P65 Protein Expression Research Articles

Hydrogen combined with tetrandrine attenuates silica-induced pulmonary fibrosis via suppressing NF-kappaB/NLRP3 signaling pathway-mediated epithelial mesenchymal transition and inflammation

Silicosis is a progressive disease characterized by interstitial fibrosis resulting from inhalation of silica particles, and currently lacks specific treatment. Hydrogen (H2) has demonstrated antioxidative, anti-inflammatory, and anti-fibrotic properties, yet its efficacy in treating silicosis remains unexplored. In this study, rats exposed to silica were administered interventions of H2 combined with tetrandrine, and euthanized at 14, 28, and 56 days post-intervention. Lung tissues and serum samples were collected for analysis. Histological examination, MDA assay, enzyme-linked immunosorbent assay, hydroxyproline assay, and Western blotting were employed to assess the impact of H2 combined with tetrandrine on pulmonary fibrosis. The results revealed that this combination significantly alleviated inflammation in silicosis-afflicted rats, effectively suppressed levels of MDA, TNF-α, and IL-1β expression, and inhibited epithelial-mesenchymal transition (EMT), thereby ameliorating pulmonary fibrosis. Notably, protein expression level of E-cadherin was increased，however protein expression levels of vimentin and α-SMA were reduced, and TGF-β were reduced, alongside a significant decrease in hydroxyproline content. Furthermore, H2 combined with tetrandrine downregulated protein expression of NF-κB p65, NF-κB p-p65, Caspase-1, ASC, and NLRP3. These findings substantiate the hypothesis that H2 combined with tetrandrine mitigates inflammation associated with silicosis and suppresses the EMT process to ameliorate fibrosis via the NF-κB/NLRP3 signaling pathway. However, the pressure of airway opening was not assessed in this study and dynamic readings of lung physiological function were not obtained, which is a major limitation of this study.

N-desmethyldauricine from Menispermum dauricum DC suppresses triple-negative breast cancer growth in 2D and 3D models by downregulating the NF-κB signaling pathway

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, for which targeted therapy regimens are lacking. The traditional Chinese medicine Menispermum dauricum DC (M. dauricum) and its compounds have been reported to have antitumor activity against various cancers; however, their anti-TNBC activity is unknown. In this work, dauricine and N-desmethyldauricine from M. dauricum were separated and identified to have anti-TNBC via a multi-component bioactivity and structure-guided method. The cell counting kit 8 assay showed that dauricine and N-desmethyldauricine inhibited the proliferation of four tested TNBC cell lines, with half maximal inhibitory concentration values ranging from 5.01 μM to 13.16 μM. Further research suggested that N-desmethyldauricine induced cell apoptosis, arrested cell cycle progression in the G0/G1 phase, and inhibited cell migration. Western blot analysis revealed that the proapoptotic protein cleaved-poly-ADP-ribose polymerase 1 was upregulated, and the G0/G1 phase-related proteins cyclin-dependent kinase 2 and cyclin D1 and the migration-related protein matrix metallopeptidase 9 were downregulated. Furthermore, N-desmethyldauricine decreased the protein expression of p65, an important subunit of nuclear factor kappa-beta (NF-κB). Moreover, an antiproliferation assay of three-dimensional (3D) tumor spheroids showed that N-desmethyldauricine diminished cell‒cell adhesion and suppressed the growth of TNBC 3D spheroids. Taken together, these findings indicate that N-desmethyldauricine inhibited the proliferation of TNBC cells and decreased the expression of p65 in the NF-κB pathway.

Effect of electroacupuncture on autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency

To observe the effects of electroacupuncture (EA) on the autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency (POI), and explore the mechanism of EA in improving POI. Thirty-two female SD rats were randomly divided into a blank group (n=8) and a model making group (n=24). The rats in the model making group were injected intraperitoneally with cyclophosphamide for 15 days to establish the POI model (the dosage on the 1st day was 50 mg/kg, and 8 mg/kg from the 2nd day to 15th day). The successfully modeled rats were then randomly divided into a model group, an EA group, and an estradiol (E2) group, with 8 rats in each group. Rats in the EA group received EA at bilateral "Gongsun" (SP 4) with continuous wave, frequency of 2 Hz, and current intensity of 0.1 to 1 mA, 20 min per treatment, once daily for 14 days. Rats in the E2 group were administered with E2 (0.01 mg/mL) by gavage (10 mL/kg), once daily for 14 days. The changes in estrous cycle were observed by rapid Giemsa staining before and after modeling. After intervention, ovarian tissue morphology was observed by HE staining; serum levels of follicle-stimulating hormone (FSH), E2, anti-Mullerian hormone (AMH), and inhibin B (INHB) were detected by ELISA; immunofluorescence staining was used to observe the expression of p62 in ovarian granulosa cells; the ultrastructure of ovarian granulosa cells was observed by transmission electron microscopy, and the number of autophagosomes and autolysosomes was compared; Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of p62, Beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3) in ovarian tissue. The results of vaginal smears in the blank group showed regular cyclical changes; the rats in the model group showed prolonged estrous cycle or cycle arrest, mostly in proestrus or metestrus, with overall ovarian atrophy, disordered structure, and decreased granulosa cells. Compared with the blank group, rats in the model group showed increased serum FSH level (P<0.01), decreased serum levels of E2, AMH, and INHB (P<0.01), decreased positive expression of p62 in ovarian granulosa cells (P<0.01), with obvious swelling of ovarian granulosa cells, mild to moderate swelling of mitochondria, slight expansion of rough endoplasmic reticulum, and hypertrophy of Golgi apparatus; the number of autophagosomes and autolysosomes in the ovaries was increased (P<0.01), the expression of p62 protein and mRNA was decreased (P<0.01), and the expression of Beclin-1 and LC3 protein and mRNA in ovarian tissue was increased (P<0.01). Compared with the model group, rats in the EA group and the E2 group showed decreased serum FSH levels (P<0.01), increased levels of E2, AMH, and INHB (P<0.01), increased positive expression of p62 in ovarian granulosa cells (P<0.01), alleviated degree of ovarian granulosa cell damage, with relatively intact organelle morphology, and decreased number of autophagosomes and autolysosomes in the ovaries (P<0.01); the rats also showed increased expression of p62 protein and mRNA (P<0.01), and decreased expression of Beclin-1 and LC3 protein and mRNA (P<0.01) in ovarian tissue. EA at "Gongsun" (SP 4) could improve ovarian reserve function in POI rats by reducing the number of autophagosomes and autolysosomes, up-regulating p62 expression, and down-regulating Beclin-1 and LC3 expression, thus inhibiting autophagy of ovarian granulosa cells, and regulating the serum levels of FSH, E2, AMH, and INHB.

