemical alterations induced by scopolamine. Furthermore, LY294002 administration reduced the memory function and GSH level, whereas, uplifted the serum TNF-α levels. Teriflunomide abrogated the memory-impairing, GSH-lowering, and TNF-α-increasing effects of LY294002. CONCLUSION: Our results delineate that the improvement in memory, locomotion, and motor coordination might be attributed to the oxidative and inflammatory stress inhibitory potential of teriflunomide. Moreover, PI3K inhibition-induced memory impairment might be attributed to reduced GSH levels and increased TNF-α levels.

SDF-1 promotes metastasis of NSCLC by enhancing chemoattraction of megakaryocytes through the PI3K/Akt signaling pathway.

Lung cancer (LC) is the leading cause of cancer-associated deaths worldwide, among which non-small-cell lung cancer (NSCLC) accounts for 80%. Stromal cell-derived factor-1 (SDF-1) inhibition results in a significant depletion of NSCLC metastasis. Additionally, SDF-1 is the only natural chemokine known to bind and activate the receptor CXCR4. Thus, we attempted to clarify the molecular mechanism of SDF-1 underlying NSCLC progression. Transwell migration, adhesion, and G-LISA assays were used to assess megakaryocytic chemotaxis in vitro and in vivo in terms of megakaryocytic migration, adherence, and RhoA activation, respectively. Western blotting was used to assess PI3K/Akt-associated protein abundances in MEG-01 cells and primary megakaryocytes under the indicated treatment. A hematology analyzer and flow cytometry were used to assess platelet counts in peripheral blood and newly formed platelet counts in Lewis LC mice under different treatments. Immunochemistry and flow cytometry were used to measure CD41+ megakaryocyte numbers in Lewis LC mouse tissue under different treatments. ELISA was used to measure serum TPO levels, and H&E staining was used to detect NSCLC metastasis.SDF-1 receptor knockdown suppressed megakaryocytic chemotaxis in Lewis LC mice. SDF-1 receptor inhibition suppressed megakaryocytic chemotaxis via the PI3K/Akt pathway. SDF-1 receptor knockdown suppressed CD41+ megakaryocyte numbers in vivo through PI3K/Akt signaling. SDF-1 receptor inhibition suppressed CD41+ megakaryocytes to hinder NSCLC metastasis. SDF-1 facilitates NSCLC metastasis by enhancing the chemoattraction of megakaryocytes via the PI3K/Akt signaling pathway, which may provide a potential new direction for seeking therapeutic plans for NSCLC.

Campothecin suppresses cell proliferation and migration in head and neck squamous cell carcinoma by blocking RAB27A-mediated phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway.

Camptothecin (CPT), a naturally occurring alkaloid derived from the Camptotheca acuminate plant, exerts anti-tumor properties. However, its specific impact on head and neck squamous cell carcinoma (HNSCC) remains uncertain. The study was to explore the action and mechanism of CPT on HNSCC cells. First, two HNSCC cell lines (FaDu and TU686) and a normal immortalized keratinocyte (HEK001) cell line, were exposed to a spectrum of CPT concentrations (ranging from 10 to 50 µM) for durations of 24 h and 48 h. Cell viability, proliferation, migration, and invasion were assessed by CCK-8 assay, EdU incorporation assay, wound healing assay and transwell assay. Subsequently, si-RAB27A or negative control (NC) was introduced into FaDu and TU686 cells through transfection, and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway was manipulated with L740Y-P, an activator of this pathway. The expression of proliferating cell nuclear antigen (PCNA), E-cadherin, PI3K/AKT signaling factors and RAB27A were determined by Western blot analysis. RAB27A was detected by immunofluorescence assay. It was found that CPT significantly hindered the viability, proliferation (p<0.01), migration (p<0.001), and invasion (p<0.001) of FaDu and TU686 cells. At the molecular level, administration of CPT caused a decline in the expression of PCNA, P-PI3K, P-AKT, and RAB27A, alongside an elevation in E-cadherin levels within HNSCC cells (p<0.05, p<0.01 and p<0.001). Reducing RAB27A expression enhanced the suppressive impacts of CPT on HNSCC cell viability (p<0.05 and p<0.01), migration (p<0.001) and invasion (p<0.01), these effects that were reversed upon treatment with L740Y-P in HNSCC cells (p<0.001). In summary, our study highlights the efficacy of CPT in HNSCC, demonstrating its influence on cell processes via the RAB27A-mediated PI3K/AKT pathway.

