Early pregnancy exposure to Microcystin-LR compromises endometrial decidualization in mice via the PI3K/AKT/FOXO1 signaling pathway.

Previous experimental studies have found that exposure to Microcystin-leucine arginine can impact pregnancy outcomes in female mice. The impact of MC-LR on early pregnancy in mammals is not yet well understood. Both mice and humans need to undergo decidualization to maintain pregnancy. In this study, we tried to evaluate whether MC-LR affects decidualization process in mice. Our research showed that MC-LR decreased maternal weight gain, uterine weight, and implantation site weight. These findings suggested that MC-LR exerted adverse effects on decidualization. In mice, we examined decreased number of polyploid decidual cells, but marked proliferation of mouse endometrial stromal cells the expression levels of prolactin (PRL)and insulin-like growth factor binding protein 1 (IGFBP1) were significantly downregulated in the decidual tissue and primary endometrial stromal cells following MC-LR treatment. Furthermore, further in vitro experiments identified that MC-LR promoted endometrial stromal cell division and cycle transition. Lastly, our study demonstrated that MC-LR impaired decidualization through the PI3K/AKT/FOXO1 pathway. Collectively, these data suggested that exposure to MC-LR impaired decidualization during early pregnancy.

Akt Inhibitors Prevent CyHV-2 Infection In Vitro.

Cyprinid herpesvirus 2 (CyHV-2) is a double-stranded DNA virus that infects goldfish (Carassius auratus) and crucian carp (C. carassius), resulting in substantial mortality rates and significant epidemiological implications. To gain deeper insights into CyHV-2-host interactions and identify potential therapeutic targets, quantitative proteomics analysis was conducted on CyHV-2-infected Ryukin goldfish fin (RyuF-2) cells. Our findings revealed significant alterations in the expression of proteins associated with the PI3K/Akt signaling pathway, which were up-regulated upon viral infection. Building on these observations, we employed LY294002, a specific inhibitor of PI3K, to investigate its impact on viral replication by inhibiting the PI3K/Akt pathway in GiCF cell line derived from the caudal fin of Carassius auratus gibelio (Bloch). Our results demonstrated the inhibition of both CyHV-2 replication and Akt phosphorylation within this pathway. Quercetin, a plant-derived analogue of LY294002, was further investigated for its anti-CyHV-2 effects in vitro as well as its underlying mechanism. The results suggested that quercetin exhibits antiviral properties against CyHV-2 and may exert its effects through mechanisms similar to those observed with LY294002. Given that aquaculture water serves as a vector for aquaculture viral diseases and the release of chemical compounds can lead to pollution of the aquatic environment, our study shifted focus to crude extracts obtained from plants. We confirmed crude quercetin extract derived from Cuminum anisum has antiviral activity against CyHV-2 in vitro. Therefore, based on our identification of the involvement of PI3K/Akt signaling pathway in CyHV-2 replication, along with verification of its antiviral mechanism using LY294002, we propose natural dietary quercetin as a promising candidate for development into a novel anti-CyHV-2 drug.

A Pair of Indicators for Characterizing Cerebral Microbleeds Based on Raman Spectrum and Two-Photon Imaging.

Cerebral microbleeds (CMBs) lead to cognitive decline, linked to the axonal structure composed of phospholipid bilayers. Current methods are difficult to obtain in situ changes of biochemical component concentration during CMB. In this study, by Raman spectrum and two-photon imaging, we achieve in situ changes in the information of biochemical components concentration during CMB. The overall concentration of phospholipids in the damaged tissue significantly decreases after CMB, forming a large region of low concentration, but the relative concentration of phosphatidylinositol (PI) increases, reflecting the inhibition role of the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway. Accordingly, two-photon images of neurons show a clear decrease in the number of axons, indicating a close correlation between phospholipid hydrolysis and axon damage, as well as cognitive impairment. Therefore, the decrease in phospholipid concentration and the increase in the PI concentration might serve as a pair of indicators for characterizing CMB and its relationship with cognitive decline.

MUC1­ND interacts with TRPV1 to promote corneal epithelial cell proliferation in diabetic dry eye mice by partly activating the AKT signaling pathway.

