P65 Protein Expression Research Articles

Astragaloside IV Protects Against IL-1β-Induced Chondrocyte Damage via Activating Autophagy.

Osteoarthritis (OA) is a chronic inflammatory condition that affects the articular cartilage. Astragaloside IV (AS-IV) constitutes the primary active component of the Chinese herbal medicine Huangqi (Radix Astragali Mongolici). AS-IV demonstrates anti-inflammatory and anti-apoptotic attributes, exhibiting therapeutic potential across various inflammatory and apoptosis-related disorders. Nevertheless, its pharmaceutical effects in OA are yet to be fully defined. This study aimed to investigate the protective impact of AS-IV on rat chondrocytes treated with IL-1β and ascertain whether autophagy plays a role in this effect. Chondrocytes were isolated and cultivated from the knee joints of neonatal SD mice. The study included the blank control group, the model group, and the AS-IV concentration gradient group (50, 100, 200 μmol/L) to intervene with chondrocytes. The MTT assay was employed to assess cell viability at varying culture periods, enabling the determination of suitable concentration and duration. Subsequently, chondrocytes were treated with the optimal AS-IV concentration and divided into three groups: the model group replicated IL-1β-induced inflammatory chondrocyte injury, the AS-IV group received a co-culture of AS-IV and IL-1β, and a blank control group was established. Changes in cell morphology and structure were observed using ghost pen cyclic peptide staining. ELISA was used to measure TNF-α and GAG levels in cell supernatants. RT-qPCR assessed p62 and LC3 mRNA expression, while Western Blot evaluated p62 and LC3Ⅱ/Ⅰ protein expression. AS-IV promoted chondrocyte proliferation and concurrently inhibited cell apoptosis. An optimal AS-IV dose of 200 μmol/L and a suitable reaction time of 48 h were identified. Ghost pen cyclic peptide staining indicated that the model group's cytoskeleton exhibited fusiform changes with reduced immunofluorescence intensity, as opposed to the blank control group. The AS-IV group displayed more polygonal cytoskeletal morphology with increased immunofluorescence intensity. AS-IV reduced TNF-α levels and elevated GAG levels in the culture supernatant. Additionally, AS-IV lowered p62 mRNA and protein expression while increasing LC3 mRNA expression in cultured chondrocytes. Our findings suggest that AS-IV mitigates inflammatory chondrocyte injury, safeguarding chondrocytes through a potential autophagy suppression mechanism. These results imply that AS-IV could offer preventive advantages for OA.

Lactiplantibacillus plantarum NMGL2 exopolysaccharide ameliorates DSS-induced IBD in mice mainly by regulation of intestinal tight junction and NF-κB p65 protein expression

Treatment of inflammatory bowel disease (IBD), a common chronic intestinal disease, by exopolysaccharides (EPSs) produced by lactic acid bacteria has raised increasing concerns. Here, the EPS produced by Lactiplantibacillus plantarum NMGL2 was evaluated for its ameliorating effect on dextran sodium sulfate (DSS)-induced IBD in mice. Administration of the EPS was shown to decrease the body weight loss and the values of disease activity index (DAI) and alleviate the colon damage as evidenced by an improvement in colonic length shortening, a reduction in colonic coefficient, and a reduction in colonic mucosal architecture and inflammatory infiltration. Cytokine assay of the blood and colon tissue samples showed that the EPS could decrease the levels of pro-inflammatory TNF-α and IL-1β, and increase anti-inflammatory IL-10. Oxidative stress assay of the colon tissue showed that the nitric oxide (NO) and malondialdehyde (MDA) levels decreased significantly (p < 0.05), while superoxide dismutase (SOD) and glutathione (GSH) levels increased significantly (p < 0.05) after the EPS intervention. These results were further confirmed by the significantly (p < 0.05) down-regulated levels of NF-κB p65, p-IKKβ, and p-IκBα, and significantly (p < 0.05) enhanced expression of ZO-1 and occludin, as evaluated by Western-blot analysis of these proteins expressed in colonic tissue. The EPS produced by L. plantarum NMGL2 alleviated IBD by suppressing the NF-κB signaling pathway, suggesting its potential as a functional food agent in the prevention of IBD.

Honokiol inhibits human osteosarcoma MG63 cell migration by upregulating FTO and Smad6 to promote autophagy

BackgroundOsteosarcoma (OS) is a common primary malignant tumor of bone, most commonly seen in children and adolescents, which has a low survival rate and is a serious threat to patients' lives. Honokiol (HKL) is the main active components of Magnolia officinalis, which have significant anti-tumor properties. The aim of this study was to observe the autophagic and migratory effects of HKL on MG63 cells and to investigate whether the mechanism of action was related to FTO and Smad6. MethodsFirstly, we cultured MG63 cells in vitro and intervened with different concentrations of HKL to detect cell activity by CCK8, apoptosis by flow cytometry, cell migration ability by scratch assay, cell invasion ability by transwell assay and MMP2, P62, LC3 I/II, FTO and Smad6 protein expression by Western blot. ResultsHKL inhibited MG63 cells activity and that this effect was dose and time dependent. Although there was no significant effect on apoptosis and invasive ability, HKL could act through effects such as promoting cell autophagy and inhibiting migration. HKL increased the protein expression levels of FTO, Smad6, MMP2, LC3 I/II and P62, and this effect was reduced after silencing of Smad6. ConclusionsHKL induced autophagy and inhibited cell migration in MG63 cells by increasing the expression of FTP and Smad6. It can be seen that HKL may be a promising drug for the treatment of OS.

