Sinapic acid modulates oxidative stress and metabolic disturbances to attenuate ovarian fibrosis in letrozole-induced polycystic ovary syndrome SD rats.

Sinapic acid (SA) is renowned for its many pharmacological activities as a polyphenolic compound. The cause of polycystic ovary syndrome (PCOS), a commonly encountered array of metabolic and hormonal abnormalities in females, has yet to be determined. The present experiment was performed to evaluate the antifibrotic properties of SA in rats with letrozole-induced PCOS-related ovarian fibrosis. SA treatment successfully mitigated the changes induced by letrozole in body weight (BW) (p < .01) and relative ovary weight (p < .05). Histological observation revealed that SA reduced the number of atretic and cystic follicles (AFs) and (CFs) (p < .01), as well as ovarian fibrosis, in PCOS rats. Additionally, SA treatment impacted the serum levels of sex hormones in PCOS rats. Luteinizing hormone (LH) and testosterone (T) levels were decreased (p < .01, p < .05), and follicle-stimulating hormone (FSH) levels were increased (p < .05). SA administration also decreased triglyceride (TG) (p < .01) and total cholesterol (TC) levels (p < .05) and increased high-density lipoprotein cholesterol (HDL-C) levels (p < .01), thereby alleviating letrozole-induced metabolic dysfunction in PCOS rats. Furthermore, SA treatment targeted insulin resistance (IR) and increased the messenger RNA (mRNA) levels of antioxidant enzymes in the ovaries of PCOS rats. Finally, SA treatment enhanced the activity of peroxisome proliferator-activated receptor-γ (PPAR-γ), reduced the activation of transforming growth factor-ß1 (TGF-ß1)/Smads, and decreased collagen I, α-smooth muscle actin (α-SMA), and connective tissue growth factor (CTGF) levels in the ovaries of PCOS rats. These observations suggest that SA significantly ameliorates metabolic dysfunction and oxidative stress and ultimately reduces ovarian fibrosis in rats with letrozole-induced PCOS.

Isolation and characterization of a novel Enterococcus phage Phi_Eg_SY1.

Enterococcus gallinarum, a gut pathobiont, is an opportunistic pathogen that carries the risk of antibiotic resistance in the clinic and has been proven to drive autoimmunity in both mice and humans. Screening for novel bacteriophages targeting Enterococcus gallinarum is expected to provide a promising strategy for controlling such infections or regulating related chronic diseases. In the present study, we isolated a novel lytic Enterococcus gallinarum phage, Phi_Eg_SY1, which presents favourable thermostability and pH stability. Further assays indicated that Phi_Eg_SY1 can efficiently adsorb and lyse the host bacteria in vitro. Genomic and phylogenetic analyses suggested that Phi_Eg_SY1 does not contain virulence or lysogeny genes and presents a novel unassigned evolutionary lineage among the related dsDNA phages. Phi_Eg_SY1 is therefore considered to be suitable for further applications.

Chrysanthemum sporopollenin: A novel vaccine delivery system for nasal mucosal immunity.

Objective: Mucosal immunization was an effective defender against pathogens. Nasal vaccines could activate both systemic and mucosal immunity to trigger protective immune responses. However, due to the weak immunogenicity of nasal vaccines and the lack of appropriate antigen carriers, very few nasal vaccines have been clinically approved for human use, which was a major barrier to the development of nasal vaccines. Plant-derived adjuvants are promising candidates for vaccine delivery systems due to their relatively safe immunogenic properties. In particular, the distinctive structure of pollen was beneficial to the stability and retention of antigen in the nasal mucosa. Methods: Herein, a novel wild-type chrysanthemum sporopollenin vaccine delivery system loaded with a w/o/w emulsion containing squalane and protein antigen was fabricated. The unique internal cavities and the rigid external walls within the sporopollenin skeleton construction could preserve and stabilize the inner proteins. The external morphological characteristics were suitable for nasal mucosal administration with high adhesion and retention. Results: Secretory IgA antibodies in the nasal mucosa can be induced by the w/o/w emulsion with the chrysanthemum sporopollenin vaccine delivery system. Moreover, the nasal adjuvants produce a stronger humoral response (IgA and IgG) compared to squalene emulsion adjuvant. Mucosal adjuvant benefited primarily from prolongation of antigens in the nasal cavity, improvement of antigen penetration in the submucosa and promotion of CD8+ T cells in spleen. Disccusion: Based on effective delivering both the adjuvant and the antigen, the increase of protein antigen stability and the realization of mucosal retention, the chrysanthemum sporopollenin vaccine delivery system has the potential to be a promising adjuvant platform. This work provide a novel idea for the fabrication of protein-mucosal delivery vaccine.

Rhamnocitrin decreases fibrosis of ovarian granulosa cells by regulating the activation of the PPARγ/NF-κB/TGF-ß1/Smad2/3 signaling pathway mediated by Wisp2.