Although both mucin1 (MUC1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) have been reported to be associated with dry eye (DE) disease, whether they interact and their regulatory roles in diabetic DE disease are unknown. Diabetic DE model mice were generated by streptozotocin induction and assessed by corneal fluorescein staining, tear ferning (TF) tests, phenol red thread tests, hematoxylin and eosin staining of corneal sections and periodic acid Schiff staining of conjunctival sections. Cell proliferation was measured by CCK8 assay. Western blotting was performed to measure protein expression. Primary mouse corneal epithelial cells (MCECs) were cultured after enzymatic digestion. Immunofluorescence staining of MCECs and frozen corneal sections was conducted to assess protein expression and colocalization. Coimmunoprecipitation was performed to detect protein­protein interactions. It was found that, compared with control mice, diabetic DE mice exhibited increased corneal epithelial defects, reduced tear production, poorer TF pattern grades and impaired corneal and conjunctival tissues. In vivo and in vitro experiments showed that hyperglycemia impaired cell proliferation, accompanied by decreased levels of the MUC1 extracellular domain (MUC1­ND) and TRPV1. Additionally, it was found that capsazepine (a TRPV1 antagonist) inhibited the proliferation of MCECs. Notably, MUC1­ND was shown to interact with the TRPV1 protein in the control group but not in the diabetic DE group. It was also found that the AKT signaling pathway was attenuated in the diabetic DE mice and downstream of TRPV1. MUC1­ND interacted with TRPV1, partly activating the AKT signaling pathway to promote MCEC proliferation. The present study found that the interaction of MUC1­ND with TRPV1 promotes MCEC proliferation by partly activating the AKT signaling pathway, providing new insight into the pathogenesis of corneal epithelial dysfunction in diabetic DE disease.

Exploring the anti-NSCLC mechanism of phillyrin targeting inhibition of the HSP90-AKT pathway.

Phillyrin (PHN), derived from the dried fruit of Forsythia suspensa (Thunb.) Vahl, is a kind of Chinese herbal medicine with the effect of clearing heat, and has been used in China for thousands of years in treating various tumors. However, the mechanism of its main components on non-small cell lung cancer (NSCLC) remains unclear. PHN is a distinct component extracted from Forsythia suspensa with promising anti-cancer activity against various tumor types. This study sought to elucidate the promising effects of PHN on NSCLC. Based on network pharmacology results, we identified potential PHN targets and pathways for NSCLC treatment. CCK-8 assay, wound healing assay, apoptosis assay, western blot, and in vivo experiments verified the inhibitory effect of PHN on NSCLC. Network pharmacology identified 160 potential PHN targets, 955 NSCLC-related targets, and 54 common targets, along with 132 pathways and 2 core genes. Biological experiments demonstrated that PHN significantly inhibited the growth and migration of A549 and LLC cells while promoting their apoptosis. Western blot analysis revealed down-regulation of AKT, HSP90AA1, and CDC37 expression, suggesting that PHN inhibits A549 and LLC cell proliferation by down-regulating the HSP90-AKT pathway. In vivo experiments confirmed that PHN significantly inhibited NSCLC growth with low toxicity. This study, using network pharmacology and biological experiments, verified the effectiveness of PHN against NSCLC through the HSP90-AKT pathway. These findings provide a foundation for further research and analysis.

Bie Jia Jian pill ameliorates BDL-induced cholestatic hepatic fibrosis in rats by regulating intestinal microbial composition and TMAO-mediated PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: As a compound of traditional Chinese medicine, Bie Jia Jian pill (BJJP) is extensively used to treat the clinical chronic liver disease. Nevertheless, the specific mechanism through which BJJP affects hepatic fibrosis (HF) remains unknown. AIM OF THE STUDY: To explore the roles and potential mechanisms of BJJP involved in treating HF. MATERIALS AND METHODS: HF model of Sprague-Dawley (SD) rats was induced by a bile duct ligation (BDL). The function of BJJP involved in the intestinal microbiota (IM) and its metabolites in BDL SD rats were explored through the 16S rRNA sequencing and untargeted metabolomics technologies. Network pharmacology was used to forecast mechanisms underlying BJJP's anti-HF effects, which were validated in BDL-induced rats and trimethylamine N-oxide (TMAO)-induced LX-2 and HSC-T6 cells. RESULTS: BJJP effectively ameliorated pathological liver damage, inflammation, and fibrosis of the BDL-induced HF rats. BJJP regulates IM diversity and composition and interferes with trimethylamine (TMA)-flavin monooxygenase 3 (FMO3)-TMAO process. In vitro, BJJP significantly inhibited the TMAO-induced activation of hepatic stellate cells (HSCs) (rat HSC cell line, HSC-T6; human HSC cell line, LX-2 cells). Network pharmacology results demonstrated that the signal pathway of PI3K/AKT is crucially involved in BJJP treatment of HF. Further research revealed that BJJP inhibited the PI3K/AKT signal pathway in BDL-induced HF rats. Moreover, TMAO activated the PI3K/AKT pathway, whereas BJJP suppressed TMAO-induced activation. Subsequent intervention with 740Y-P (the PI3K agonist) successfully neutralized the repression effect on the pathway of PI3K/AKT by BJJP. CONCLUSION: These results clearly show that BJJP attenuates HF by regulating the IM, as well as inhibiting PI3K/AKT pathway mediated by TMAO.