Indigo alleviates psoriasis through the AhR/NF-κB signaling pathway: an in vitro and in vivo study.

Psoriasis is a chronic inflammatory skin disease. A strong association between the AhR/ NFκB axis and the inflammatory response in psoriasis. Indigo (IDG) has demonstrated significant anti-inflammatory properties. This study aimed to assess the anti-psoriatic efficacy of IDG while investigating the underlying mechanisms involved. In the in vitro experiments, cell viability was assessed using the CCK-8. qRT-PCR was employed to measure the mRNA levels of NF-κB, TNF-α, IL-1β, AhR, and CYP1A1. Western blotting was conducted to examine alterations in cytoplasmic and nuclear AhR protein levels. Additionally, an IDG nanoemulsion (NE) cream was prepared for the in vivo experiments. A psoriasis-like skin lesion mice model was induced using IMQ (62.5 mg/day for 7 days). The severity of psoriasis was evaluated using PASI, and skin lesions were scored while epidermal thickness was assessed via HE staining. The expression of inflammatory markers, including IL-6, IL-13, IL-17A, MCP-1, and TNF-α, was detected in skin lesions using Luminex. The levels of CYP1A1, p65, and p-p65 proteins were determined by Western blotting. LPS stimulation significantly elevated TNF-α, IL-6, and NF-κB mRNA levels, which were notably reduced by IDG treatment. Additionally, IDG significantly enhanced the expression of AhR and CYP1A1 mRNA. Further investigation revealed that IDG facilitated AhR translocation from the cytoplasm to the nucleus. In the IMQ-induced psoriasis-like mouse model, IDG NE substantially ameliorated the severity of skin lesions. Moreover, IDG NE treatment reduced the upregulation of inflammatory cytokines such as IL-6, IL-17A, MCP-1, and TNF-α in IMQ-induced skin lesions. It was also observed that IDG NE treatment increased CYP1A1 protein expression while inhibiting p65 and p-p65 protein expression. IDG emerges as a promising treatment for psoriasis, demonstrating effective therapeutic outcomes. Its mechanism of action is likely linked to the modulation of the AhR/NFκB signaling pathway.

Effects of wheat-grain moxibustion on autophagy and endoplasmic reticulum stress in rats with cervical spondylotic radiculopathy

To observe the effects of wheat-grain moxibustion on autophagy and endoplasmic reticulum stress (ERS) in the spinal cord and nerve root tissues of rats with cervical spondylotic radiculopathy (CSR), and to explore the potential mechanisms by which wheat-grain moxibustion alleviates neuropathic pain in CSR. Forty-eight SPF-grade SD rats were randomly divided into a sham operation group, a model group, a wheat-grain moxibustion group, and a wheat-grain moxibustion + 3-methyladenine (3MA) group, with 12 rats in each group. The CSR model was established in the model group, the wheat-grain moxibustion group, and the wheat-grain moxibustion + 3MA group using the spinal canal insertion method. From the third day after successful modeling, the wheat-grain moxibustion group received wheat-grain moxibustion at "Dazhui" (GV 14), once daily, six cones each session. The wheat-grain moxibustion + 3MA group received intraperitoneal injection of 3MA (2.5 mg/kg), followed by the same wheat-grain moxibustion intervention as the wheat-grain moxibustion group. Interventions were performed once daily for seven consecutive days. The gait disturbance scores and peripheral nerve mechanical pain thresholds were observed before and after the intervention. Western blot was used to detect the expression of ERS apoptosis factors C/EBP-homologous protein (CHOP) and Caspase-12, as well as autophagy substrate P62 in spinal cord and nerve root tissues. Transmission electron microscopy was used to observe autophagosomes and cellular ultrastructure in spinal cord and nerve root tissues. After modeling, compared with the sham operation group, gait disturbance scores were increased (P<0.05) and peripheral nerve mechanical pain thresholds were decreased (P<0.05) in the model group, the wheat-grain moxibustion group, and the wheat-grain moxibustion + 3MA group. After intervention, compared with the sham operation group, gait disturbance scores were increased (P<0.05) and peripheral nerve mechanical pain thresholds were decreased (P<0.05) in the model group; compared with the model group, gait disturbance score was decreased (P<0.05) and peripheral nerve mechanical pain threshold was increased (P<0.05) in the wheat-grain moxibustion and the wheat-grain moxibustion + 3MA group; compared with the wheat-grain moxibustion + 3MA group, gait disturbance score was decreased (P<0.05) and peripheral nerve mechanical pain threshold was increased (P<0.05) in the wheat-grain moxibustion group. Compared with the sham operation group, the expression of CHOP and Caspase-12 proteins in spinal cord and nerve root tissues was increased in the model group (P<0.05); compared with the model group and the wheat-grain moxibustion + 3MA group, the expression of CHOP, Caspase-12, and P62 proteins in spinal cord and nerve root tissues was decreased in the wheat-grain moxibustion group (P<0.05). Compared with the model group and the wheat-grain moxibustion + 3MA group, the number of autophagosomes in spinal cord and nerve root tissues was increased in the wheat-grain moxibustion group, and the cellular ultrastructure was clear and intact, similar to the sham operation group. Wheat-grain moxibustion at "Dazhui" (GV 14) could effectively alleviate neuropathic pain in CSR model rats. The analgesic mechanism may be related to promoting autophagy, inhibiting ERS, reducing ERS-mediated apoptosis, and repairing damaged nerves.

