The effect of biological mechanisms of melatonin on the proliferation of spermatogonial stem cells: a systematic review.

In the last decade, melatonin has gained recognition as a potent scavenger and an effective antioxidant capable of neutralizing free radicals, including reactive oxygen species. Additionally, it exhibits anti-apoptotic properties. In this review, we will examine a compilation of articles that explore the cellular signaling function of melatonin on spermatogonial stem cells (SSCs) and adjacent cells such as Sertoli and Leydig cells. These cells play a crucial role in the proliferation of SSCs both in vitro and in vivo. In this review, we analyze the function of melatonin in the proliferation of SSCs from other aspects. For this purpose, we examine the articles based on the presence of melatonin on SSCs in four groups: As a supplement in SSCs medium culture, SSCs three-dimensional culture system, SSCs freezing medium, and as a therapeutic factor in vivo. Mechanisms of growth and proliferation of SSCs were considered. The purpose of this study is to investigate the potential effects of melatonin as a powerful antioxidant or growth stimulant for SSCs, both in vivo and in vitro.

Bacillus subtilis alleviates excessive apoptosis of intestinal epithelial cells in intrauterine growth restriction suckling piglets via the members of Bcl-2 and caspase families.

BACKGROUND: The intestine is a barrier resisting various stress responses. Intrauterine growth restriction (IUGR) can cause damage to the intestinal barrier via destroying the balance of intestinal epithelial cells' proliferation and apoptosis. Bacillus subtilis has been reported to regulate intestinal epithelial cells' proliferation and apoptosis. Thus, the purpose of this study was to determine if B. subtilis could regulate intestinal epithelial cells' proliferation and apoptosis in intrauterine growth restriction suckling piglets. RESULTS: Compared with the normal birth weight group, the IUGR group showed greater mean optical density values of Ki-67-positive cells in the ileal crypt (P < 0.05). IUGR resulted in higher ability of proliferation and apoptosis of intestinal epithelial cells, by upregulation of the messenger RNA (mRNA) or proteins expression of leucine rich repeat containing G protein coupled receptor 5, Caspase-3, Caspase-7, ß-catenin, cyclinD1, B-cell lymphoma-2 associated agonist of cell death, and BCL2 associated X (P < 0.05), and downregulation of the mRNA or protein expression of B-cell lymphoma-2 and B-cell lymphoma-2-like 1 (P < 0.05). However, B. subtilis supplementation decreased the mRNA or proteins expression of leucine rich repeat containing G protein coupled receptor 5, SPARC related modular calcium binding 2, tumor necrosis factor receptor superfamily member 19, cyclinD1, Caspase-7, ß-catenin, B-cell lymphoma-2 associated agonist of cell death, and Caspase-3 (P < 0.05), and increased the mRNA expression of B-cell lymphoma-2 (P < 0.05). CONCLUSION: IUGR led to excessive apoptosis of intestinal epithelial cells, which induced compensatory proliferation. However, B. subtilis treatment prevented intestinal epithelial cells of IUGR suckling piglets from excessive apoptosis. © 2024 Society of Chemical Industry.

Physicochemical Characterization, Antioxidant, and Proliferative Activity of Colombian Propolis Extracts: A Comparative Study.