Compound Sophorae Decoction mitigates DSS-induced ulcerative colitis by activating autophagy through PI3K-AKT pathway：a integrative research combining network pharmacology and in vivo animal model validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Compound sophora decoction (CSD), a widely used Chinese herbal formula, has been shown to effectively alleviate symptoms ulcerative colitis (UC), including of bloody diarrhea, tenesmus, abdominal pain, and fever. Despite its clinical use, the precise pharmacological mechanisms of CSD remain enigmatic. AIM OF THE STUDY: This study aims to investigate the potential efficacy and underlying mechanisms of CSD in the treatment of UC by employing an integrative pharmacology-based approach, molecular docking analysis and experimental validation. MATERIALS AND METHODS: In this study, an integrative pharmacology-based approach was employed to predict the primary pathway through which CSD treats UC. The mechanism of CSD was further validated using a DSS-induced UC mouse model. Disease severity was assessed by monitoring stool property, body weight, colon length, and colon histopathology. Colonic pathological changes were examined using hematoxylin and eosin (H&E) staining. The concentration of cytokines was measured via ELISA, while key molecules in the PI3K/AKT pathway and autophagy-related markers were evaluated using Western blotting. Autophagy in intestinal epithelial cells was observed using electron microscopy. RESULTS: The results demonstrated that CSD alleviated DSS-induced UC by inhibiting the activation of PI3K/AKT pathway, reducing the release of inflammatory cytokines, down-regulating oxidative mediators, and enhancing autophagy. Moreover, the protective effects of CSD were diminished by bpV, a PTEN inhibitor, further supporting the involvement of the PI3K/AKT pathway. CONCLUSIONS: The underlying mechanism of CSD's therapeutic effect on UC may involve significant attenuation of DSS-induced intestinal inflammation by promoting autophagy through the inhibition of PI3K-AKT pathway activation.

Three new coumarins from Notopterygium incisum with potential anti-inflammatory activity.

Three previously undescribed coumarins (1-3) were obtained from the roots of Notopterygium incisum. Their chemical structures were elucidated using a variety of spectroscopic techniques and chemical calculations. The inhibitory effects of these new compounds on NO production and pro-inflammatory factors (IL-1ß, IL-6, and TNF-α) in LPS-stimulated RAW 264.7 cells were investigated. Further studies revealed that compound 1 suppressed the expression of COX-2 and iNOS while also reduced ROS accumulation. Western blot analysis demonstrated that compound 1 could inhibit the PI3K/AKT pathway by decreasing the levels of p-PI3K and p-AKT. Collectively, these findings suggest that compounds 1-3 exhibit promising anti-inflammatory properties.

Mitigation of allergic asthma in mice: A compound mixture comprising luteolin, arbutin, and marmesin from Gerbera Piloselloides Herba by suppression of PI3K/Akt pathway.

Background: Gerberae Piloselloidis Herba (GPH) exhibits notable efficacy in alleviating allergic asthma. Previous studies in our research have identified a mixture of luteolin, arbutin, and marmesin as effective components of GPH in treating allergic asthma. However, the underlying mechanism remains unclear. This study aims to elucidate the molecular mechanism of these active components. Method: Using an ovalbumin (OVA)-induced allergic asthma mouse model, various treatment groups were administered, including GPH, the active component mixture (termed "Mixture") containing luteolin, arbutin, and marmesin, and a positive drug (dexamethasone, DEX). Relevant indices were assessed, including behavioral characteristics, inflammatory cell counts, cytokine levels, histopathological examination of lung tissue, apoptosis, and expression of key proteins such as Caspase-3, Bax, Bcl-2, PI3K, p-PI3K, Akt, and p-Akt. The effect of the Mixture on the PI3K/Akt signaling pathway was further verified using the PI3K inhibitor LY294002. Results: The Mixture significantly alleviated asthma symptoms, decreased IgE levels, cytokine levels (IL-4, IL-5, IL-13 and TNF-α), and the number of inflammatory cells in serum or bronchoalveolar lavage fluid (BALF), leading to the alleviation of lung pathological lesions. Additionally, the Mixture reduced the expression of Bax and Caspase-3 while increasing Bcl-2 expression, resulting in mitigated apoptosis in lung tissue. Furthermore, there appeared a decrease in the levels of PI3K and p-PI3K, as well as the ratio of p-Akt to Akt in the Mixture group, indicating the suppression of PI3K and Akt phosphorylation. Interestingly, the effects of the Mixture were comparable to those of GPH, LY294002, or the combination of LY294002 with the Mixture. Conclusion: The study confirms that the Mixture containing luteolin, arbutin, and marmesin indeed alleviates allergic asthma induced by OVA in mice by suppressing the PI3K/Akt signaling pathway. These findings highlight the potential of the GPH-derived Mixture as a novel therapeutic for the treatment of allergic asthma.