Effects of acupuncture on fecal short-chain fatty acids and TLR4/MyD88/NF-κB signaling pathway in spontaneously hypertensive rats

To observe the effects of acupuncture on blood pressure, fecal short-chain fatty acids (SCFAs) and toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway in spontaneously hypertensive rats (SHR), and to explore the mechanism of acupuncture for anti-hypertension. Twenty-four male SHR of SPF grade were randomly divided into a model group, a western medication group, an acupuncture group and a sham acupuncture group, with 6 rats in each group, and 6 male Wistar-Kyoto rats were selected as the blank group additionally. Hydrochlorothiazide solution was given by gavage in the western medication group; acupuncture was applied at bilateral "Renying" (ST 9) and "Zusanli" (ST 36) in the acupuncture group, 20 min a time; acupuncture was applied at the non-meridian and non-acupoint points close to bilateral "Renying" (ST 9) and "Zusanli" (ST 36) in the sham acupuncture group, 20 min a time. The intervention was adopted once a day for 4 weeks continuously in each group. The systolic blood pressure (SBP) of the caudal artery was measured before intervention and after 1, 2, 3 and 4 weeks of intervention. After intervention, the morphology of colonic tissue was observed by HE staining; the fecal level of SCFAs was detected by gas chromatography; the serum levels of interleukin (IL)-6, IL-1βand tumor necrosis factor-α (TNF-α) were detected by ELISA; the protein expression of TLR4, MyD88 and NF-κB p65 in the mesenteric artery was detected by Western blot. Compared with the blank group, in the model group, the SBP was increased (P<0.05), significant pathological changes could be found in the colonic tissue, the fecal SCFAs level was decreased (P<0.05), the serum levels of IL-6, IL-1β and TNF-α were increased (P<0.05), the protein expression of TLR4, MyD88 and NF-κB p65 in the mesenteric artery was increased (P<0.05). Compared with the model group, the SBP after 2, 3 and 4 weeks of intervention was decreased (P<0.05), the serum levels of IL-6, IL-1β and TNF-α were decreased (P<0.05) in the acupuncture group and the western medication group; the mucosal epithelium of colonic tissue was intact, the number of intestinal glands was abundant, the fecal SCFAs level was increased (P<0.05), and the protein expression of TLR4, MyD88 and NF-κB p65 in the mesenteric artery was decreased (P<0.05) in the acupuncture group. Compared with the sham acupuncture group, the SBP after 2, 3 and 4 weeks of intervention was decreased (P<0.05), the fecal SCFAs level was increased (P<0.05), the serum levels of IL-6, IL-1β and TNF-α were decreased (P<0.05), the protein expression of TLR4, MyD88 and NF-κB p65 in the mesenteric artery was decreased (P<0.05) in the acupuncture group. Acupuncture at bilateral "Renying" (ST 9) and "Zusanli" (ST 36) can effectively play an anti-hypertensive role in SHR. Its mechanism may be related to regulating fecal SCFAs level and inhibiting the TLR4/MyD88/NF-κB signaling pathway.

Mechanism of electroacupuncture regulating nuclear factor-κB pathway to improve the dedifferentiation of pancreatic β-cells in rats with T2DM

To observe the effects of electroacupuncture (EA) on the expression of serum interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and the pancreatic nuclear factor-κB (NF-κB) pathway in type 2 diabetes mellitus (T2DM) rats, and to explore the possible mechanism by which EA improving the dedifferentiation of pancreatic β-cells in the treatment of T2DM. Among 18 SPF-grade male Wistar rats, 6 rats were randomly selected as the control group, and the remaining 12 rats were fed with high-sugar and high-fat diet combined with intraperitoneal injection of 2% streptozotocin solution (35 mg/kg) to establish T2DM model. After successful modeling, the 12 rats were randomly divided into a model group and an EA group, with 6 rats in each group. The EA group received EA at bilateral "Zusanli" (ST 36), "Sanyinjiao" (SP 6), "Weiwanxiashu" (EX-B 3), and "Pishu" (BL 20), with continuous wave, frequency of 15 Hz, current intensity of 2 mA, for 20 min each time, once a day, 6 times a week, for a total of 6 weeks. Fasting blood glucose (FBG) levels were measured before modeling and before and after intervention. After intervention, ELISA was used to detect the serum fasting insulin (FINS), IL-1β and TNF-α levels, and the β-cell function index (HOMA-β) and insulin resistance index (HOMA-IR) were calculated; HE staining was used to observe the morphology of the pancreatic islets; Western blot was used to detect the protein expression of pancreatic forkhead box protein O1 (FoxO1), pancreatic and duodenal homeobox 1 (PDX-1), neurogenin 3 (NGN3), and NF-κB p65. After intervention, the FBG in the model group was higher than that in the control group (P<0.01), and the FBG in the EA group was lower than that in the model group (P<0.01). Compared with the control group, the model group had increased levels of serum FINS, IL-1β, TNF-α, and HOMA-IR (P<0.01), and decreased HOMA-β (P<0.01), reduced protein expression of pancreatic FoxO1 and PDX-1 (P<0.01), and increased protein expression of pancreatic NGN3 and NF-κB p65 (P<0.01, P<0.05). Compared with the model group, the EA group had lower serum FINS, IL-1β, TNF-α levels, and HOMA-IR (P<0.01), higher HOMA-β (P<0.05), increased protein expression of pancreatic FoxO1 and PDX-1 (P<0.01, P<0.05), and decreased protein expression of pancreatic NGN3 and NF-κB p65 (P<0.01, P<0.05). The control group's pancreatic islets showed no obvious abnormalities; the model group's pancreatic islets were irregular in shape and had unclear boundaries with the surrounding area, with immune cell infiltration, reduced β-cell nuclei, disordered arrangement of islet cells, and increased intercellular spaces; the EA group showed improvements in islet morphology, immune cell infiltration, β-cell nuclei count, and the arrangement and spacing of islet cells approaching normal. EA could lower the blood glucose levels in T2DM rats, alleviate chronic inflammatory responses in the islets, and improve the dedifferentiation of pancreatic β-cells, which may be related to the inhibition of pancreatic NF-κB pathway expression.