Background: Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility in women. Rhamnocitrin (Rha) has anti-inflammatory and antioxidant actions. The WNT1-inducible-signaling pathway protein 2 (Wisp2) and nuclear factor (NF)-κB are involved in fibrosis in many diseases. We aimed to elucidate the role of Rha in fibrosis of PCOS and the underlying mechanisms. Methods: Dehydroepiandrosterone (DHEA)-incubated ovarian granulosa KGN cells were treated by Rha. Cell proliferation was detected with cell counting kit-8 (CCK-8) and 5-ethynyul-2'-deoxyuridine (EdU) staining. The levels of Wisp2 and transforming growth factor-ß1 (TGF-ß1) in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). We observed α-smooth muscle actin (α-SMA) protein by immunofluorescence (IF). The levels of fibrosis factors were determined using Western blot. We observed p65 nuclear translocation with confocal microscopy. We used Wisp2 overexpression and knockdown in cells treated with DHEA or Rha to validate Wisp2 function. Interaction between Wisp2 and NF-κB, as well as Wisp2 and PPARγ, were assessed by co-immunoprecipitation assay, luciferase reporter assay and chromatin immunoprecipitation (ChIP). Results: The results showed that Rha elevated the reduced proliferation of DHEA-treated cells. In addition, Rha reversed the decreased Wisp2 and the increased TGF-ß1 in supernatant. The proteins CTGF, α-SMA, Collagen I, TGF-ß1, p-Smad2, and p-Smad3 were up-regulated while Wisp2, Sirt1, and PPARγ were down-regulated by DHEA treatment, which were reversed by Rha. Meanwhile, DHEA up-regulated p-IKBa and p-p65 and promoted p65 nuclear translocation, which were inhibited by Rha. These effects of Rha were antagonized by Wisp2 knockdown and were mimicked by Wisp2 overexpression. We confirmed the protein interaction between Wisp2 and NF-κB, along with Wisp2 and PPARγ. Conclusions: Wisp2-mediated PPARγ/NF-κB/TGF-ß1/Smad2/3 signaling contributes to Rha-improved ovarian granulosa cells fibrosis, suggesting Rha as a novel agent for the treatment of PCOS.

Rhamnocitrin Attenuates Ovarian Fibrosis in Rats with Letrozole-Induced Experimental Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine-related cause of infertility in women and has an unknown etiology. Studies have shown that rhamnocitrin (Rha) exhibits positive effects on the reproductive system. This study investigated Rha's antifibrotic effects on PCOS rats and revealed its underlying mechanisms. Female SD rats were randomized into 4 groups (n = 8, each); the control group received tea oil by intraperitoneal injection and 1% w/v CMC by oral gavage; the PCOS group received letrozole (1 mg/kg); the PCOS+Rha group received letrozole and Rha (5 mg/kg); the PCOS+Met group received letrozole and Met (265 mg/kg) for 21 days. At the study end, Rha treatment restored letrozole-induced alterations in the relative ovarian weights, body weight, and relative weights of uterine and visceral adipose tissues. Histological observation showed that Rha ameliorates ovarian structure and fibrosis in PCOS. Administration of Rha reduced letrozole-induced metabolic dysfunction by ameliorating the levels of TC, TG, and HDL-C in the PCOS rats. Rha treatment also modulated the serum levels of sex hormones, which decreased T, E2, and LH and increased FSH in PCOS rats. In addition, Rha treatment modulated insulin resistance and increased gene expression of antioxidant enzymes (Cat, Sod2, Gpx3, Mgst1, Prdx3, Gsta4, Gsr, and Sod1) in the ovaries of the PCOS rats. Finally, Rha treatment appeared to increase the activity of PPAR-γ and inhibit the TGF-ß1/Smad pathway in the ovaries of the PCOS rats. Our findings suggest that Rha significantly ameliorated metabolic disturbances and ovarian fibrosis in the PCOS rats. Rha perhaps is an effective compound for preventing ovarian fibrosis in the future.

Effects of Sinapic Acid Combined with Cisplatin on the Apoptosis and Autophagy of the Hepatoma Cells HepG2 and SMMC-7721.

Sinapic acid (Sa) is a small-molecule phenolic acid compound predominant in fruits, vegetables, and grains. This study investigated the antitumor effects of cisplatin (DDP) combined with Sa (Sa/DDP) on the hepatic cancer cells (HCC), HepG2 and SMMC-7721. The HepG2 and SMMC-7721 cells were treated with Sa or Sa/DDP, and the cell proliferation and cell cycle were detected using the MTT assay. The cell migration was detected using the transwell and scratch assays, while apoptosis and autophagy were detected using Hoechst, MDC, and Annexin V-FITC/PI staining. The protein expression was quantitated using the western blot. Sa/DDP was found to not only inhibit cancer cell proliferation and migration but also induce cell apoptosis. Simultaneously, the Sa/DDP combination was found to activate autophagy, and the HCQ autophagy inhibitor enhanced the apoptosis in the Sa/DDP-induced liver cancer cells. The combined use of Sa and DDP makes it an attractive adjuvant therapy strategy for tumors, establishing the prospect of phenolic acid compounds for the adjuvant treatment of hepatocellular carcinoma.