ZDHHC20 Activates AKT Signaling Pathway to Promote Cell Proliferation in Hepatocellular Carcinoma.

Background: Liver cancer is the sixth most common cancer worldwide, and hepatocellular carcinoma (HCC) presents one of the most challenging global health issues. ZDHHC20, a member of the ZDHHC palmitoyltransferase (ZDHHC-PAT) family, is involved in a reversible lipid modification known as palmitoylation, which contributes to the occurrence and progression of various tumors. However, the specific mechanisms underlying the involvement of ZDHHC20 in this process are unclear. Methods: The effects of both ZDHHC20 knockdown and overexpression on hepatocellular carcinoma cell proliferation were evaluated using PCR, Western blotting, CCK-8 assay, colony formation assay, cell cycle analysis, apoptosis analysis, and EDU assay. The TCGA-LIHC dataset was analyzed bioinformatically, and the phosphorylation level of PI3K and AKT in SK-Hep1 and Huh7 cells was assessed using Western blotting. Nude mouse subcutaneous xenograft experiments were conducted to evaluate the effects of different treatment conditions on mouse tumor growth. Results: ZDHHC20 knockdown inhibited cell proliferation and promoted apoptosis, while overexpression of ZDHHC20 promoted cell proliferation and inhibited apoptosis. Knockdown of ZDHHC20 also decreased phosphorylation of PI3K and AKT in HCC, whereas overexpression of ZDHHC20 increased phosphorylation of PI3K and AKT. The PI3K-AKT pathway inhibitors, LY294002 and MK2206, effectively inhibited the promotional effects of ZDHHC20 on the proliferation and growth of HCC. Conclusion: High expression of ZDHHC20 promotes the proliferation and tumor growth of HCC by activating the PI3K-AKT signaling pathway. The PI3K inhibitor LY294002 and the AKT inhibitor MK2206 inhibit the promotional effects of ZDHHC20 on the proliferation of HCC and the growth of tumors.

Fenitrothion induces glucose metabolism disorders in rat liver BRL cells by inhibiting AMPKα and IRS1/PI3K/AKT signaling pathway.

Fenitrothion (FNT) is a common organophosphorus pesticide that is widely used in both agricultural and domestic pest control. FNT has been frequently detected in various environmental media, including the human body, and is a notable contaminant. Epidemiological investigations have recently shown the implications of exposure to FNT in the incidence of various metabolic diseases, such as diabetes mellitus in humans, indicating that FNT may be a potential endocrine disruptor. However, the effects of FNT exposure on glucose homeostasis and their underlying mechanisms in model organisms remain largely unknown, which may limit our understanding of the health risks of FNT. In this study, FNT (4 5, 90, 180, and 4 50 µM) exposure model of rat hepatocytes (Buffalo Rat Liver, BRL cells) was established to investigate the effects and potential mechanisms of its toxicity on glucose metabolism. Several key processes of glucose metabolism were detected in this study. The results showed significantly increased glucose levels in the culture medium and decreased glycogen content in the FNT-exposed BRL cells. The results of quantitative real-time PCR and enzymology showed the abnormal expression of genes and activity/content of glucose metabolic enzymes involved in glucose metabolism, which might promote gluconeogenesis and inhibit glucose uptake, glycolysis, and glycogenesis. Furthermore, gluconeogenesis and glycolytic were carried out in the mitochondrial membrane. The abnormal of mitochondrial membrane potential may be a potential mechanism underlying FNT-induced glucose metabolism disorder. In addition, the mRNA and protein expression implicated that FNT may disrupt glucose metabolism by inhibiting the AMPKα and IRS1/PI3K/AKT signaling pathways. In conclusion, results provide in vitro evidence that FNT can cause glucose metabolism disorder, which emphasizes the potential health risks of exposure to FNT in inducing diabetes mellitus.

Effect of irisin on ovarian phosphatidylinositol-3-kinase/protein kinase B signaling pathway and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways of rats with polycystic ovary syndrome.