PI3K/mTOR inhibition induces tumour microenvironment remodelling and sensitises pS6high uterine leiomyosarcoma to PD-1 blockade.

BACKGROUND: Uterine leiomyosarcomas (uLMS) are aggressive tumours with poor prognosis and limited treatment options. Although immune checkpoint blockade (ICB) has proven effective in some 'challenging-to-treat' cancers, clinical trials showed that uLMS do not respond to ICB. Emerging evidence suggests that aberrant PI3K/mTOR signalling can drive resistance to ICB. We therefore explored the relevance of the PI3K/mTOR pathway for ICB treatment in uLMS and explored pharmacological inhibition of this pathway to sensitise these tumours to ICB. METHODS: We performed an integrated multiomics analysis based on TCGA data to explore the correlation between PI3K/mTOR dysregulation and immune infiltration in 101 LMS. We assessed response to PI3K/mTOR inhibitors in immunodeficient and humanized uLMS patient-derived xenografts (PDXs) by evaluating tumour microenvironment modulation using multiplex immunofluorescence. We explored response to single-agent and a combination of PI3K/mTOR inhibitors with PD-1 blockade in humanized uLMS PDXs. We mapped intratumoural dynamics using single-cell RNA/TCR sequencing of serially collected biopsies. RESULTS: PI3K/mTOR over-activation (pS6high) associated with lymphocyte depletion and wound healing immune landscapes in (u)LMS, suggesting it contributes to immune evasion. In contrast, PI3K/mTOR inhibition induced profound tumour microenvironment remodelling in an ICB-resistant humanized uLMS PDX model, fostering adaptive anti-tumour immune responses. Indeed, PI3K/mTOR inhibition induced macrophage repolarisation towards an anti-tumourigenic phenotype and increased antigen presentation on dendritic and tumour cells, but also promoted infiltration of PD-1+ T cells displaying an exhausted phenotype. When combined with anti-PD-1, PI3K/mTOR inhibition led to partial or complete tumour responses, whereas no response to single-agent anti-PD-1 was observed. Combination therapy reinvigorated exhausted T cells and induced clonal hyper-expansion of a cytotoxic CD8+ T-cell population supported by a CD4+ Th1 niche. CONCLUSIONS: Our findings indicate that aberrant PI3K/mTOR pathway activation contributes to immune escape in uLMS and provides a rationale for combining PI3K/mTOR inhibition with ICB for the treatment of this patient population.

PM2.5 toxicity in blood impairs cardiac redox balance and promotes mitochondrial dysfunction in rat heart that further aggravates ischemia reperfusion injury by modulating PI3K/AKT/mTOR/NF-kB signaling axis.

According to the pathophysiological mechanisms linking particulate matter (PM2.5) exposure and cardiovascular diseases, PM2.5 may directly translocate into the blood stream and remote target organs and thereby induce cardiovascular effects. The toxicity of PM2.5 is known to induce oxidative stress in pulmonary tissue, but its impact on the redox state in heart (distant organ) is unknown and how it modulates the cardiac response to ischemia reperfusion (IR) remains unclear. In the present study, we evaluated the toxic effect of PM2.5 on cardiac physiology in the presence and absence of IR after introducing PM2.5 into the blood. Female Wistar rats were injected with diesel particulate matter (DPM) via i.p & i.v routes at a concentration of 10 µg/ml. The toxic impact of PM2.5 not only adversely affects the cardiac ultra-structure (leading to nuclear infiltration, edema, irregularities in heart muscle and nuclear infiltration), but also altered the cellular redox balance, elevated inflammation and promoted the upregulation of proapoptotic mediator genes at the basal level of myocardium. The results showed alterations in cardiac ultrastructure, elevated oxidative stress and significant redox imbalance, increased inflammation and proapoptotic mediators at the basal level of myocardium. Moreover, the cardioprotective pro survival signaling axis was declined along with an increased NF-kB activation at the basal level. IR inflicted further injury with deterioration of cardiac hemodynamic indices (Heart rate [HR], Left ventricular developed pressure [LVDP], Left ventricular end-diastolic pressure [LVEDP] and rate pressure product [RPP]) along with prominent inactivation of signaling pathways. Furthermore, the levels of GSH/GSSG, NADH/NAD, NADPH/NADP were significantly low along with increased lipid peroxidation in mitochondria of PM2.5 treated IR rat hearts. This observation was supported by downregulation of glutaredoxin and peroxiredoxin genes in the myocardium. Similarly the presence of oxidative stress inducing metals was found at a higher concentration in cardiac mitochondria. Thus, the toxic impact of PM2.5 in heart augment the IR associated pathological changes by altering the physiological response, initiating cellular metabolic alterations in mitochondria and modifying the signaling molecules.