A high-throughput screen identifies inhibitors of lung cancer stem cells.

Metastasis is the main cause of cancer morbidity and mortality. Cancer stem cells (CSCs) are a rare subpopulation of cancer cells that can drive metastasis. The identification of CSC inhibitors and CSC-related genes is an alluring strategy for suppressing metastasis. Here, we established a simple and repeatable high-throughput CSC inhibitor screening platform that combined tumor sphere formation assays and cell viability assays. Human lung cancer cells were cocultured with 1280 pharmacologically active compounds (FDA-approved). Fifty-four candidate compounds obtained from our screening system completely or partially inhibited tumor sphere formation. A total of 5 of these 54 compounds (prochlorperazine dimaleate, thioridazine hydrochloride, ciproxifan hydrochloride, Ro 25-6981 hydrochloride, and AMN 082) completely inhibited the self-renewal of CSCs without cytotoxicity in vitro via their targets and suppressed lung cancer metastasis in vivo, suggesting that our screening platform is selective and reliable. DRD2, HRH3, and GRIN2B exhibited potent genes promoting CSCs in vitro experiments and clinical datasets. Further validation of the top hit (DRD2) and previously published studies demonstrate that our screening platform is a useful tool for CSC inhibitor and CSC-related gene screening.

Dietary proanthocyanidins alleviated ovarian fibrosis in letrozole-induced polycystic ovary syndrome in rats.

This study investigated the effects of proanthocyanidins (PCs) on ovarian fibrosis in letrozole-induced polycystic ovary syndrome (PCOS) in rats. The administration of PCs effectively reduced the body weight (BW) and relative ovarian weight in PCOS rats. ELISA results revealed that PCs significantly reduced the level of serum T, LH, LH/FSH in the PCOS group. In addition, qRT-PCR results revealed that treatment with PCs significantly increased the main antioxidant enzymes (Cat, Sod2, Gpx3, Mgst1, Gsta4, Sod1 and Prdx3) in PCOS rats. Also, the expression analysis of proteins by Western blotting revealed that PCs significantly decreased the level of TGF-ßR1, p-Smad3, p-Smad2 and Smad4 and reversed the downregulation of Smad7 in PCOS rats. The study suggested that PCs improved ovarian fibrosis in PCOS rats by regulating the serum hormone level, inhibiting oxidative stress and suppressing the activation of the TGF-ß1/Smads signaling pathway. PRACTICAL APPLICATIONS: Currently, plant extracts are being widely used to treat female reproductive and metabolic disorders. Particularly, proanthocyanidins (PCs), the well-known natural polyphenolic compounds, which are a significant source of antioxidants present in many colored fruits, are consumed as fruits as well as a dietary supplement to prevent many disorders. Recent pharmacological studies have reported that PCs have many health beneficial properties, such as antioxidant activity, improving cholesterol homeostasis, blood lipid regulatory properties, microcirculation improvement effect, antitumor activity and anti-aging activity. Despite these properties of PCs, the antifibrosis effect of PCs has not been studied to date. The main purpose of this study was to research the role and the mechanisms of PCs in ovarian fibrosis in PCOS rats.

SWI/SNF complex subunit BAF60a represses hepatic ureagenesis through a crosstalk between YB-1 and PGC-1α.

OBJECTIVE: Ureagenesis predominantly occurs in the liver and functions to remove ammonia, and the dysregulation of ureagenesis leads to the development of hyperammonemia. Recent studies have shown that ureagenesis is under the control of nutrient signals, but the mechanism remains elusive. Therefore, intensive investigation of the molecular mechanism underlying ureagenesis will shed some light on the pathology of metabolic diseases related to ammonia imbalance. METHODS: Mice were fasted for 24 h or fed a high-fat diet (HFD) for 16 weeks. For human evaluation, we obtained a public data set including 41 obese patients with and without hepatic steatosis. We analyzed the expression levels of hepatic BAF60a under different nutrient status. The impact of BAF60a on ureagenesis and hyperammonemia was assessed by using gain- and loss-of-function strategies. The molecular chaperons mediating the effects of BAF60a on ureagenesis were validated by molecular biological strategies. RESULTS: BAF60a was induced in the liver of both fasted and HFD-fed mice and was positively correlated with body mass index in obese patients. Liver-specific overexpression of BAF60a inhibited hepatic ureagenesis, leading to the increase of serum ammonia levels. Mechanistically, BAF60a repressed the transcription of Cps1, a rate-limiting enzyme, through interaction with Y-box protein 1 (YB-1) and by switching the chromatin structure of Cps1 promoter into an inhibitory state. More importantly, in response to different nutrient status, PGC-1α (as a transcriptional coactivator) and YB-1 competitively bound to BAF60a, thus selectively regulating hepatic fatty acid ß-oxidation and ureagenesis. CONCLUSION: The BAF60a-YB-1 axis represses hepatic ureagenesis, thereby contributing to hyperammonemia under overnutrient status. Therefore, hepatic BAF60a may be a novel therapeutic target for the treatment of overnutrient-induced urea cycle disorders and their associated diseases.