OBJECTIVE: To investigate the independent effects of irisin on insulin resistance (IR) in ovary of polycystic ovary syndrome (PCOS) and explore possible pathways. METHODS: We established PCOS medel using Poretsky L's method, then PCOS rats were randomly divided into model group (M) and irisin group (I), and normal rats (N) were used as the control. Then rats in the group I were injected with recombinant irisin. Then the levels of circulating fasting blood glucose (FBG), fasting insulin (FINS), homeostasis model assessment of IR (HOMA-IR) and PI3K/AKT and MAPK/ERK pathways in each group were observed, as well as the effects of irisin on the levels of circulating HOMA-IR and PI3K/AKT and MAPK/ERK pathways in ovary of PCOS rats were evaluated. RESULTS: Compared with normal group, levels of FBG, FINS, and HOMA-IR of model group were significantly increased (p < 0.001, p < 0.001, and p < 0.001, respectively), levels of average optical density by IHC of p-PI3K, PI3K, p-AKT, and AKT (p = 0.015, p = 0.010, p = 0.005, and p = 0.009, respectively) and levels of mRNA concentration of PI3K and AKT (p = 0.001, and p = 0.005, respectively) were decreased, while the levels of average optical density of p-ERK, ERK (p = 0.011, and p = 0.013, respectively) and level of mRNA concentration of ERK (p < 0.001) were increased in ovary. After irisin intervention, compared with model group, levels of FBG, FINS, and HOMA-IR of rats in irisin group were significantly decreased (p = 0.001, p < 0.001, and p < 0.001, respectively), levels of average optical density by IHC of p-PI3K, PI3K, p-AKT, and AKT (p = 0.030, p = 0.024, p = 0.012, and p = 0.025, respectively) and levels of mRNA concentration of PI3K and AKT (p = 0.002, and p = 0.003, respectively) were significantly increased, while the levels of average optical density of p-ERK, ERK (p = 0.004, and p = 0.026, respectively) and level of mRNA concentration of ERK (p = 0.001) were significantly decreased. CONCLUSION: Our study demonstrated that irisin could not only improve circulating insulin resistance, but may also improve ovarian IR through an increase in the activity of PI3K/AKT signaling and a decrease of MAPK/ERK signaling.

KIF18A promotes cervical squamous cell carcinoma progression by activating the PI3K/AKT pathway through upregulation of CENPE.

BACKGROUND: Cervical cancer is a prevalent malignancy that significantly contributes to morbidity and mortality rates among women in developing nations. Although the association of KIF18A with various cancers has been established, its role in cervical squamous cell carcinoma (CESC) remains elusive. METHODS: The KIF18A impact on the progression of CESC and its underlying mechanism were investigated through comprehensive bioinformatics analysis utilizing publicly available datasets. The levels of KIF18A and CENPE were assessed in clinical CESC samples through western blotting and qRT-PCR. To discover the role and molecular pathways of KIF18A in CESC, a combination of experimental approaches, including wound-healing, flow cytometry, CCK-8, and Transwell assay, were employed. RESULTS: Our results demonstrate a significant KIF18A expression upregulation in CESC tissues in contrast to healthy tissues. In vitro, KIF18A upregulation was found to enhance cell growth, migration, and invasion and activate the PI3K/AKT signaling pathway while concurrently suppressing apoptosis. Conversely, downregulating KIF18A exhibited contrasting effects. Mechanistically, we observed a positive significant connection between KIF18A and CENPE in CESC cells. CONCLUSION: KIF18A promotes tumor growth in CESC by modulating the PI3K/AKT signaling pathway through regulation of CENPE, making it a potential biomarker for diagnosis and prognosis as well as a therapeutic target.

The role of ESM1 in the lipids metabolic reprogramming and angiogenesis of lung adenocarcinoma cells.

Background: Lung adenocarcinoma (LUAD) is one of the respiratory diseases with high mortality and incidence. As an important angiogenic factor, (Endothelial cell-specific molecule 1) ESM1 plays an important role in the occurrence and development of LUAD. However, the role and molecular mechanism of ESM1 on LUAD metabolic reprogramming and angiogenesis remain unclear. Methods: We used multiple databases to analyze the prognostic significance and potential function of ESM1 in patients with LUAD. The expression of ESM1 in LUAD cells was down-regulated/overexpressed by RNA interference, and the effects of ESM1 on the proliferation, migration, lipid metabolism and angiogenesis of LUAD cells in vitro and in vivo were analyzed using MTT, EdU, wound healing, oil red O, tubule formation, xenograft tumor model and chicken embryo allantoic model. Results: ESM1 is closely associated with poor prognosis in LUAD patients. ESM1 promotes LUAD proliferation, migration, fatty acid synthesis and angiogenesis. It also accelerates the proliferation, migration, lipid synthesis and tubule formation of endothelial cells in the tumor microenvironment in the form of secreted protein. Mechanically, ESM1 can promote the activation of AKT signaling pathway and up-regulate the expression of SCD1 and FASN. Conclusion: Our results suggest that ESM1 promotes the proliferation, migration, lipid reprogramming, and angiogenesis of LUAD cells by activating the AKT signaling pathway, suggesting that ESM1 may be a potential therapeutic target and prognostic marker in LUAD patients.