Aspirin use and changes in circulating tumor DNA levels in patients with metastatic colorectal cancer.

The study aimed to investigate the effects of aspirin on patients with metastatic colorectal cancer, focusing on circulating tumor DNA levels and bone tissue. Two groups (A and B) of ten patients with osteoporosis were selected for the study. Bone tissue samples were obtained from the patients and cultured under sterile conditions. The aspirin group showed a significant decrease in circulating tumor DNA levels and an increase in bone tissue density compared to the control group. Additionally, osteoblast apoptosis was reduced, while proliferation was enhanced in the aspirin group. The protein pAkt related to the PI3K/Akt signaling pathway was upregulated in the aspirin group. These results indicate that aspirin can effectively lower circulating tumor DNA levels, promote bone tissue proliferation, inhibit apoptosis, and activate the PI3K/Akt signaling pathway, thereby influencing bone cell function. These findings provide a basis for aspirin's potential application in treating metastatic colorectal cancer and encourage further research on its mechanism and clinical use.

Coenzyme Q10 prevents RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways.

Coenzyme Q10 (CoQ10) is a potent antioxidant that is implicated in the inhibition of osteoclastogenesis, but the underlying mechanism has not been determined. We explored the underlying molecular mechanisms involved in this process. RAW264.7 cells received receptor activator of NF-κB ligand (RANKL) and CoQ10, after which the differentiation and viability of osteoclasts were assessed. After the cells were treated with CoQ10 and/or H2O2 and RANKL, the levels of reactive oxygen species (ROS) and proteins involved in the PI3K/AKT/mTOR and MAPK pathways and autophagy were tested. Moreover, after the cells were pretreated with or without inhibitors of the two pathways or with the mitophagy agonist, the levels of autophagy-related proteins and osteoclast markers were measured. CoQ10 significantly decreased the number of TRAP-positive cells and the level of ROS but had no significant impact on cell viability. The relative phosphorylation levels of PI3K, AKT, mTOR, ERK, and p38 were significantly reduced, but the levels of FOXO3/LC3/Beclin1 were significantly augmented. Moreover, the levels of FOXO3/LC3/Beclin1 were significantly increased by the inhibitors and mitophagy agonist, while the levels of osteoclast markers showed the opposite results. Our data showed that CoQ10 prevented RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways in RAW264.7 cells.

Sinomenine attenuates pulmonary fibrosis by downregulating TGF-ß1/Smad3, PI3K/Akt and NF-κB signaling pathways.

BACKGROUND: Since COVID-19 became a global epidemic disease in 2019, pulmonary fibrosis (PF) has become more prevalent among persons with severe infections, with IPF being the most prevalent form. In traditional Chinese medicine, various disorders are treated using Sinomenine (SIN). The SIN's strategy for PF defense is unclear. METHODS: Bleomycin (BLM) was used to induce PF, after which inflammatory factors, lung histological alterations, and the TGF-/Smad signaling pathway were assessed. By administering various dosages of SIN and the TGF- receptor inhibitor SB-431,542 to human embryonic lung fibroblasts (HFL-1) and A549 cells, we were able to examine proliferation and migration as well as the signaling molecules implicated in Epithelial-Mesenchymal Transition (EMT) and Extra-Cellular Matrix (ECM). RESULTS: In vivo, SIN reduced the pathological changes in the lung tissue induced by BLM, reduced the abnormal expression of inflammatory cytokines, and improved the weight and survival rate of mice. In vitro, SIN inhibited the migration and proliferation by inhibiting TGF-ß1/Smad3, PI3K/Akt, and NF-κB pathways, prevented the myofibroblasts (FMT) of HFL-1, reversed the EMT of A549 cells, restored the balance of matrix metalloenzymes, and reduced the expression of ECM proteins. CONCLUSION: SIN attenuated PF by down-regulating TGF-ß/Smad3, PI3K/Akt, and NF-κB signaling pathways, being a potential effective drug in the treatment of PF.