Propolis extracts have been widely studied due to their popularity in traditional medicine, presenting incredible biodiversity. This study aimed to analyze propolis extracts' phytochemical, physicochemical, and biological activities from four different biogeographic zones of the Huila region (Colombia). The raw material samples were collected by the scraping method and the ethanolic extracts (EEPs) were obtained by cold maceration with ethanol (96%). The physicochemical and sensory characterization was carried out according to the protocols recommended by the Brazilian Ministry of Agriculture and the main components of the EEPs were identified by LC-HRMS analysis. The determination of total phenols and flavonoids was carried out using colorimetric techniques. The antioxidant activity, cytotoxicity, and cell cycle regulation analyses in L929 and HGnF cells were evaluated using DPPH, Alamar Blue, and 7-amino actinomycin D (7-AAD) assays. The propolis samples presented an average yield of 33.1%, humidity between 1.6 and 2.8%, melting point between 54 and 62 °C, ashes between 1.40 and 2.19%, and waxes of 6.6-17.9%, respectively. The sensory characteristics of all samples were heterogeneous, complying with the quality specifications established by international standards. The polyphenolic and total flavonoid content was representative in the samples from Quebradon (255.9 ± 9.2 mg GAE/g, 543.1 ± 8.4 mg QE/g) and Arcadia (543.1 ± 8.4 mg GAE/g, 32.5 ± 1.18 g QE/g) (p < 0.05) that correlated with high antioxidant activity (Quebradon: 37.2 ± 1.2 µmol/g, Arcadia: 38.19 ± 0.7 µmol/g). In the chemical composition analysis, 19 compounds were characterized as phenolic acids and flavonoids, the most representative being chrysoeriol-O-methyl-ether, ellagic acid, and 3,4-O-dimethylcaffeic acid. Regarding biological activity, Quebradon and Arcadia propolis presented low toxicity with IC50 of 2.83 ± 2.3 mg/mL and 4.28 ± 1.4 mg/mL in HGnF cells, respectively, and an arrest of the cell cycle in the G2/M phase of 71.6% and 50.8% compared to the control (11.9%) (p < 0.05). In general, the results of this study contribute to the identification of valid quality criteria to evaluate Colombian propolis, contributing to its study and chemical and biological characterization as a source of raw material for industrial and pharmaceutical use. In addition, Quebradon and Arcadia propolis can be important sources of bioactive molecules for the development of new drugs.

The Difference of Milk-Derived Extracellular Vesicles from Cow Colostrum and Mature Milk on miRNAs Expression and Protecting Intestinal Epithelial Cells against Lipopolysaccharide Damage.

Intestinal epithelial cells (IECs) play crucial roles in forming an essential barrier, providing host defense against pathogens and regulating nutrients absorption. Milk-derived extracellular vesicles (EVs) within its miRNAs are capable of modulating the recipient cell function. However, the differences between colostrum and mature milk EVs and their biological function in attenuating intestinal epithelial cell injury remain poorly understood. Thus, we carried out the present study to characterize the difference between colostrum and mature milk-derived miRNA of EVs and the effect of colostrum and mature milk EVs on the proliferation, apoptosis, proinflammatory cytokines and intestinal epithelial barrier related genes in IEC-6 induced by LPS. Differential expression of 329 miRNAs was identified between colostrum and mature milk EVs, with 185 miRNAs being downregulated and 144 upregulated. In addition, colostrum contains a greater number and protein concentration of EVs than mature milk. Furthermore, compared to control, EVs derived from colostrum significantly inhibited the expression of apoptosis- (Bax, p53, and caspase-3) and proinflammatory-related genes (TNFα, IL6, and IL1ß). EVs derived from mature milk did not affect expression of apoptosis-related genes (Bax, p53, bcl2, and caspase-3). The EVs derived from mature milk significantly inhibited the expression of proinflammatory-related genes (TNFα and IL6). Western blot analysis also indicated that colostrum and mature milk EVs significantly decreased the apoptosis of IEC-6 cells. The EdU assay results showed that colostrum and mature milk EVs significantly increased the proliferation of IEC-6 cells. The expression of intestinal barrier-related genes (TJP1, CLDN1, OCLN, CDX2, MUC2, and IGF1R) was significantly promoted in IEC-6 cells after colostrum and mature milk EVs addition. Importantly, colostrum and mature milk EVs significantly relieved the LPS-induced inhibition of proliferation and intestinal barrier-related genes expression and attenuated apoptosis and proinflammatory responses induced by LPS in IEC-6 cells. Flow cytometry and Western blot analysis also indicated that colostrum and mature milk EVs significantly affect the apoptosis of IEC-6 cells induced by LPS. The results also indicated that EVs derived from colostrum had better effects on inhibiting the apoptosis- and proinflammatory cytokines-related genes expression. However, the EVs derived from mature milk exhibited beneficial effects on intestinal epithelial barrier protection. The present study will provide a better understanding of the role of EVs derived from colostrum and milk in dairy cows with different responses in the regulation of intestinal cells function, and also presents new evidence for the change of EVs cargos during various stages of lactation.