Interaction of NF-κB and FOSL1 drives glioma stemness

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor; GBM’s inevitable recurrence suggests that glioblastoma stem cells (GSC) allow these tumors to persist. Our previous work showed that FOSL1, transactivated by the STAT3 gene, functions as a tumorigenic gene in glioma pathogenesis and acts as a diagnostic marker and potential drug target in glioma patients. Accumulating evidence shows that STAT3 and NF-κB cooperate to promote the development and progression of various cancers. The link between STAT3 and NF-κB suggests that NF-κB can also transcriptionally regulate FOSL1 and contribute to gliomagenesis. To investigate downstream molecules of FOSL1, we analyzed the transcriptome after overexpressing FOSL1 in a PDX-L14 line characterized by deficient FOSL1 expression. We then conducted immunohistochemical staining for FOSL1 and NF-κB p65 using rabbit polyclonal anti-FOSL1 and NF-κB p65 in glioma tissue microarrays (TMA) derived from 141 glioma patients and 15 healthy individuals. Next, mutants of the human FOSL1 promoter, featuring mutations in essential binding sites for NF-κB were generated using a Q5 site-directed mutagenesis kit. Subsequently, we examined luciferase activity in glioma cells and compared it to the wild-type FOSL1 promoter. Then, we explored the mutual regulation between NF-κB signaling and FOSL1 by modulating the expression of NF-κB or FOSL1. Subsequently, we assessed the activity of FOSL1 and NF-κB. To understand the role of FOSL1 in cell growth and stemness, we conducted a CCK-8 assay and cell cycle analysis, assessing apoptosis and GSC markers, ALDH1, and CD133 under varying FOSL1 expression conditions. Transcriptome analyses of downstream molecules of FOSL1 show that NF-κB signaling pathway is regulated by FOSL1. NF-κB p65 protein expression correlates to the expression of FOSL1 in glioma patients, and both are associated with glioma grades. NF-κB is a crucial transcription factor activating the FOSL1 promoter in glioma cells. Mutual regulation between NF-κB and FOSL1 contributes to glioma tumorigenesis and stemness through promoting G1/S transition and inhibiting apoptosis. Therefore, the FOSL1 molecular pathway is functionally connected to NF-κB activation, enhances stemness, and is indicative that FOSL1 may potentially be a novel GBM drug target.

Salidroside Inhibits α-Amanitin-Induced AML-12 Cell Apoptosis via the Regulation of PINK1/Parkin-Mediated Mitophagy and Mitochondrial Function.

Poisoning caused by the mushroom Amanita phalloides, due to the toxin α-amanitin, accounts for approximately 90% of food poisoning deaths in China with no specific antidotes. To investigate the role of salidroside (Sal) in α-amanitin (α-AMA)-induced mitophagy, mouse liver cells AML-12 were exposed to α-AMA in the presence of Sal or not. Intracellular reactive oxygen species (ROS) levels were measured using a ROS detection kit, mitochondrial activity was evaluated using a mitochondrial red fluorescent probe kit or JC-1 dye, and protein expression levels of PINK1, Parkin, LC3 II, P62, Bax, Bcl-2, Caspase 3, Cleaved-Caspase 3, PARP I, and Cleaved-PARP I were detected through Western blot. Results demonstrated that α-AMA led to increased intracellular ROS levels, cell apoptosis, and decreased mitochondrial membrane potential. Notably, expression levels of mitophagy-related proteins PINK1, Parkin, and LC3 increased significantly while the P62 protein expression decreased remarkably. Furthermore, Sal reversed the α-AMA-induced decrease in cell viability and mitochondrial membrane potential and increase in intracellular ROS level. In addition, Sal promoted expression levels of PINK1, Parkin, and LC3 II while suppressing the Bax/Bcl-2 ratio, Cleaved-Caspase 3, and Cleaved-PARP I as well as P62. The results above proved that salidroside alleviates α-AMA-induced mouse liver cells damage via promoting PINK1/Parkin-mediated mitophagy and reducing cell apoptosis.

Glucocorticoids promote joint microenvironment alteration of GABBR1 expression associated with mitigating rheumatoid arthritis.