Transcranial Magneto-Acoustic Stimulation Protects Synaptic Rehabilitation from Amyloid-Beta Plaques via Regulation of Microglial Functions.

Transcranial magneto-acoustic stimulation (TMAS), which is characterized by high spatiotemporal resolution and high penetrability, is a non-invasive neuromodulation technology based on the magnetic-acoustic coupling effect. To reveal the effects of TMAS treatment on amyloid-beta (Aß) plaque and synaptic plasticity in Alzheimer's disease, we conducted a comparative analysis of TMAS and transcranial ultrasound stimulation (TUS) based on acoustic effects in 5xFAD mice and BV2 microglia cells. We found that the TMAS-TUS treatment effectively reduced amyloid plaque loads and plaque-associated neurotoxicity. Additionally, TMAS-TUS treatment ameliorated impairments in long-term memory formation and long-term potentiation. Moreover, TMAS-TUS treatment stimulated microglial proliferation and migration while enhancing the phagocytosis and clearance of Aß. In 5xFAD mice with induced microglial exhaustion, TMAS-TUS treatment-mediated Aß plaque reduction, synaptic rehabilitation improvement, and the increase in phospho-AKT levels were diminished. Overall, our study highlights that stimulation of hippocampal microglia by TMAS treatment can induce anti-cognitive impairment effects via PI3K-AKT signaling, providing hope for the development of new strategies for an adjuvant therapy for Alzheimer's disease.

Cinnamaldehyde Protects against P. gingivalis Induced Intestinal Epithelial Barrier Dysfunction in IEC-6 Cells via the PI3K/Akt-Mediated NO/Nrf2 Signaling Pathway.

Porphyromonas gingivalis (Pg), a Gram-negative oral pathogen, promotes and accelerates periodontitis-associated gut disorders. Intestinal epithelial barrier dysfunction is crucial in the pathogenesis of intestinal and systemic diseases. In this study, we sought to elucidate the protective role of cinnamaldehyde (CNM, an activator of Nrf2) against P. gingivalis (W83) and Pg-derived lipopolysaccharide (Pg-LPS) induced intestinal epithelial barrier dysfunction via antioxidative mechanisms in IEC-6 cells. IEC-6 (ATCC, CRL-1592) cells were pretreated with or without CNM (100 µM), in the presence or absence of P. gingivalis (strain W83, 109 MOI) or Pg-LPS (1, 10, and 100 µg/mL), respectively, between 0-72 h time points by adopting a co-culture method. Intestinal barrier function, cytokine secretion, and intestinal oxidative stress protein markers were analyzed. P. gingivalis or Pg-LPS significantly (p < 0.05) increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels expressing oxidative stress damage. Pg-LPS, as well as Pg alone, induces inflammatory cytokines via TLR-4 signaling. Furthermore, infection reduced Nrf2 and NAD(P)H quinone dehydrogenase 1 (NQO1). Interestingly, inducible nitric oxide synthase (iNOS) protein expression significantly (p < 0.05) increased with Pg-LPS or Pg infection, with elevated levels of nitric oxide (NO). CNM treatment suppressed both Pg- and Pg-LPS-induced intestinal oxidative stress damage by reducing ROS, MDA, and NO production. Furthermore, CNM treatment significantly upregulated the expression of tight junction proteins via increasing the phosphorylation levels of PI3K/Akt/Nrf2 suppressing inflammatory cytokines. CNM protected against Pg infection-induced intestinal epithelial barrier dysfunction by activating the PI3K/Akt-mediated Nrf2 signaling pathway in IEC-6 cells.