Honokiol Suppresses Cell Proliferation and Tumor Migration through ROS in Human Anaplastic Thyroid Cancer Cells.

BACKGROUND: Honokiol is a natural polyphenolic compound extracted from Magnolia officinali, which is commonly used material in Chinese herbal medicine, has a variety of biological functions, including anti-tumor, anti-oxidant, anti-inflammation, anti-microbial and anti-allergy. Although honokiol has numerous beneficial effects on human diseases, the underlying mechanisms of tumor metastasis are still unclear. Previously, we reported that honokiol suppresses thyroid cancer cell proliferation with cytotoxicity through cell cycle arrest, apoptosis, and dysregulation of intracellular hemostasis. Herein, we hypothesized that the antioxidant effect of honokiol might play a critical role in thyroid cancer cell proliferation and migration. METHODS: The cell viability assays, cellular reactive oxygen species (ROS) activity, cell migration, and immunoblotting were performed after cells were treated with honokiol. RESULTS: Based on this hypothesis, we first demonstrated that honokiol suppresses cell proliferation in two human anaplastic thyroid carcinoma (ATC) cell lines, KMH-2 and ASH-3, within a dosage- and time-dependent manner by cell counting kit-8 (CCK-8) assay. Next, we examined that honokiol induced ROS activation and could be suppressed by pre-treated with an antioxidant agent, N-acetyl-l-cysteine (NAC). Furthermore, the honokiol suppressed cell proliferation can be rescued by pre-treated with NAC. Finally, we demonstrated that honokiol inhibited ATC cell migration by modulating epithelial-mesenchymal transition (EMT)-related markers by Western blotting. CONCLUSION: Taken together, we provided the potential mechanism for treating ATC cells with honokiol, which significantly suppresses tumor proliferation and inhibits tumor metastasis in vitro through reactive oxygen species (ROS) induction.

Influence of Maternal Nutrition and One-Carbon Metabolites Supplementation during Early Pregnancy on Bovine Fetal Small Intestine Vascularity and Cell Proliferation.

To investigate the effects of nutrient restriction and one-carbon metabolite (OCM) supplementation (folate, vitamin B12, methionine, and choline) on fetal small intestine weight, vascularity, and cell proliferation, 29 (n = 7 ± 1 per treatment) crossbred Angus beef heifers (436 ± 42 kg) were estrous synchronized and conceived by artificial insemination with female sexed semen from a single sire. Then, they were allotted randomly to one of four treatments in a 2 × 2 factorial arrangement with the main factors of nutritional plane [control (CON) vs. restricted feed intake (RES)] and OCM supplementation [without OCM (-OCM) or with OCM (+OCM)]. Heifers receiving the CON level of intake were fed to target an average daily gain of 0.45 kg/day, which would allow them to reach 80% of mature BW by calving. Heifers receiving the RES level of intake were fed to lose 0.23 kg/heifer daily, which mimics observed production responses in heifers that experience a diet and environment change during early gestation. Targeted heifer gain and OCM treatments were administered from d 0 to 63 of gestation, and then all heifers were fed a common diet targeting 0.45 kg/d gain until d 161 of gestation, when heifers were slaughtered, and fetal jejunum was collected. Gain had no effect (p = 0.17) on the fetal small intestinal weight. However, OCM treatments (p = 0.02) displayed less weight compared to the -OCM groups. Capillary area density was increased in fetal jejunal villi of RES - OCM (p = 0.02). Vascular endothelial growth factor receptor 2 (VEGFR2) positivity ratio tended to be greater (p = 0.08) in villi and was less in the crypts (p = 0.02) of the RES + OCM group. Cell proliferation decreased (p = 0.02) in villi and crypts of fetal jejunal tissue from heifers fed the RES + OCM treatment compared with all groups and CON - OCM, respectively. Spatial cell density increased in RES - OCM compared with CON + OCM (p = 0.05). Combined, these data show OCM supplementation can increase expression of VEGFR2 in jejunal villi, which will promote maintenance of the microvascular beds, while at the same time decreasing small intestine weight and crypt cell proliferation.