Angptl8 mediates food-driven resetting of hepatic circadian clock in mice.

Diurnal light-dark cycle resets the master clock, while timed food intake is another potent synchronizer of peripheral clocks in mammals. As the largest metabolic organ, the liver sensitively responds to the food signals and secretes hepatokines, leading to the robust regulation of metabolic and clock processes. However, it remains unknown which hepatokine mediates the food-driven resetting of the liver clock independent of the master clock. Here, we identify Angptl8 as a hepatokine that resets diurnal rhythms of hepatic clock and metabolic genes in mice. Mechanistically, the resetting function of Angptl8 is dependent on the signal relay of the membrane receptor PirB, phosphorylation of kinases and transcriptional factors, and consequently transient activation of the central clock gene Per1. Importantly, inhibition of Angptl8 signaling partially blocks food-entrained resetting of liver clock in mice. We have thus identified Angptl8 as a key regulator of the liver clock in response to food.

Silibinin A decreases statin­induced PCSK9 expression in human hepatoblastoma HepG2 cells.

Hypercholesterolemia is one of the major risk factors for the occurrence and development of atherosclerosis. The most common drugs used to treat hypercholesterolemia are 3­hydroxy­3­methyl­glutaryl­CoA reductase inhibitors, known as statins. Statins induce a beneficial increase in the levels of the low density lipoprotein receptor (LDLR) and additionally upregulate proprotein convertase subtilisin/kexin type 9 (PCSK9), which leads to LDLR degradation. This process causes a negative feedback response that attenuates the lipid lowering effects of statins. Therefore, the development of PCSK9 inhibitors may increase the lipid­lowering functions of statins. In the present study, a drug­screening assay was developed using the human PCSK9 promoter, based on data from a dual­luciferase reporter assay, and the efficacies of various compounds from Traditional Chinese Medicine were examined. Among the compounds examined, SIL was demonstrated to function by targeting PCSK9. It was identified that SIL treatment decreased the expression levels of PCSK9 in HepG2 cells by decreasing the activity of the PCSK9 promoter in a dose­and time­dependent manner. Notably, SIL antagonized the statin­induced phosphorylation of the p38 MAPK signaling pathway. The present study suggested that SIL may be developed as a novel PCSK9 inhibitor that may increase the efficiency of statin treatment.

Cloxiquine, a traditional antituberculosis agent, suppresses the growth and metastasis of melanoma cells through activation of PPARγ.

Melanoma is one of the most aggressive skin cancers and 5-year survival rate is only 4.6% for metastatic melanoma patients. Current therapies, especially those involving clinical chemotherapy drugs, have achieved remarkable advances. However, their side effects, such as bone marrow suppression, limit the effectiveness of available pharmacological therapies. Therefore, exploring new antimelanoma drugs with less toxicity is critical for the treatment of melanoma. In the present study, we aimed to identify the antimelanoma drugs with ability to repress the proliferation of melanoma cells by using a high-content screening of FDA-approved drug libraries. We found that cloxiquine (CLQ), a traditional antituberculosic drug, exhibited strong inhibitory effects on the growth and metastasis of melanoma cells both in vivo and in vitro. In contrast, CLQ at the tested doses did not show any apparent toxicity in normal melanocytes and in the liver. At the metabolic level, treatment with CLQ decreased glycolysis, thus potentially inhibiting the "Warburg effect" in B16F10 cells. More importantly, combination of CLQ and 2-deoxyglucose (2-DG), a well-known glycolysis inhibitor, did not show a synergistic effect on the tumor growth and metastasis, indicating that inhibition of glycolysis is potentially involved in mediating CLQ's antimelanoma function. Bioinformatics analyses revealed that peroxisome proliferator-activated receptor-gamma (PPARγ) served as a potential CLQ target. Mechanistically, CLQ stimulated the transcription and nuclear contents of PPARγ. Furthermore, the specific PPARγ inhibitor GW9662 or PPARγ shRNA partially abolished the effects of CLQ. Collectively, our findings demonstrate that CLQ has a great potential in the treatment of melanoma through activation of PPARγ.