Polysaccharides from Ostrea rivularis alleviate type II diabetes induced-retinopathy and VGEF165-induced angiogenesis via PI3K/AKT signaling pathway.

The purpose of this study was to investigate the role of polysaccharides from Ostrea rivularis Gloud (ORPs) in the progression of diabetic retinopathy (DR) and its anti-angiogenic effect on endothelial cell. Transgenic db/db mice with DR model were used to evaluate the protective effect of ORPs on retinal damage. It was found that ORPs could down-regulated levels of random blood glucose and fasting insulin, and further ameliorate retinal structure abnormalities as well as vascular network structure. Moreover, ORPs could reduce the expression of VEGF in retinal tissue and lessen pathological angiogenesis, thus slowing the progression of DR. In vitro, the proliferation, migration and tube formation of VGEF165-induced EA.hy926 cells were inhibited with ORPs administration. Furthermore, the expression of related proteins in the PI3K/AKT pathway and angiogenesis related factors were improved after ORPs intervention. Overall, these findings suggested that ORPs could effectively control the development of DR, and inhibit VGEF165-induced EA.hy926 cells proliferation, migration and tube formation, which effects might work through blocking the activation of PI3K/AKT signaling pathway.

Schisandra chinensis lignans improve insulin resistance by targeting TLR4 and activating IRS-1/PI3K/AKT and NF-κB signaling pathways.

Schisandra chinensis, a traditional Chinese medicine, has been widely applied in China to treat diabetes and its complications. The aim of this study was to discover the active compounds and explain related molecular mechanism contributing to the anti-diabetic effect of Schisandra chinensis. Herein, the therapeutic effects of Schisandra chinensis extracts on type 2 diabetes mellitus (T2DM) were firstly confirmed in vivo. Subsequently, various lignans were isolated from Schisandra chinensis and tested for hypoglycemic activity in palmitic acid-induced insulin-resistant HepG2 (IR-HepG2) cells. Among these lignans, R-biar-(7S,8R)-6,7,8,9-tetrahydro-1,2,3,12,13,14-hexamethoxy-7,8-dimethyl-7-dibenzo [a, c] cyclooctenol (compound 2) and Gomisin A (compound 4) were identified significantly increased the glucose consumption in IR-HepG2 cells. Meanwhile, compounds 2 and 4 activated the insulin receptor substrate-1 (IRS-1)/phosphoinositide 3-kinase (PI3K)/Ak strain transforming (AKT) pathway, which regulates glucose transporter 2 (GLUT2) and glucose-6-phosphatase (G6Pase), essential for gluconeogenesis and glucose uptake. These compounds also inhibited the nuclear factor-κB (NF-κB) signaling pathway, reducing interleukin-6 (IL-6) levels. Importantly, the hypoglycemic effects of compounds 2 and 4 were diminished after Toll-like receptor 4 (TLR4) knockdown. Cellular thermal shift assays confirmed increased TLR4 protein stability upon treatment with these compounds, indicating direct binding to TLR4. Furthermore, TLR4 knockdown reversed the effects of compounds 2 and 4 on the NF-κB and IRS-1/PI3K/AKT pathways. Taken together, compounds 2 and 4 alleviate IR by targeting TLR4, thereby modulating the NF-κB and IRS-1/PI3K/AKT pathways. These findings suggest that compounds 2 and 4 could be developed as therapeutic agents for T2DM.

miRNA omics reveal neferine induces apoptosis through Ca2+mediated endoplasmic reticulum stress pathway in human endometrial cancer.