Molecular dissection of PI3Kß synergistic activation by receptor tyrosine kinases, GßGγ, and Rho-family GTPases.

Phosphoinositide 3-kinase (PI3K) beta (PI3Kß) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kß prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kß when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kß membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

LAMC2 regulates the proliferation, invasion, and metastasis of gastric cancer via PI3K/Akt signaling pathway.

OBJECTIVES: Gastric cancer (GC) is a prevalent malignant tumor widely distributed globally, exhibiting elevated incidence and fatality rates. The gene LAMC2 encodes the laminin subunit gamma-2 chain and is found specifically in the basement membrane of epithelial cells. Its expression is aberrant in multiple types of malignant tumors. This research elucidated a link between LAMC2 and the clinical characteristics of GC and investigated the potential involvement of LAMC2 in GC proliferation and advancement. MATERIALS AND METHODS: LAMC2 expressions were detected in GC cell lines and normal gastric epithelial cell lines via qRT-PCR. Silencing and overexpression of the LAMC2 were conducted by lentiviral transfection. A xenograft mouse model was also developed for in vivo analysis. Cell functional assays were conducted to elucidate the involvement of LAMC2 in cell growth, migration, and penetration. Further, immunoblotting was conducted to investigate the impact of LAMC2 on the activation of signal pathways after lentiviral transfection. RESULTS: In the findings, LAMC2 expression was markedly upregulated in GC cell lines as opposed to normal gastric epithelial cells. In vitro analysis showed that sh-LAMC2 substantially inhibited GC cell growth, migration, and invasion, while oe-LAMC2 displayed a contrasting effect. Xenograft tumor models demonstrated that oe-LAMC2 accelerated tumor growth via high expression of Ki-67. Immunoblotting analysis revealed a substantial decrease in various signaling pathway proteins, PI3K, p-Akt, and Vimentin levels upon LAMC2 knockdown, followed by increased E-cadherin expression. Conversely, its overexpression exhibited contrasting effects. Besides, epithelial-mesenchymal transition (EMT) was accelerated by LAMC2. CONCLUSION: This study provides evidence indicating that LAMC2, by stimulating signaling pathways, facilitated EMT and stimulated the progression of GC cells in laboratory settings and mouse models. Research also explored that the abnormal LAMC2 expression acts as a biomarker for GC.

Fuzhengjiedu San inhibits porcine reproductive and respiratory syndrome virus by activating the PI3K/AKT pathway.

BACKGROUND: Traditional Chinese medicine (TCM) has been garnering ever-increasing worldwide attention as the herbal extracts and formulas prove to have potency against disease. Fuzhengjiedu San (FZJDS), has been extensively used to treat viral diseases in pigs, but its bioactive components and therapeutic mechanisms remain unclear. METHODS: In this study, we conducted an integrative approach of network pharmacology and experimental study to elucidate the mechanisms underlying FZJDS's action in treating porcine reproductive and respiratory syndrome virus (PRRSV). We constructed PPI network and screened the core targets according to their degree of value. GO and KEGG enrichment analyses were also carried out to identify relevant pathways. Lastly, qRT-PCR, flow cytometry and western blotting were used to determine the effects of FZJDS on core gene expression in PRRSV-infected monkey kidney (MARC-145) cells to further expand the results of network pharmacological analysis. RESULTS: Network pharmacology data revealed that quercetin, kaempferol, and luteolin were the main active compounds of FZJDS. The phosphatidylinositol-3-kinase (PI3K)/Akt pathway was deemed the cellular target as it has been shown to participate most in PRRSV replication and other PRRSV-related functions. Analysis by qRT-PCR and western blotting demonstrated that FZJDS significantly reduced the expression of P65, JNK, TLR4, N protein, Bax and IĸBa in MARC-145 cells, and increased the expression of Bcl-2, consistent with network pharmacology results. This study provides that FZJDS has significant antiviral activity through its effects on the PI3K/AKT signaling pathway. CONCLUSION: We conclude that FZJDS is a promising candidate herbal formulation for treating PRRSV and deserves further investigation.