GABBR1 receptors have been implicated in the progression of rheumatoid arthritis (RA), and p38 MAP kinase (MAPK) was shown to be downregulated by GABA and result in unchecked production of pro-inflammatory cytokine. GABBR1 is a member of GABA receptors, and it is known to be upregulated and plays a vital role in RA. Glucocorticoids are efficient therapeutics in rheumatoid arthritis (RA) and are known to regulate GABA actions; therefore, we intended to investigate the potential of glucocorticoids in RA concerning the potential pathway GABBR1/MAPK. Joint specimens were obtained from collagen-induced arthritis mouse model. A double-blind semi-quantitative analysis of vascularity, cell infiltration, as well as lining thickness by help of a 4-point scale setting was used to assess joint inflammation. Expression of GABBR1 and p38 was evaluated immunohistochemically. In vitro peripheral blood (PB), synovial fluid (SF), and mononuclear cells (MCs) were acquired from RA mice. Western blotting was used for detecting expression of GABBR1 and p38 proteins. The presence of high levels of GABBR1 and p38 was prevalent in RA joints relative to healthy joints and related to the inflammation level. Glucocorticoid treatment alters GABBR1 along with p38 protein expression in joints while reducing joint inflammation. Ex vivo and in vitro assays revealed glucocorticoids have a direct impact on p38, such as the decreased GABBR1 expression level after dexamethasone incubation with SFMC. GABBR1 together with p38 expression in RA joints depends on local inflammation and can be targeted by glucocorticoids.

Mechanism of Guigan Longmu Decoction in the Treatment of Arrhythmias Based on Network Pharmacology and Untargeted Metabolomics Assays.

Guigan longmu decoction (GGLM), a traditional Chinese medicine compound, has demonstrated efficacy in treating rapid arrhythmia clinically. Nevertheless, its mechanism of action remains elusive. This study aims to elucidate the molecular mechanism underlying the efficacy of GGLM in treating arrhythmia utilizing non-targeted metabolomics, widely-targeted metabolomics, and network pharmacology, subsequently validated through animal experiments. Initially, network pharmacology analysis and widely-targeted metabolomics were performed on GGLM. Subsequent to that, rats were administered GGLM intervention, and nontargeted metabolomics assays were utilized to identify metabolites in rat plasma postadministration. The primary signaling pathways, core targets, and key active ingredients of GGLM influencing arrhythmia were identified. Additionally, to validate the therapeutic efficacy of GGLM on arrhythmia rat models, a rat model of rapid arrhythmia was induced via subcutaneous injection of isoproterenol, and alterations in pertinent pathogenic pathways and proteins in the rat model were assessed through qRT-PCR and Western blot following GGLM administration. The results of network pharmacology showed that 99 active ingredients in GGLM acted on 249 targets and 201 signaling pathways, which may be key to treating arrhythmia. Widelytargeted metabolic quantification analysis detected a total of 448 active ingredients in GGLM, while non-targeted metabolomics identified 279 different metabolites and 10 major metabolic pathways in rats. A comprehensive analysis of the above results revealed that the core key active ingredients of GGLM in treating arrhythmia include calycosin, licochalcone B, glabridin, naringenin, medicarpin, formononetin, quercetin, isoliquiritigenin, and resveratrol. These active ingredients mainly act on the relevant molecules and proteins upstream and downstream of the MAPK pathway to delay the onset of arrhythmia. Animal experimental results showed that the heart rate of rats in the model group increased significantly, and the mRNA and protein expression of p38, MAPK, JNK, ERK, NF-kb, IL-1β, and IL-12 in myocardial tissue also increased significantly. However, after intervention with GGLM, the heart rate of rats in the drug group decreased significantly, while the mRNA and protein expression of p38 MAPK, JNK, ERK1, NF-kb, IL-1β, and IL-12 in myocardial tissue decreased significantly. GGLM, as an adjunctive therapy in traditional Chinese medicine, exhibits favorable therapeutic efficacy against arrhythmia. This can be attributed to the abundant presence of bioactive compounds in the formulation, including verminin, glycyrrhizin B, glabridine, naringenin, ononin, quercetin, isorhamnetin, and kaempferol. The metabolites derived from these active ingredients have the potential to mitigate myocardial inflammation and decelerate heart rate by modulating the expression of proteins associated with the MAPK signaling pathway in vivo.

Acupuncture at "Die E acupoint" alleviates inflammatory reaction via inhibiting TLR4/MyD88/NF-κB signaling in rats with allergic rhinitis.

To observe effects of acupuncture at "Die E acupoint" on the protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear transcription factor κB (NF-κB), transcription factor T-bet (T-bet), and GATA-binding protein-3 (GATA-3) in the nasal mucosa and the serum contents of related inflammatory cytokines in rats with allergic rhinitis, so as to explore the mechanism of acupuncture in treating allergic rhinitis. Twenty-four healthy SD rats were randomly divided into blank, model, acupuncture, and sham acupuncture groups, with 6 rats in each group. The rat model of allergic rhinitis was established by using ovalbumin induction. The rats in the acupuncture group received bilateral acupuncture at the "Die E acupoint" with a depth of 15-20 mm, while the rats in the sham acupuncture group received only sham acupuncture (light and shallow acupunture of the skin at the "Die E acupoint" ). Both interventions were performed once daily for a total of 6 days. Behavioral scores of rats in each group were recorded. Pathological changes of nasal mucosa were observed by H.E. staining. Serum contents of IgE, ovalbumin-specific IgE (OVA-sIgE), interferon(IFN)-γ, interleukin(IL)-4, IL-10 and IL-17 were measured by ELISA and the protein expression levels of T-bet, GATA-3, TLR4, MyD88 and NF-κB p65 in the nasal mucosa were detected by Western blot. After modeling, compared with the blank group, rats in the model group showed increased behavioral scores, serum IgE, OVA-sIgE, IL-4, and IL-17 contents, and nasal mucosal GATA-3, TLR4, MyD88, and NF-κB p65 protein expression levels (P<0.05), whereas the contents of serum IFN-γ, IL-10 and the protein expression level of T-bet in the nasal mucosa were decreased (P<0.05). Comparison between the EA and model groups showed that acupuncture intervention can decrease the behavioral scores of rats with allergic rhinitis, the contents of serum IgE, OVA-sIgE, IL-4, IL-17, and the protein expression levels of GATA-3, TLR4, MyD88, and NF-κB p65 in the nasal mucosa (P<0.05), and up-regulate the contents of serum IFN-γ, IL-10, and the nasal mucosal T-bet protein expression level. Sham acupuncture did not have a significant modulating effect on the above indicators. Inflammatory infiltration of nasal mucosa was seen in the model group and sham acupuncture, and the inflammatory reaction was milder in the acupuncture group. Acupuncture at "Die E acupoint" can alleviate the symptoms of allergic rhinitis and suppress the inflammation of nasal mucosa in rats, which may be related to inhibiting the TLR4/MyD88/NF-κB signaling and balancing the levels of cytokines of Th1/Th2 and Treg/Th17, and T-bet/GATA-3.