Upregulated DNMT3a coupling with inhibiting p62-dependent autophagy contributes to NNK tumorigenicity in human bronchial epithelial cells

NNK, formally known as 4-(methyl nitrosamine)-1-(3-pyridyl)-1-butanoe, is a potent chemical carcinogen prevalent in cigarette smoke and is a key contributor to the development of human lung adenocarcinomas. On the other hand, autophagy plays a complex role in cancer development, acting as a "double-edged sword" whose impact varies depending on the cancer type and stage. Despite this, the relationship between autophagy and NNK-induced lung carcinogenesis remains largely unexplored. Our current study uncovers a marked reduction in p62 protein expression in both lung adenocarcinomas and lung tissues of mice exposed to cigarette smoke. Interestingly, this reduction appears to be contingent upon the activity of extrahepatic cytochrome P450 (CYP450), revealing that NNK metabolic activation by CYP450 enzyme escalates its potential to induce p62 downregulation. Further mechanistic investigations reveal that NNK suppresses autophagy by accelerating the degradation of p62 mRNA, thereby promoting the malignant transformation of human bronchial epithelial cells. This degradation process is facilitated by the hypermethylation of the Human antigen R (HuR) promoter, resulting in the transcriptional repression of HuR - a key regulator responsible for stabilizing p62 mRNA through direct binding. This hypermethylation is triggered by the activation of ribosomal protein S6, which is influenced by NNK exposure and subsequently amplifies the translation of DNA methyltransferase 3 alpha (DNMT3a). These findings provide crucial insights into the nature of p62 in both the development and potential treatment of tobacco-related lung cancer.

9317 Diabetogenic Stress-Mediated Beta Cell Extracellular Vesicle:Autophagy Pathway Crosstalk

Abstract Disclosure: A. Roy: None. G. Ariyaratne: None. A. Matveyenko: None. N. Javeed: None. Diabetogenic stressors including elevated free fatty acids (lipotoxicity), low-grade systemic inflammation, and proteotoxicity are central to the development of Type 2 Diabetes (T2D). Due to their high protein synthetic burden, β-cells employ autophagy and endo/lysosomal pathways to reduce protein aggregates, and protect β-cell mass and function. Recently, extracellular vesicles (EVs; 30-150 nm sized nanoparticles) have been shown to work in tandem with autophagy to facilitate cellular adaptation and intercellular communication. As diabetogenic stressors have been shown to perturb autophagy via lysosomal defects, our group has noted enhanced EV secretion. Thus, we hypothesize that diabetogenic factors enhance EV secretion through activation of an EV-based secretory autophagy pathway and that re-balancing of both pathways could improve β-cell function in T2D. To test this, MIN6 cells were exposed to free fatty acid palmitate (PAL; 24h), or pro-inflammatory cytokines IL-1β, TNFα, and IFNγ (CYTO; 48h). Expression of autophagic markers, LC3 and p62 were increased in CYTO and PAL conditions suggesting defects in auto/lysosomal function. Upon PAL/CYTO treatment in MIN6 cells, conditioned media EVs were isolated using differential ultracentrifugation. We noted enhanced protein expression of LC3-II and p62 in PAL- and cytoEVs, suggesting a diversion of immature autophagosome components into EVs due to lysosomal inhibition. To restore balance between autophagy:EV flux, we used EV biogenesis inhibitor, GW4869 (GW; 24h) or autophagy inducer, rapamycin (Rapa; 24h) on MIN6 cells exposed to PAL or CYTO. Isolated EVs were subjected to Nanoparticle Tracking Analysis (NTA) which showed a significant reduction in EV concentration with GW or Rapa addition (p&amp;lt;.05). Isolated mouse islet exposed to CYTO+GW/Rapa or PAL+GW/Rapa showed significant improvements in glucose stimulated insulin secretion (vs. CYTO or PAL; p&amp;lt;.05). Taken together, these data implicate activation of EV-based secretory autophagy in response to diabetogenic stressors and thus, potential targets to improve functional β-cell mass. Presentation: 6/2/2024

Drug pairs of Huangqi and Danggui alleviates pyroptosis by promoting autophagy activity via AMPK/mTOR signaling pathway in middle-cerebral artery occlusion/reperfusion in rats