Lycopene promoted M2 macrophage polarization via inhibition of NOTCH1-PI3K-mTOR-NF-κB-JMJD3-IRF4 pathway in response to Escherichia coli infection in J744A.1 cells.

Escherichia coli (E. coli) can induce severe clinical bovine mastitis, which is to blame for large losses experienced by dairy farms. Macrophage polarization into various states is in response to pathogen infections. Lycopene, a naturally occurring hydrocarbon carotenoid, relieved inflammation by controlling M1/M2 status of macrophages. Thus, we wanted to explore the effect of lycopene on polarization states of macrophages in E. coli-induced mastitis. Macrophages were cultivated with lycopene for 24, before E. coli inoculation for 6 h. Lycopene (0.5 µmol/L) significantly enhanced cell viabilities and significantly reduced lactic dehydrogenase (LDH) levels in macrophages, whereas 2 and 3 µmol/L lycopene significantly enhanced LDH activities. Lycopene treatment significantly reduced the increase in LDH release, iNOS, CD86, TNF-α, IL-1ß and phosphatase and tensin homolog (PTEN) expressions in E. coli group. 0.5 µmol/L lycopene significantly increased E. coli-induced downregulation of CD206, arginase I (ARG1), indoleamine 2,3-dioxygenase (IDO), chitinase 3-like 3 (YM1), PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, jumonji domain-containing protein-3 (JMJD3) and interferon regulatory factor 4 (IRF4) levels. Moreover, Ginkgolic acid C17:1 (a specific PTEN inhibitor), 740YPDGFR (a specific PI3K activator), SC79 (a specific AKT activator) or CHPG sodium salt (a specific NF-κB activator) significantly decreased CD206, AGR1, IDO and YM1 expressions in lycopene and E. coli-treated macrophages. Therefore, lycopene increased M2 macrophages via inhibiting NOTCH1-PI3K-mTOR-NF-κB-JMJD3-IRF4 pathway in response to E. coli infection in macrophages. These results contribute to revealing the pathogenesis of E. coli-caused bovine mastitis, providing the new angle of the prevention and management of mastitis.

Ti3C2 nanosheet-induced autophagy derails ovarian functions.

BACKGROUND: Two-dimensional ultrathin Ti3C2 (MXene) nanosheets have gained significant attention in various biomedical applications. Although previous studies have described the accumulation and associated damage of Ti3C2 nanosheets in the testes and placenta. However, it is currently unclear whether Ti3C2 nanosheets can be translocated to the ovaries and cause ovarian damage, thereby impairing ovarian functions. RESULTS: We established a mouse model with different doses (1.25, 2.5, and 5 mg/kg bw/d) of Ti3C2 nanosheets injected intravenously for three days. We demonstrated that Ti3C2 nanosheets can enter the ovaries and were internalized by granulosa cells, leading to a decrease in the number of primary, secondary and antral follicles. Furthermore, the decrease in follicles is closely associated with higher levels of FSH and LH, as well as increased level of E2 and P4, and decreased level of T in mouse ovary. In further studies, we found that exposure toTi3C2 nanosheets increased the levels of Beclin1, ATG5, and the ratio of LC3II/Ι, leading to autophagy activation. Additionally, the level of P62 increased, resulting in autophagic flux blockade. Ti3C2 nanosheets can activate autophagy through the PI3K/AKT/mTOR signaling pathway, with oxidative stress playing an important role in this process. Therefore, we chose the ovarian granulosa cell line (KGN cells) for in vitro validation of the impact of autophagy on the hormone secretion capability. The inhibition of autophagy initiation by 3-Methyladenine (3-MA) promoted smooth autophagic flow, thereby partially reduced the secretion of estradiol and progesterone by KGN cells; Whereas blocking autophagic flux by Rapamycin (RAPA) further exacerbated the secretion of estradiol and progesterone in cells. CONCLUSION: Ti3C2 nanosheet-induced increased secretion of hormones in the ovary is mediated through the activation of autophagy and impairment of autophagic flux, which disrupts normal follicular development. These results imply that autophagy dysfunction may be one of the underlying mechanisms of Ti3C2-induced damage to ovarian granulosa cells. Our findings further reveal the mechanism of female reproductive toxicity induced by Ti3C2 nanosheets.