BACKGROUND: Endometrial cancer (EC) as one of the most prevalent malignancies in the female reproductive system, usually has a poor diagnosis and unfavorable health effects. Neferine (Nef), derived from the edible and medicinal lotus seed, has been known for its functional activity; however, its anti-cancer mechanism for EC remains elusive. PURPOSE: We explored the potential anti-cancer effects and underlying molecular mechanisms of Nef on EC. METHODS: The cytotoxicity was tested using MTT, and the cell cycle, apoptosis, Ca2+ levels, and the mitochondrial membrane potential (MMP) were observed through flow cytometry. After Nef treatment, differences in miRNA expression were identified using miRNA-seq data. Furthermore, western blot and immunohistochemistry (IHC) were employed to identify the proteins associated with apoptosis in both mice and cells. RESULTS: Nef treatment led to Ishikawa cell apoptosis and blocked cell proliferation in the G2/M phase. In total, 101 significantly different miRNA (p 〈 0.05 and |logFC| 〉 1) were obtained and subjected to GO and KEGG enrichment analysis, which revealed the Ca2+ and PI3K/AKT signaling pathways pertaining to apoptosis. Nef treatment significantly changed intracellular Ca2+ levels and MMP, activating the endoplasmic reticulum stress (ERS) pathway and the expression of key proteins in the mitochondrial pathway. In addition, Nef also inhibited the expression of key proteins in the PI3K/AKT pathway, causing cell apoptosis. Moreover, in mouse tumor tissues, the expression of CHOP, Bcl-2, Caspase 3, Cyto-c, and p-AKT was also consistent with the results in vitro. CONCLUSION: Nef could block the cell cycle and induce the activation of the mitochondrial apoptotic pathway involving the Ca2+-mediated ERS pathway and the PI3K/AKT pathway, thereby inducing apoptosis in EC cells, confirming the potential role of Nef in the prevention and treatment of EC.

Combining systems pharmacology, metabolomics, and transcriptomics to reveal the mechanism of Salvia miltiorrhiza-Cortex moutan herb pair for the treatment of ischemic stroke.

Ischemic stroke (IS), predominantly triggered by blockages in cerebral blood flow, is increasingly recognized as a critical public health issue. The combination of Salvia miltiorrhiza (SM) and Cortex moutan (CM), traditional herbs in Eastern medicine, are frequently used for managing heart and brain vascular conditions. However, the exact mechanisms by which this herb pair (SC) combats IS remain largely unexplored. This investigation focuses on pinpointing the active constituents in SC that contribute to its protective role and deciphering the mechanisms countering cerebral ischemia, particularly in a middle cerebral artery occlusion (MCAO) rat model. We employed UPLC-Q-TOF-MS/MS alongside network pharmacology for predicting SC's target actions against IS. Key ingredients were examined for their interaction with principal targets using molecular docking. The therapeutic impact was gauged through H&E, TUNEL, and Nissl staining, complemented by transcriptomic and metabolomic integration for mechanistic insights, with vital genes confirmed via western blot. UPLC-Q-TOF-MS/MS analysis revealed that the main components of SC included benzoylpaeoniflorin, salvianolic acid B, oxypaeoniflora, salvianolic acid A, and others. Network pharmacology analysis indicated that SC's mechanism in treating IS primarily involves inflammation, angiogenesis, and cell apoptosis-related pathways, potentially through targets such as AKT1, TNF, PTGS2, MMP9, PIK3CA, and VEGFA. Molecular docking underscored strong affinities between these constituents and their targets. Our empirical studies indicated SC's significant role in enhancing neuroprotection in IS, with transcriptomics suggesting the involvement of the VEGFA/PI3K/AKT pathway and metabolomics revealing improvements in various metabolic processes, including amino acids, glycerophospholipids, sphingomyelin, and fatty acids metabolisms.

Electroacupuncture promotes angiogenesis by regulating miR-142-5p and activating ADAMTS1/PI3K/AKT pathway in ischemic stroke rats. / 电针调控miR-142-5p和ADAMTS1/PI3K/AKTé€šè·¯ä¿ƒè¿›ç¼ºè¡€æ€§è„‘å’ä¸­å¤§é¼ è¡€ç®¡æ–°ç”Ÿçš„æœºåˆ¶ç ”ç©¶.