Ethnopharmacological relevanceCerebral ischemia-reperfusion (I/R) injury is a common complication of ischemic stroke, with autophagy and pyroptosis playing key roles. Huangqi and Danggui (HQDG) are a commonly used drug pair of Chinese traditional medicine for clinical treatment of ischemic stroke. Aim of the studyThe study aims to investigate the interaction between autophagy and pyroptosis regulated by HQDG through the AMPK/mTOR signaling pathway during cerebral I/R injury. Materials and methodsModel of middle-cerebral artery occlusion/reperfusion (MCAO/R) in SD rats was established using the Longa suture method. The components of traditional Chinese medicine were detected by liquid chromatography coupled to quadrupole orbitrap high resolution mass spectrometry (LC/MS). Neurological deficits were evaluated by neurological function score. Changes of cerebral blood flow were detected by a laser speckle blood flow imaging instrument. The volume of cerebral infarction was observed by 2,3,5-Chlorotriphenyltetrazolium (TTC) staining. The permeability of the blood-brain barrier was measured by Evans blue test. Neurovascular unit and autophagosomes in brain tissue were assessed by transmission electron microscopy. Neuronal pyroptosis was detected by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL)/Caspase-1 staining. The expression of autophagy related proteins, pyroptosis related proteins, and AMPK/mTOR pathway related proteins were detected by Western blot. ResultsAfter cerebral I/R injury, autophagy and pyroptosis, were characterized by increased number of autophagosomes and pyroptosis cells, upregulated expression of Beclin 1, LC3-II/LC3-I, NLRP3, cleaved Caspase-1, IL-1beta, IL-18 proteins, and downregulated expression of P62 proteins. HQDG significantly improved neurological function, reduced the volume of cerebral infarction, increased cerebral blood flow, improved blood-brain barrier permeability and the function of neurovascular units. Autophagy was further activated and pyroptosis was significantly inhibited by HQDG, which promoted increased number of autophagosomes, enhanced expression of Beclin 1, LC3-II/LC3-I proteins, reduced expression of P62, NLRP3, cleaved Caspase-1, IL-1beta, and IL-18 proteins, and downregulated the number of pyroptosis cells. On the other hand, after administering 3-Methyladenine (3-MA) to inhibit autophagy, the above effects of HQDG were significantly inhibited. Besides, HQDG promoted AMPK phosphorylation, and weakened mTOR phosphorylation. However, after the administration of AMPK inhibitor Compound C, HQDG caused increase in Beclin 1 and LC3-II/LC3-I, reduced P62 and NLRP3, and cleaved Caspase-1 protein expression, whereas cerebral blood flow decreased. ConclusionHQDG alleviated pyroptosis by promoting autophagy via AMPK/mTOR signaling pathway after middle-cerebral artery occlusion/reperfusion in rats, showing its potential for treatment of cerebral I/R injury in humans.

Naringin alleviates fluoride-induced neurological impairment: A focus on the regulation of energy metabolism mediated by mitochondrial permeability transition pore

The neurological impairment induced by fluoride is associated with mitochondrial dysfunction. Normal mitochondrial permeability transition pore (mPTP) opening plays a pivotal role in mitochondrial function. However, it remains unclear whether p53-dependent mPTP-related mitochondrial apoptosis is associated with fluoride-induced neurological impairment, and the alleviation of naringin on those. In vivo, NaF-treated rats had impaired learning and memory abilities, damaged hippocampal structure, and higher respiratory exchange rates (RER). In vitro, the increased apoptosis rates, excessive opening of mPTP, and decreased mitochondrial membrane potential (MMP) were observed in PC12 cells treated with NaF. The protein expressions of p53, CytoC, and cleaved caspase 3 were significantly increased in hippocampi of rats treated with 50 mg/L and 100 mg/L NaF and in 40 mg/L and 80 mg/L NaF-treated PC12 cells, while the protein expression of CypD remains stable. And the changes of p53 and CypD were also confirmed by the immunofluorescence staining in vivo. After inhibiting the expression of p53 with pifithrin-α and p53-siRNA, the decreased apoptosis rates and mPTP opening, increased MMP, and decreased protein expressions of p53, CytoC, and cleaved caspase 3 were observed in NaF-treated PC12 cells. Rats, treated with NaF and naringin, had alleviated impaired neurological function, and had lower RER than rats treated with NaF alone. And compared with those in the NaF group, the decreased apoptosis rates and mPTP opening, and increased MMP were also found in PC12 cells treated with NaF and naringin. Furthermore, hippocampi of rats and PC12 cells treated with NaF and naringin had decreased protein expressions of p53, CytoC, and cleaved caspase 3. Our results indicate that fluoride activates the p53-dependent mPTP-related mitochondrial apoptosis, which then affects energy metabolism, resulting in neurological impairment. Additionally, naringin can alleviate this damage, and further studies on the potential health benefits of naringin are needed.

BPIFA1 alleviates allergic rhinitis by regulating the NF-κB signaling pathway and Treg/Th17 balance.

Allergic rhinitis (AR) is an allergic condition characterized by inflammation of the nasal mucosa. Bacterial permeability-increasing family member A1 (BPIFA1) exhibits anti-inflammatory properties; however, its impact on AR remains unclear. Aim of this study is to investigate the expression and function of BPIFA1 in AR and its influence on inflammation and immune regulation in a mouse model of AR induced by ovalbumin (OVA). The expression of BPIFA1 was analyzed using quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Morphological assessments of nasal mucosal tissues were conducted. Levels of inflammatory mediators in nasal lavage fluid (NALF) and serum were quantified using enzyme-linked immunosorbent assay (ELISA) kits. Protein expressions of BPIFA1, phosphorylated and total p65 (p-p65/p65), and IκBα were evaluated through Western blot analysis. The total cell counts, including epithelial cells, eosinophils, and lymphocytes in NALF, were determined using a hemocytometer. A mouse model of AR was established by OVA management. BPIFA1 expression was found to be reduced in the nasal mucosa tissues of patients with AR, suggesting a potential role in the disease's progression. We successfully developed a mouse model of AR, where BPIFA1 was similarly downregulated, indicating its possible involvement in modulating the NF-κB signaling pathway. Overexpression of BPIFA1 in this model attenuated inflammation and allergic responses by inhibiting the NF-κB pathway. Additionally, overexpression of BPIFA1 promoted the differentiation of regulatory T cells (Treg) and inhibited the differentiation of T helper 17 cells (Th17) in the NALF of AR mice, further demonstrating its regulatory impact on immune responses. The study confirmed that BPIFA1 upregulation reduced the levels of inflammatory cytokines TNF-α and IL-6, decreased infiltration of inflammatory cells, and modulated antigen-specific immunoglobulin levels and histamine in serum. BPIFA1 mitigated both inflammatory and allergic responses in AR mice induced by OVA through the modulation of the NF-κB signaling pathway and the balance between regulatory T cells (Treg) and T helper 17 cells (Th17). These findings suggest that BPIFA1 could serve as a novel biomarker and therapeutic target for AR, offering potential for the development of targeted treatments to improve patient outcomes.