Shikonin-loaded PLGA nanoparticles: A promising strategy for psoriasis treatment

Psoriasis is an inflammation-based skin illness marked by aggravated proliferation of epidermal cells. Shikonin is a natural naphthoquinone obtained from Arnebiae radix. It exerts anti-inflammatory and immunosuppressive effects. However, the poor water solubility and low bioavailability of shikonin limit its application. In this study, shikosin-loaded PLGA nanoparticle hydrogel was prepared and used to deliver the drug to the epidermis of psoriasis mice through local administration. The results demonstrated that shikosin-loaded PLGA nanoparticles inhibited HaCaT cell multiplication, increased drug uptake, and induced apoptosis of HaCaT cells. Results from Western blotting assays indicated that shikosin down-regulated the protein expressions of p65 and p-p65. Furthermore, shikonin mitigated psoriasis and decreased the concentrations of inflammation-inducing cytokines, i.e., IL17A, IL-17F, IL-22, IL-1β, and TNF-α. Taken together, these results suggest that shikonin-PLGA nanoparticles loaded in hydrogel system possess promising therapeutic potential for psoriasis.

The biphasic activity of autophagy and heat shock protein response in peripheral blood mononuclear cells following acute resistance exercise in resistance-trained males.

Autophagy and heat shock protein (HSP) response are proteostatic systems involved in the acute and adaptive responses to exercise. These systems may upregulate sequentially following cellular stress including acute exercise, however, currently few data exist in humans. This study investigated the autophagic and HSP responses to acute intense lower body resistance exercise in peripheral blood mononuclear cells (PBMCs) with and without branched-chain amino acids (BCAA) supplementation. Twenty resistance-trained males (22.3 ± 1.5yr; 175.4 ± .7cm; 86.4 ± 15.6kg) performed a bout of intense lower body resistance exercise and markers of autophagy and HSP70 were measured immediately post- (IPE) and 2, 4, 24, 48, and 72h post-exercise. Prior to resistance exercise, 10 subjects were randomly assigned to BCAA supplementation of 0.22g/kg/d for 5days pre-exercise and up to 72h following exercise while the other 10 subjects consumed a placebo (PLCB). There were no difference in autophagy markers or HSP70 expression between BCAA and PLCB groups. LC3II protein expression was significantly lower 2 and 4h post-exercise compared to pre-exercise. LC3II: I ratio was not different at any time point compared to pre-exercise. Protein expression of p62 was lower IPE, 2, and 4h post-exercise and elevated 24h post-exercise. HSP70 expression was elevated 48 and 72h post-exercise. Autophagy and HSP70 are upregulated in PBMCs following intense resistance exercise with autophagy increasing initially post-exercise and HSP response in the latter period. Moreover, BCAA supplementation did not affect this response.

Galectin-receptor interaction: a key player in liver fibrosis induced by Schistosoma japonicum infection

BackgroundSchistosoma japonicum eggs lodge in the liver and induce a fibrotic granulomatous immune response in the liver of host. Galectin 3 (Gal-3) is a protein implicated in fibrosis in multiple organs. However, the pathology and molecular mechanisms promoting hepatic granuloma formation remain poorly understood.MethodsTo investigate the effect of blocking galectin-receptor interactions by α-lactose on liver immunopathology in mice with S. japonicum infection, C57BL/6 mice were infected with S. japonicum and alpha (α)-lactose was intraperitoneally injected to block the interactions of galectins and their receptors.ResultsCompared with S. japonicum-infected mice, there were significantly decreased Gal-3 mRNA and protein expression levels, decreased intensity of Gal-3 fluorescence in the liver, decreased serum ALT and AST levels, decreased egg numbers of S. japonicum in the liver section, attenuated hepatic and spleen pathology, and alleviated liver fibrosis accompanied with decreased protein expression levels of fibrosis markers [α-smooth muscle actin (α-SMA), collagen I, and collagen IV] in the liver of S. japonicum-infected mice blocked galectin-receptor interactions with hematoxylin-eosin staining, Masson’s trichrome staining, immunohistochemistry, or Western blot analysis. Compared with S. japonicum-infected mice, blocking galectin-receptor interactions led to increased eosinophil infiltration and higher eosinophil cationic protein (ECP) expression in the liver, accompanied by increased mRNA levels of eosinophil granule proteins [ECP and eosinophil peroxidase (EPO)], IL-5, CCL11, and CCR3 in the liver and decreased mRNA levels of Gal-3 and M2 macrophage cytokines (TGF-β, IL-10, and IL-4) in the liver and spleen by using quantitative real-time reverse transcription-polymerase chain reaction. In addition, there were increased Beclin1 protein expression and protein expression ratio of LC3B-II/LC3B-I and decreased p62 protein expression and protein expression ratios of phospho-mTOR/mTOR and phospho-AKT/AKT by Western blot; increased double-labeled F4/80+/LC3B+ cells by immunofluorescence staining; increased M1 macrophage polarization in the liver of S. japonicum-infected mice blocked galectin-receptor interactions by flow cytometric analysis and immunofluorescence staining.ConclusionsOur data found that blockage of galectin-receptor interactions downregulated Gal-3, which in turn led to reduced liver functional damage, elevated liver eosinophil recruitment, promoted macrophage autophagy through the Akt/mTOR signaling pathway, and alleviated liver pathology and fibrosis. Therefore, Gal-3 plays a pivotal role during S. japonicum infection and could be a target of pharmacologic potential for liver fibrosis induced by S. japonicum infection.Graphical

Acteoside and isoacteoside alleviate renal dysfunction and inflammation in lipopolysaccharide-induced acute kidney injuries through inhibition of NF-κB signaling pathway.