OBJECTIVES: To observe the effect of electroacupuncture on miR-142-5p and ADAMTS1/PI3K/AKT pathway in rats with ischemic stroke, so as to explore the regulatory mechanism of electroacupuncture on angiogenesis after ischemic stroke. METHODS: This study was divided into two parts. The first part of the experiment：SD rats were randomly divided into sham operation group, model group and electroacupuncture group. There were 20 rats in each group. The middle cerebral artery occlusion (MCAO) rat model was prepared using a modified Longa's method. In the electroacupuncture group, "Shuigou" (GV26) was selected for electroacupuncture intervention (4 Hz/20 Hz) for 30 min each time. The rats in the electroacupuncture group were given electroacupuncture immediately after successful modeling, once a day for 4 times. Hunter score and TTC staining were used to observe the neurological deficits and infarct volumes respectively；HE staining was used to observe the cortical pathological changes；immunohistochemistry was used to determine the changes of cerebral microvascular density. Real-time quantitative PCR and Western blot were used to observe the miR-142-5p expression, mRNA and protein expression levels of ADAMTS1, VEGF, PI3K, AKT, eNOS in ischemic cortex. The second part of the experiment：The rats were randomly divided into electroacupuncture+control group and electroacupuncture+miR-142-5p Antagomir group with 8 rats in each group. MCAO model was established after injection. Electroacupuncture+control group was given 0.9% sodium chloride solution injected into the right ventricle.The rats in the electroacupuncture+miR-142-5p Antagomir group were injected with miR-142-5p inhibitor into the right ventricle 30 min before modeling. Rats in electroacupuncture+control group and electroacupuncture+miR-142-5p Antagomir group were all given the same electroacupuncture treatment. Real-time fluorescence quantitative PCR was used to observe the effect of miR-142-5p Antagomir on the expression of miR-142-5p and ADAMTS1 mRNA. The effect of miR-142-5p Antagomir on ADAMTS1 protein was observed by Western blot. RESULTS: In the first part of the experiment, compared with the sham operation group, the Hunter score in the model group was significantly increased (P<0.01)；the volume of cerebral infarction in the model group was significantly increased (P<0.01)；the degree of brain edema and neuronal necrosis and the density of cerebral microvessels was increased；the cerebral microvascular density was significantly increased (P<0.01)；the expression levels of miR-142-5p and the mRNA expression levels of VEGF, AKT and eNOS were significantly decreased (P<0.01, P<0.05), and the protein expression levels of VEGF, p-AKT and eNOS were significantly down-regulated (P<0.01), while the mRNA expression levels of ADAMTS1 and PI3K, and the protein expression levels of ADAMTS1 and p-PI3K were all up-regulated (P<0.01, P<0.05) in the model group. Compared with the model group, after intervention, the Hunter score in the electroacupuncture group was decreased (P<0.01), the volume of cerebral infarction was significantly decreased (P<0.01)；the degree of brain edema and neuronal necrosis were alleviated；the cerebral microvascular density was significantly increased (P<0.01)；the expression of miR-142-5p and the mRNA expression of VEGF, PI3K, AKT and eNOS were increased (P<0.01), the protein expressions of VEGF, p-PI3K, p-AKT and eNOS were increased (P<0.01, P<0.05), while the mRNA and protein expression of ADAMTS1 were decreased (P<0.05, P<0.01). After injection of miR-142-5p inhibitor, compared with electroacupuncture+control group, the expression of miR-142-5p in electroacupuncture+miR-142-5p Antagomir group was decreased(P<0.05), while the mRNA and protein expression of ADAMTS1 were increased (P<0.01, P<0.05). CONCLUSIONS: Electroacupuncture at GV26 can improve the neurological damage of ischemic stroke rats, reduce the volume of cerebral infarction and promote angiogenesis. The mechanism may be associated with the function of electroacupuncture in promoting the expression of miR-142-5p, so as to inhibit the expression of its target gene ADAMTS1, mediate the up-regulation of VEGF expression, activate PI3K/AKT pathway, promote the release of eNOS, and participate in promoting angiogenesis in ischemic stroke rats.

Integrating network pharmacology, molecular docking and experimental verification to explore the therapeutic effect and potential mechanism of nomilin against triple-negative breast cancer.

BACKGROUND: Nomilin is a limonoid compound known for its multiple biological activities, but its role in triple negative breast cancer (TNBC) remains unclear. This study aims to uncover the potential therapeutic effect of nomilin on TNBC and elucidate the specific mechanism of its action. METHODS: We employed weighted gene co-expression network analysis (WGCNA), differential expression analysis, and the GeneCards database to identify potential targets for TNBC. Simultaneously, we utilized the Swiss Target Prediction, ChEMBL, and STITCH databases to identify potential targets of nomilin. The core targets and mechanisms of nomilin against TNBC were predicted through protein-protein interaction (PPI) network analysis, molecular docking, and enrichment analysis. The results of the network pharmacology were corroborated by conducting experiments. RESULTS: A total of 17,204 TNBC targets were screened, and 301 potential targets of nomilin were identified. Through the PPI network, eight core targets of nomilin against TNBC were pinpointed, namely BCL2, Caspase3, CyclinD1, EGFR, HSP90AA1, KRAS, PARP1, and TNF. Molecular docking, molecular dynamics simulation and proteome microarray revealed that nomilin exhibits strong binding activity to these core proteins. Enrichment analysis results indicated that the anti-TNBC effect of nomilin is associated with PI3K/Akt pathway. In vitro and in vivo experiments have demonstrated that nomilin inhibits TNBC cell proliferation and migration while promoting cell apoptosis through the PI3K/Akt pathway. CONCLUSION: For the first time, the research effectively discovered the objectives and mechanisms of nomilin in combating TNBC using network pharmacology, molecular docking, molecular dynamics simulation, proteome microarray and experimental confirmation, presenting a hopeful approach for treating TNBC.