Exploring the Mechanism of Action of Sericin in Ameliorating non-Alcoholic Fatty Liver Disease in Mice Based on the Nrf2- GPX4/ NF-κB p65 Pathway

Objective: To explore the ameliorative effect and mechanism of action of sericin on liver injury in Non-Alcoholic Fatty Liver Disease (NAFLD) model mice. Methods: Mice were fed a high-fat diet for 8 weeks to establish NAFLD model mice. The NAFLD mice were then randomly divided into 6 groups and given 0, 250, 500, and 1000 mg/kg body weight of sericin, 1000 mg/kg sericin + ML385 (a protein inhibitor of Nrf2), and Zhibitai Capsules (a positive drug), by gavage, respectively. The levels of TC, TG, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in mouse serum and the levels of MDA, GSH, SOD, TNF-α, and IL-6 in liver tissues were measured. Pathological changes in liver tissues were evaluated using hematoxylin-eosin (HE) and Oil Red O staining. Protein expression of Nrf2, Keap1, HO-1, GPX4, and NF-κB p65 in liver tissues was detected by Western blotting, and the expression level of GPX4 in the liver was examined by immunohistochemistry. Results: Sericin and Zhibitai Capsules improved hepatic steatosis and lipid accumulation in NAFLD model mice. TC, TG, LDL-C, MDA, and TNF-α, IL-6 levels were down-regulated, and HDL-C and SOD levels were up-regulated. There was no significant difference in the expression of AST and ALT in all the groups. In addition, Sericin and Zhibitai Capsules upregulated the expression levels of Nrf2, keap1, HO-1, and GPX4 proteins and inhibited the expression of NF-κB p65 in the liver of NAFLD model mice. There was no significant difference in serum lipids and liver indices between the 1000 mg/kg sericin + ML385 group and the sericin dose groups. Conclusion: Sericin has an ameliorative effect on liver injury in NAFLD model mice by alleviating oxidative stress and inflammatory responses in liver injury. It remains to be explored whether this effect is regulated through the Nrf2- GPX4/ NF-κB p65 signaling pathway.

Shisandra Decoction Alleviates Parkinson's Disease Symptoms in a Mouse Model Through PI3K/AKT/mTOR Signalling Pathway.

The present study aimed to characterize neuroprotective effects of Schisandra Decoction (Sch D) treatment in a mouse model of Parkinson's disease (PD), and to explore underlying mechanisms focused on the mammalian target of rapamycin (mTOR) signaling pathway. 50 male C57 BL/6 mice were randomly assigned to either control (n = 10) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model (n = 40) groups. PD mice were further divided into four groups of ten mice each: MPTP group, LY294002 group, Sch D group, and LY2940002 + Sch D group. Mice from each group were assessed in pole climbing, rotary rod and open field tests. Brain Tyrosine hydroxylase (TH) protein was observed using immunohistochemistry. mRNA levels of PTEN, PI3K and LC3 in brain tissue were measured using RT-PCR. Protein levels of PTEN, PI3K, Akt, p-Akt, mTOR, p-mTOR, p70s6K, p62, LC3II / I, α-synuclein (α-syn), TH in brain tissue were assessed by Western blotting (WB). In behavioral tests, PD mice treated with Sch D showed reduced pole climbing time, longer rotarod duration, and greater distance traveled. In terms of neuroprotection, PD mice in the Sch D group exhibited higher levels of TH protein and enhanced α-syn clearance. Regarding autophagy, compared to the control group, mice in the MPTP group had elevated PTEN protein expression, which inhibited PI3K, p-AKT/AKT, and p-mTOR/mTOR protein levels, decreased LC3II/I protein expression, and increased P62 protein expression. Treatment with Sch D reversed these effects. Sch D reduces α-syn aggregation in the brains of MPTP-induced PD model mice, exerts neuroprotective effects, and improves motor function. Additionally, Sch D inhibits autophagy through the PI3K/AKT/mTOR pathway. The neuroprotective effect of Sch D may involve the suppression of abnormal autophagy and its antioxidant properties, which indirectly reduces α-syn accumulation. Future studies should assess the impact of Sch D on oxidative stress markers to evaluate its antioxidant effects.