Acute kidney injury (AKI) is a sudden loss of renal function with a high mortality rate and inflammation is thought to be the underlying cause. The phenylpropanoid components acteoside (ACT) and isoacteoside (ISO), which were isolated from Cistanche deserticola Y.C.Ma, have been reported to have preventive effects against kidney disorders. This study aimed to investigate the anti-inflammatory properties and protective mechanisms of ACT and ISO. In this investigation, kidney function was assessed using a semi-automatic biochemical analyzer, histopathology was examined using Hematoxylin-Eosin staining and immunohistochemistry, and the concentration of inflammatory cytokines was assessed using an enzyme-linked immunosorbent assay (ELISA) test. In addition, using Western blot and q-PCR, the expression of proteins and genes connected to the NF-κB signaling pathway in mice with lipopolysaccharide (LPS)-induced AKI was found. The findings showed that under AKI intervention in LPS group, ACT group and ISO group, the expression of Rela (Rela gene is responsible for the expression of NFκB p65 protein) and Tlr4 mRNA was considerably elevated (P<0.01), which led to a significant improvement in the expression of MyD88, TLR4, Iκ-Bɑ and NF-κB p65 protein (P<0.001). The levels of Alb, Crea and BUN (P<0.001) increased along with the release of downstream inflammatory factors such as IL-1β, IL-6, Cys-C, SOD1 and TNF-α (P<0.001). More importantly, the study showed that ISO had a more favorable impact on LPS-induced AKI mice than ACT. In conclusion, by inhibiting NF-κB signaling pathway, ACT and ISO could relieve renal failure and inflammation in AKI, offering a fresh possibility for the therapeutic management of the condition.

AMPK regulates immature boar Sertoli cell proliferation through affecting CDK4/Cyclin D3 pathway and mitochondrial function

Sertoli cell (SC) proliferation plays an important role in sperm production and quality; however, the regulatory mechanism of SC proliferation is not well understood. This study investigated the role of adenosine monophosphate-activated protein kinase (AMPK) in the regulation of immature boar SC activity. Cell counting kit-8, Seahorse XFe96, mitochondrial respiratory enzyme-related assay kits, and transmission electron microscopy were used to detect SC proliferative viability, oxygen consumption rate (OCR), mitochondrial respiratory enzyme activity, and the ultrastructure of primary cultured SCs in vitro from the testes of 21-day-old boars. A dual luciferase reporter assay was performed to determine the miRNA-mRNA target interaction. Western blotting was used to analyze cell proliferation-related protein expression of p38, p21, proliferating cell nuclear antigen (PCNA), Cyclin-dependent kinase 4 (CDK4), Cyclin D3, and phosphorylated retinoblastoma protein (Rb). Each experiment had a completely randomized design, with three replicates in each experiment. The results showed that the AMPK inhibitor (Compound C, 20 μM-24 h) increased cell proliferation viability, ATP production, and maximal respiration of SCs by 0.64-, 0.12-, and 0.08-fold (p < 0.05), respectively; increased the SC protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.13-, 0.09-, 0.88-, and 0.12-fold (p < 0.05), respectively; and decreased the SC protein expression of p38 and p21 by 0.36- and 0.27-fold (p < 0.05), respectively. The AMPK agonist AICAR (2 mM-6 h) significantly inhibited SC ultrastructure, OCR, mitochondrial respiratory enzyme activity, and cell proliferation-related protein levels. AMPK was validated to be a target gene of miR-1285 based on the result in which the miR-1285 mimic inhibited the luciferase activity of wild-type AMPK by 0.54-fold (p < 0.001). MiR-1285 mimic promoted the OCR of SCs, with 0.45-, 0.15-, 0.21-, and 0.30-fold (p < 0.01) increases in ATP production, basal and maximal respiration, and spare capacity, respectively. MiR-1285 mimic increased the mitochondrial respiratory enzyme activity of SCs, with 0.63-, 0.70-, and 0.97-fold (p < 0.01) increases in NADH-Q oxidoreductase, cytochrome c oxidase, and ATP synthase, respectively. Moreover, the miR-1285 mimic increased the protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.24-, 0.30-, 0.22-, and 0.13-fold (p < 0.05), respectively, and reduced the protein expression of p38 and p21 by 0.58- and 0.66-fold (p < 0.001). MiR-1285 inhibitor showed opposite effects on the above indicators and induced numerous autophagosomes and large lipid droplets in SCs. A high dose of estradiol (10 μM-6 h, showed a promotion of AMPK activation in a previous study) significantly inhibited SC ultrastructure, mitochondrial function, and proliferation-related pathways, while these adverse effects were weakened by Compound C treatment or miR-1285 mimic transfection. Our findings suggest that the activation and inhibition of AMPK induced by specific drugs or synthesized targeted miRNA fragments could regulate immature boar SC proliferative activity by influencing the CDK4/Cyclin D3 pathway and mitochondrial function; this helps to provide a basis for the prevention and treatment of male sterility in clinical practice.

NOVEL ITACONATE DERIVATIVE ATTENUATES ISOPROTERENOL INDUCED CARDIOTOXICITY IN EXPERIMENTAL ANIMALS: A MOLECULAR PATHWAY INSIGHT

Objective: 4-octyl itaconate (4-OI) is a potential drug for the treatment of myocardial infarction on account of its potent anti-inflammatory, antioxidant and anti-apoptotic properties. Thus, we hypothesise that 4-OI may attenuate isoproterenol(ISO) induced cardiotoxicity by modulating several molecular pathways in Wistar albino rats. Design and method: After getting approval from animal ethics committee(278/IAEC/2021), the experimental work was started. ISO 5mg/kg s.c., was administered daily for 15 days. Adult male rats were randomly divided into 5 groups (n=6): normal control group, ISO control group, treatment control groups. 4-OI was administered i.p. at 3 different doses (2.5,5,10 mg/kg) to rats from day 1 and continued for 15 days along with ISO. On the 16th day, surgery was performed, right carotid artery was cannulated for recording of hemodynamic parameters. Blood was drawn using cardiac puncture and rats were then sacrificed, one portion of the heart was immediately stored in 10% formalin for histopathological evaluation and second portion was kept in -80 degree C for further studies. Serum was stored for biochemical, pro-inflammatory cytokines estimation (TNF, IL-6, IL1 beta) and cardiac injury markers (CKMB, LDH). Results: 4-OI improved cardiac function and attenuated pathological changes as seen microscopically in the H&amp;E stained heart tissue sections. On ultrastructural evaluation, 4-OI treatment (5, 10mg/kg) groups presented with lesser nuclear condensation and mitochondrial swelling, as compared to the ISO control group. A significant decline in the serum and tissue levels of cardiac injury markers (CK-MB, LDH) and inflammatory markers were observed in 4-OI (5,10mg/kg) treated rats, as compared to ISO control group. Additionally, 4-OI suppressed oxidative stress and apoptosis, along with an increased PPAR-gamma expression. Molecular pathway assessment revealed that at the dose of 10mg/kg, 4-OI reduced the expression of NFkappaB, IKK-beta and enhanced the expression of Nrf-2 and HO-1. On analysis of the inflammatory MAPK pathway, expression of the pro-survival kinase ERK was also increased while JNK and p38 protein expressions were reduced. Conclusions: 4-OI at a dose of 10mg/kg mitigates myocardial toxicity in ISO treated rats by maintaining morphological structure of heart tissue with oxidative stress profile and reduced inflammation

Mechanism of total saponins of Panax japonicus against liver injury induced by acetaminophen in mice based on ERK/NF-κB/COX-2 signaling pathway

This study investigated the effects and mechanisms of total saponins of Panax japonicus(TSPJ) against liver injury induced by acetaminophen(APAP). Male Kunming mice were randomly divided into a blank control group, TSPJ group(200 mg·kg~(-1), ig), model group, APAP+ TSPJ low-dose group(50 mg·kg~(-1), ig), APAP+ TSPJ medium-dose group(100 mg·kg~(-1), ig), APAP+ TSPJ high-dose group(200 mg·kg~(-1), ig), and APAP+ N-acetyl-L-cysteine group(200 mg·kg~(-1), ip). The administration group received the corresponding medications via ig or ip once a day for 14 consecutive days. After the last administration for one hour, except for the blank control group and TSPJ group, all groups of mice were given 500 mg·kg~(-1) APAP by gavage. After 24 hours, mouse serum and liver tissue were collected for serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), reactive oxygen species(ROS), tumor necrosis factor alpha(TNF-α), interleukin-1 beta(IL-1β), cyclooxygenase-2(COX-2), IL-6, IL-4, IL-10, as well as lactate dehydrogenase(LDH), glutathione(GSH), superoxide dismutase(SOD), catalase(CAT), total antioxidant capacity(T-AOC), malondialdehyde(MDA), and myeloperoxidase(MPO) liver tissue. Hematoxylin-eosin staining was used to observe the morphological changes of liver tissue. The mRNA expression levels of lymphocyte antigen 6G(Ly6G), galectin 3(Mac-2), TNF-α, IL-1β, COX-2, IL-6, IL-4, and IL-10 in liver tissue were determined by quantitative real-time polymerase chain reaction(PCR). Western blot was utilized to detect the protein expression levels of Ly6G, Mac-2, extracellular regulated protein kinases(ERK), phosphorylated extracellular regulated protein kinases(p-ERK), COX-2, inhibitor of nuclear factor κB protein α(IκBα), phosphorylated inhibitor of nuclear factor κB protein α(p-IκBα), and nuclear factor-κB subunit p65(NF-κB p65) in cytosol and nucleus in liver tissue. The results manifested that TSPJ dramatically reduced liver coefficient, serum ALT, AST, ROS, TNF-α, IL-1β, IL-6, and COX-2 levels, LDH, MPO, and MDA contents in liver tissue, and mRNA expressions of TNF-α, IL-1β, and IL-6 in APAP-induced liver injury mice. It prominently elevated serum IL-4 and IL-10 levels, GSH, CAT, SOD, and T-AOC contents, and mRNA expressions of IL-4 and IL-10 in liver tissue, improved the degree of liver pathological damage, and suppressed neutrophil infiltration and macrophage recruitment in liver tissue. In addition, TSPJ lessened the mRNA and protein expressions of neutrophil marker Ly6G, macrophage marker Mac-2, and COX-2 in liver tissue, protein expressions of p-ERK, p-IκBα, and NF-κB p65 in nuclear, and p-ERK/ERK and p-IκBα/p-IκBα ratios and hoisted protein expression of NF-κB p65 in cytosol. These results suggest that TSPJ has a significant protective effect on APAP-induced liver injury in mice, and it can alleviate APAP-induced oxidative damage and inflammatory response. Its mechanism may be related to suppressing ERK/NF-κB/COX-2 signaling pathway activation, thus inhibiting inflammatory cell infiltration, cytokine production, and liver cell damage.

To Investigate the Mechanism of Qinpi Tongfeng Formula in Treating Acute Gouty Arthritis by UHPLC-Q-Orbitrap-MS, Network Pharmacology and Experimental Validation.