Integrating network pharmacology and experimental verification to study the mechanism of Polygonum hydropiper total flavonoids against stress-induced gastric mucosal damage

ContextPolygonum hydropiper L. (P. hydropiper) has outstanding clinical efficacy in treating both acute and chronic gastroenteritis. However, the definite mechanism remains unclear. ObjectiveThis study aimed to explore the potential mechanisms of the total flavonoid of P. hydropiper (FPH) in stress-induced gastric mucosal damage (SGMD) rats through a combination of network pharmacology, molecular docking, and animal experiments. MethodsNetwork pharmacology and molecular docking were utilized to predict the potential mechanisms of FPH against SGMD. In experimental studies, SGMD rat models were established using water-immersion restraint stress (WIRS). FPH was administered at doses of 140, 70, and 35 mg/kg, with ranitidine serving as a positive control, through gavage once daily for 6 consecutive days after model establishment. Stomach and serum specimens were analyzed using HE staining, Western blotting, qPCR, and ELISA to investigate the protective mechanism of FPH in SGMD. ResultsThe network pharmacology analysis identified 16 active ingredients and 183 common targets, with potential pathways including PI3K/Akt, MAPK and Keap1/Nrf2. In vivo experiments demonstrated that FPH intervention alleviated SGMD pathological changes, reduced elevated serum IL-6 and TNF-α levels, and enhanced SOD and GSH activity in rats. Additionally, FPH increased the protein expression of p62, Nrf2, HO-1, PI3K, and p-Akt, along with mRNA levels of Nrf2, p62, and HO-1. ConclusionsFPH exerts a gastric mucosal protective effect by upregulating antioxidant gene expression through the PI3K/Akt and Keap1/Nrf2 pathways. This study provides an experimental basis for the potential clinical treatment of SGMD with the traditional Chinese medicine P. hydropiper.

Salvia Miltiorrhiza Injection Inhibited the Proliferation of AML Cells by Inducing Apoptosis through the p38MAPK Pathway.

The purpose of this study was to explore the antitumor effect and mechanism of Salvia miltiorrhiza injection (SMI) on acute myeloid leukemia (AML) cells in vitro and in vivo. Bioinformatics was used to detect c-Myc mRNA expression in AML patients in the Oncomine database. qRT‒PCR and western blotting were used to detect the mRNA and protein expression of c-Myc and HOXA5 in clinical samples. Different concentrations (6.25, 12.5, 25, 50 and 100 μg/mL) of SMI were added to KG1a and HL60 cells for 24, 48 and 72 h to determine the IC50 value of SMI. A CCK-8 assay was used to detect the effects of different concentrations of SMI and different treatment times on the proliferation of KG1a and HL60 cells. The indicated concentrations of SMI and SB203580 were used to treat KG1a and HL60 cells. The cell cycle distribution was determined by flow cytometry. The percentage of apoptotic cells was detected by Hoechst 33258 staining and flow cytometry. qRT‒PCR was performed to detect the mRNA expression of p38, c-Myc and HOXA5 in KG1a and HL60 cells. Western blotting was used to detect the protein expression of p38, p-p38, c-Myc, HOXA5, cCaspase 3 and cPARP in KG1a and HL60 cells. AutoDock software was used to analyze the molecular docking of the three main active components of SMI with c-Myc. AutoDock analysis revealed that the binding effect of molecular leisure was evaluated by binding energy, and a binding energy <-5 kcal/mol was considered good. SMI decreased the mRNA and protein expression of c-Myc and HOXA5. SMI significantly inhibited the proliferative activity of KG1a and HL60 cells and induced their apoptosis. However, SMI had no significant effect on the cell cycle distribution of KG1a and HL60 cells. With increasing SMI concentrations, the p-p38/p38 ratio increased, while the protein expression of c-Myc and HOXA5 decreased, and the protein expression of cCaspase and cPARP increased. However, SB203580 intervention in addition to SMI reversed these changes. Tanshinone IIA, cryptanshinone and salvianolic acid B can bind to multiple sites of c-Myc. In summary, SMI could be used for the treatment of acute leukemia, and its mechanism may be related to activation of the p38MAPK signaling pathway.

Electroacupuncture inhibits TLR4/NF-κB signaling in the dorsal root ganglion of rats with spared nerve injury.

Neuropathic pain can be provoked by high mobility group box 1 (HMGB1) activation of toll-like receptor (TLR)4/nuclear factor (NF)-κB signaling in the dorsal root ganglion (DRG). Electroacupuncture (EA) has been reported to effectively alleviate neuropathic pain with few side effects, but its precise mechanism of action remains unknown. The aim of this study was to explore whether 2 Hz EA stimulation suppresses TLR4/NF-κB signaling in the DRG following spared nerve injury (SNI) in a rat model. In this experiment, SNI rats were given 2 Hz EA once every other day for a total of 21 days. Paw withdrawal threshold (PWT) was measured to assess SNI-induced mechanical hypersensitivity, and western blotting and immunofluorescence staining were used to determine the levels of pain-related signaling molecules and pro-inflammatory mediators in the DRG. SNI up-regulated HMGB1, TLR4, myeloid differentiation factor-88 adaptor protein (MyD88) and NF-κB p65 protein expression in the DRG. In addition, immunofluorescence staining demonstrated that SNI induced higher levels of TLR4 and MyD88 in the DRG. We also demonstrated co-localization of TLR4 and MyD88 with both calcitonin gene-related peptide (CGRP) and isolectin GS-IB4 in the DRG of SNI rats, respectively. Meanwhile, 2 Hz EA stimulation effectively reversed the elevations of HMGB1, TLR4, MyD88 and NF-κB p65 induced by SNI in the DRG, which was coupled with amelioration of SNI-induced mechanical hypersensitivity. The results of this study suggested that inhibition of the TLR4/NF-κB signaling pathway in the DRG by 2 Hz EA might be exploited as a therapeutic option for neuropathic pain.