Acute gouty arthritis (AGA) is characterized by the accumulation of monosodium urate crystals within the joints, leading to inflammation and severe pain. Western medicine treatments have limitations in addressing this condition. Previous studies have shown the efficacy of Qinpi Tongfeng formula (QPTFF) in treating AGA, but further investigation is needed to understand its mechanism of action. We used ultra-high-performance liquid chromatography tandem Q-Exactive Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-MS) to identify compounds in QPTFF. Target proteins regulated by these compounds were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, Chemistry Database, and Swiss Target Prediction Database. AGA-related targets were searched and screened from various databases, including Genecards, PharmGKB, Drugbank, etc. Intersection targets of QPTFF and AGA were analyzed for protein-protein interaction networks, GO function enrichment, and KEGG pathway enrichment. We then verified QPTFF's mechanism of action using an AGA rat model, assessing pathological changes via H&E staining and target expression via ELISA, RT-qPCR, and Western blot. UHPLC-Q-Orbitrap-MS identified 207 compounds in QPTFF, with 55 selected through network pharmacology. Of 589 compound-regulated targets and 1204 AGA-related targets, 183 potential targets were implicated in QPTFF's treatment of AGA. Main target proteins included IL-1β, NFKBIA, IL-6, TNF, CXCL8, and MMP9, with the IL-17 signaling pathway primarily regulated by QPTFF. Experimental results showed that medium and high doses of QPTFF significantly reduced serum inflammatory factors and MMP-9 expression, and inhibited IL-17A, IL-6, IKK-β, and NF-κB p65 mRNA and protein expression in AGA rats compared to the model group. Key targets of QPTFF include IL-1β, NFKBIA, IL-6, TNF-α, CXCL8, and MMP9. QPTFF effectively alleviates joint inflammation in AGA rats, with high doses demonstrating no liver or kidney toxicity. Its anti-inflammatory mechanism in treating AGA involves the IL-17A/NF-κB p65 signaling pathway.

Ginsenoside Rg_1 ameliorates OGD/R-induced PC12 cell injury by inhibiting autography via IRE1-JNK-CHOP pathway

This study investigated the protective effect of ginsenoside Rg_1(GRg_1) on oxygen and glucose deprivation/reoxygenation(OGD/R)-injured rat adrenal pheochromocytoma(PC12) cells and whether the underlying mechanism was related to the regulation of inositol-requiring enzyme 1(IRE1)-c-Jun N-terminal kinase(JNK)-C/EBP homologous protein(CHOP) signaling pathway. An OGD/R model was established in PC12 cells, and PC12 cells were randomly classified into control, model, OGD/R+GRg_1(0.1, 1, 10 μmol·L~(-1)), OGD/R+GRg_1+rapamycin(autophagy agonist), OGD/R+GRg_1+3-methyladenine(3-MA,autophagy inhibitor), OGD/R+GRg_1+tunicamycin(endoplasmic reticulum stress agonist), OGD/R+GRg_1+4-phenylbutyric acid(4-PBA, endoplasmic reticulum stress inhibitor), and OGD/R+GRg_1+3,5-dibromosalicylaldehyde(DBSA, IRE1 inhibitor) groups. Except the control group, the other groups were subjected to OGD/R treatment, i.e., oxygen and glucose deprivation for 6 h followed by reoxygenation for 6 h. Cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT) assay. Apoptosis was detected by Hoechst 33342 staining, and the fluorescence intensity of autophagosomes by the monodansylcadaverine(MDC) assay. Western blot was employed to determine the expression of autophagy-related proteins(Beclin1, LC3-Ⅱ, and p62) and the pathway-related proteins [IRE1, p-IRE1, JNK, p-JNK, glucose-regulated protein 78(GRP78), and CHOP]. The results showed that GRg_1 dose-dependently increased the viability of PC12 cells and down-regulated the expression of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, compared with the model group. Furthermore, GRg_1 decreased the apoptosis rate and MDC fluorescence intensity and up-regulated the expression of p62 protein. Compared with the OGD/R+GRg_1(10 μmol·L~(-1)) group, OGD/R+GRg_1+rapamycin and OGD/R+GRg_1+tunicamycin groups showed increased apoptosis rate and MDC fluorescence intensity, up-regulated protein levels of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, decreased relative cell survival rate, and down-regulated protein level of p62. The 3-MA, 4-PBA, and DBSA groups exerted the opposite effects. Taken together, GRg_1 may ameliorate OGD/R-induced PC12 cell injury by inhibiting autophagy via the IRE1-JNK-CHOP pathway.

Betulinic acid protects against ovarian impairment by decreasing F-2 toxin-induced oxidative stress and inflammation associated with the downregulation of p38 expression in mice

F-2 toxin is an estrogenic mycotoxin that causes reproductive disorders in animals. Betulinic acid (BA) is a natural pentacyclic lupane-structure triterpenoid that has diverse pharmacological activities. In this study, the antioxidative and anti-inflammatory effects of BA and its underlying mechanism are explored in F-2 toxin-triggered mouse ovarian damage. We found that BA alleviated the F-2 toxin-induced ovarian impairment by stimulating follicle growth, reducing inflammatory cell infiltration, repairing damaged mitochondria and endoplasmic reticulum. Simultaneously, BA not only reversed F-2 toxin-induced reduction of follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels in the serum, but also restrained the protein expression of the estrogen receptors α (ERα) and ERβ. Moreover, BA restored the balance of F-2 toxin-induced ovarian redox system disorders. Subsequently, we found that 0.25 mg/kg BA played an anti-inflammatory role in the F-2 toxin-induced ovarian impairment by decreasing interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) mRNA expression, as well as inhibiting p38 protein expression. These data demonstrated that BA exerts its protective effect on F-2 toxin-induced ovarian oxidative impairment and inflammation by inhibiting p38 expression, which implies a natural product-based medicine to ameliorate F-2 toxin-caused female reproductive toxicity and provides a detoxifying method for food contaminated by mycotoxin.