Supplementing ageing male laying breeders with lycopene alleviates oxidative stress in testis and improves testosterone secretion

BackgroundReproductive performance is a crucial aspect of poultry production and is carefully controlled by endocrine, paracrine, and autocrine factors. This study aimed to investigate the effect of lycopene on testosterone synthesis in Leydig cells of laying breeder roosters, clarify the mechanism of lycopene improving Leydig cells function and promoting testosterone production, and explore the role of related signal transduction pathways in testosterone synthesis. ResultsA total of 96 healthy 55-week-old breeding roosters were randomly assigned to one of five dietary treatments. They were provided with a corn-soybean meal-based diet containing different levels of lycopene: 0 mg/kg (control), 50 mg/kg, 100 mg/kg, or 200 mg/kg. The experiment lasted for 6 weeks. With the increase in lycopene levels, the testosterone content in the plasma was significantly higher than in the control group. Testicular Leydig cells were isolated and cultured from fresh testicular tissue of 45-wk-old to 60-wk-old breeding roosters. Various doses of lycopene were administered to Leydig cells, and subsequently, cells were collected for the detection of cell viability and testosterone content. The optimal concentration of lycopene to be added was determined, and changes in mRNA expression and protein levels of key proteins involved in testosterone synthesis were investigated. The results showed that lycopene treatment significantly increased testosterone secretion, mRNA expression, and protein levels of steroid-producing enzymes. Cells were collected to measure the activity of antioxidant enzymes, the mRNA transcription level of apoptotic factors, and the protein expression of apoptotic factors after treatment with lycopene. The results showed that lycopene significantly increased the activities of antioxidant enzymes, and the ability to inhibit oxygen radicals, and decreased the content of malondialdehyde. Apoptosis was inhibited by regulating the expression of apoptosis-inducing and anti-apoptosis factors. After that, the MAPK signaling pathway and downstream SF-1, Nrf2 gene, and protein expression levels were detected. The results showed that lycopene treatment significantly increased the gene and protein expression of JNK, SF-1, and Nrf2, and significantly decreased the gene and protein expression of p38. ConclusionsLycopene treatment could promote testosterone synthesis of testicular Leydig cells by activating MAPK-SF-1 (increasing steroid-producing enzyme level) and MAPK-Nrf2 pathways (resisting oxidative damage).

Enhancement of cognitive function in mice with Alzheimer's disease through hyperbaric oxygen-induced activation of cellular autophagy.

In this study, we examined the effectiveness of hyperbaric oxygen (HBO) therapy in ameliorating cognitive deficits in mice with Alzheimer's disease (AD), while also assessing its impact on the autophagic pathway within the context of AD. 20 double-transgenic mice expressing the amyloid precursor protein and presenilin 1 (APP/PS1) were purposefully selected and randomly assigned to groups A and B. Concurrently, 20 C57BL/6 mice were chosen and randomly categorized into groups C and D, each consisting of 10 mice. Mice in groups B and D received HBO treatment. The Morris water maze assay was used to assess changes in mouse behavior. Immunohistochemistry techniques were used to quantify the expression levels of amyloid-beta 42 (Aβ42) and microtubule-associated protein 1A/1B-light chain 3 (LC3) in hippocampal tissues, while western blot analysis was used to investigate the levels of LC3-II, p62, phosphoinositide 3-kinase (PI3K), and mammalian target of rapamycin (mTOR) proteins within hippocampal tissues. Mice allocated to group B exhibited reduced escape latency and prolonged dwell time in the target quadrant compared to other groups. Histological examination revealed conspicuous plaque-like deposits of Aβ42 in the hippocampal tissues of mice in groups A and B. Group B displayed diminished Aβ42-positive reactants and augmented microtubule-associated protein 1A/1B-LC3-positive reactants compared to group A. LC3-positive reactants were also detected in the hippocampal tissues of mice in groups C and D, surpassing the levels observed in groups A and B. Furthermore, group B demonstrated significantly lower expression of mTOR protein and markedly higher expression of LC3-II protein in mouse hippocampal tissues when compared to group A (P < 0.05). Conversely, there were no significant disparities noted in PI3K and p62 protein expression between groups B and A. Notably, no discernible discrepancies were observed in the expression levels of mTOR, PI3K, LC3-II, and p62 proteins between groups C and D within mouse hippocampal tissues. HBO treatment demonstrates efficacy in enhancing cognitive function in mice with AD and holds promise as a potential therapeutic intervention for AD by facilitating the activation of the mTOR pathway-mediated autophagy.

Resistin alleviates lipopolysaccharide-induced inflammation in bovine alveolar macrophages by activating the AMPK/mTOR signaling pathway and autophagy

ObjectiveResistin (RETN) is an adipocyte-specific hormone that participates in metabolism and modulates cellular inflammation. Our study aimed to assess the effects of RETN treatment on autophagy and the underlying molecular and biological mechanisms in bovine alveolar macrophages (BAMs). MethodsThe optimal concentration of RETN + lipopolysaccharide (LPS) on macrophages was screened and then used to co-culture with alveolar macrophages. Autophagosomes in BAMs were examined using a transmission electron microscope (TEM). Quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression of microtubule-associated protein light chain 3 (LC3) and p62. Western blot (WB) was used to detect the protein expressions of LC3 and p62. The distribution of LC3 and p62 proteins in the cells was observed by immunofluorescence (IF). The concentrations of interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) were detected using enzyme-linked immunosorbent assay (ELISA). The protein expression of adenosine-monophosphate-activated protein kinase (AMPK), p-AMPK, mammalian target of rapamycin (mTOR), and p-mTOR was detected using WB. ResultsThe treatment of BAMs with RETN or LPS increased the number of autophagosomes and the ratio of LC3II/LC3I and decreased the expression level of p62 protein. RETN treatment significantly triggered autophagy compared to LPS treatment. Moreover, the ratios of p-AMPK/AMPK and p-mTOR/mTOR were upregulated and downregulated, respectively, after RETN treatment, suggesting that AMPK/mTOR signaling pathway activation is required for RETN-mediated autophagy in BAMs. Additionally, the ratio of LC3-II/LC3-I was lower, and the concentrations of IL-1β, IL-6, and TNF-α significantly decreased in the LPS and RETN co-treatment groups compared to the single LPS treatment group. However, both autophagy- and LPS-induced inflammation were partially alleviated by RETN treatment. ConclusionRETN can promote autophagy in BAMs by activating the AMPK/mTOR signaling pathway, it may help prevent LPS-induced inflammation.

Methionine deficiency inhibited pyroptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella): possibly via activating the ROS-AMPK-autophagy axis

BackgroundMethionine (Met) is the only sulfur-containing amino acid among animal essential amino acids, and methionine deficiency (MD) causes tissue damage and cell death in animals. The common modes of cell death include apoptosis, autophagy, pyroptosis, necroptosis. However, the studies about the major modes of cell death caused by MD have not been reported, which worth further study.MethodsPrimary hepatocytes from grass carp were isolated and treated with different doses of Met (0, 0.5, 1, 1.5, 2, 2.5 mmol/L) to examine the expression of apoptosis, pyroptosis, autophagy and necroptosis-related proteins. Based on this, we subsequently modeled pyroptosis using lipopolysaccharides and nigericin sodium salt, then autophagy inhibitors chloroquine (CQ), AMP-activated protein kinase (AMPK) inhibitors compound C (CC) and reactive oxygen species (ROS) scavengers N-acetyl-L-cysteine (NAC) were further used to examine the expression of proteins related to pyroptosis, autophagy and AMPK pathway in MD-treated cells respectively.ResultsMD up-regulated B-cell lymphoma protein 2 (Bax), microtubule-associated protein 1 light chain 3 II (LC3 II), and down-regulated the protein expression levels of B-cell lymphoma-2 (Bcl-2), sequestosome 1 (p62), cleaved-caspase-1, cleaved-interleukin (IL)-1β, and receptor-interacting protein kinase (RIP) 1 in hepatocytes, while it did not significantly affect RIP3. In addition, MD significantly increased the protein expression of liver kinase B1 (LKB1), p-AMPK, and Unc-51-like kinase 1 (ULK1) without significant effect on p-target of rapamycin. Subsequently, the use of CQ increased the protein expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cleaved-caspase-1, and cleaved-IL-1β inhibited by MD; the use of CC significantly decreased the protein expression of MD-induced LC3 II and increased the protein expression of MD-suppressed p62; then the use of NAC decreased the MD-induced p-AMPK protein expression.ConclusionMD promoted autophagy and apoptosis, but inhibited pyroptosis and necroptosis. MD inhibited pyroptosis may be related regarding the promotion of autophagy. MD activated AMPK by inducing ROS production which in turn promoted autophagy. These results could provide partial theoretical basis for the possible mechanisms of Met in ensuring the normal structure and function of animal organs. Furthermore, ferroptosis is closely related to redox states, it is worth investigating whether MD affects ferroptosis in hepatocytes.

Metformin Improves the Function of Abdominal Aortic Aneurysm Patient Derived Aortic Smooth Muscle Cells

Type 2 diabetes mellitus (T2DM) is a cardiovascular risk factor. Paradoxically, a decreased risk of abdominal aortic aneurysm (AAA) presence and growth rate is described among patients with T2DM, associated with metformin use. This study aimed to investigate the effect of metformin on AAA patient derived aortic smooth muscle cell (SMC) function. Aortic biopsies were obtained from patients with AAA (n = 21) and controls (n = 17) during surgery. The SMCs of non-pathological aortic controls, non-diabetic patients with AAA, and diabetic patients with AAA were cultured from explants and treated with or without metformin. The SMC contractility was measured upon ionomycin stimulation, as well as metabolic activity, proliferation, and migration. Then, mRNA and protein expression of markers for contraction, metabolic activity, proliferation, and inflammation were measured. The mRNA expression of KLF4 and GYS1, genes involved in metabolic activity, differed between the SMCs from non-diabetic and diabetic patients with AAA before metformin stimulation (p < .041). However, the effect of metformin on the various SMC functions was similar between non-diabetic and diabetic patients with AAA. Upon stimulation, metformin increased the contractility of AAA patient SMCs (p = .001). The mRNA expression of smoothelin, a marker for the contractile phenotype, increased in the SMCs of patients with AAA after treatment with metformin (p = .006). An increase in metabolic activity (p < .001) and a decrease in proliferation (p < .001) and migration were found in the SMCs of controls and patients with AAA with metformin. Increased mRNA expression of PPARγ, a nuclear receptor involved in mitochondrial biogenesis (p < .009), and a decrease in gene expression of Ki-67, a marker for proliferation (p < .005), were observed. Gene expression of inflammation markers MCP-1 and IL-6, and protein expression of NF-κB p65 decreased after treatment with metformin in patients with AAA. This study found that metformin increases contractility and metabolic activity, and reduces proliferation, migration, and inflammation in aortic SMCs in vitro.