Phytochemical analysis and anticancer effect of Camellia oleifera bud ethanol extract in non-small cell lung cancer A549 cells.

Camellia oleifera is a medicine food homology plant widely cultivated in the Yangtze River Basin and southern China due to its camellia oil. Camellia oleifera bud and fruit exist simultaneously, and its bud is largely discarded as waste. However, C. oleifera bud has been used in traditional Chinese medicine to treat a variety of ailments. Thus, the purpose of this study was to identify the chemical components of C. oleifera bud ethanol extract (EE) and first evaluate its anticancer effects in non-small cell lung cancer A549 cells. Based on UHPLC-Q-Orbitrap-MS analysis, seventy components were identified. For anticancer activity, C. oleifera bud EE had remarkable cytotoxic effect on non-small cell lung cancer A549 (IC50: 57.53 ± 1.54 µg/mL) and NCI-H1299 (IC50: 131.67 ± 4.32 µg/mL) cells, while showed lower cytotoxicity on non-cancerous MRC-5 (IC50 > 320 µg/mL) and L929 (IC50: 179.84 ± 1.08 µg/mL) cells. It dramatically inhibited the proliferation of A549 cells by inducing cell cycle arrest at the G1 phase. Additionally, it induced apoptosis in A549 cells through a mitochondria-mediated pathway, which decreased mitochondrial membrane potential, upregulated Bax, activated caspase 9 and caspase 3, and resulted in PARP cleavage. Wound healing and transwell invasion assays demonstrated that C. oleifera bud EE inhibited the migration and invasion of A549 cells in a dose-dependent manner. The above findings indicated that C. oleifera bud EE revealed notable anticancer effects by inhibiting proliferation, inducing apoptosis, and suppressing migration and invasion of A549 cells. Hence, C. oleifera bud ethanol extract could serve as a new source of natural anticancer drugs.

Circ_0003764 Regulates the Osteogenic Differentiation of Periodontal Ligament Stem Cells.

INTRODUCTION AND AIMS: Specific circular RNAs (circRNAs) have been proven to play crucial roles in osteogenesis in vitro and in vivo. This study aims to identify a certain circRNA involved in the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and explore its regulatory role. METHODS: The expression of 5 candidate circRNAs (circ_0026344, circ_ACAP2, circ_0003764, circ_0008259, and circ_0060731) was detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) after PDLSCs were cultured in the osteogenic induction medium or medium supplemented with tumour necrosis factor-α (TNF-α, 10 ng/mL) for 3 and 7 days. The circRNA significantly decreased in both 3 and 7 days of osteogenic induction in PDLSCs and markedly increased in TNF-α-induced PDLSCs for 3 and 7 days screened. Identified circRNA was knocked down or overexpressed, and the effect on the osteogenic differentiation of PDLSCs was investigated by qRT-PCR, western blot, alkaline phosphatase (ALP) staining, and alizarin red S (ARS) staining. Cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were applied to detect the effect of the circRNA on the proliferation of PDLSCs. RESULTS: qRT-PCR results showed that the expression of circ_0003764 was significantly decreased when PDLSCs were cultured in the osteogenic induction medium for 3 or 7 days, whereas it was dramatically increased in TNF-α-induced PDLSCs. Knockdown of circ_0003764 promoted the expression of the osteogenesis-related genes (RUNX2, ALP, OCN) and proteins (RUNX2, OCN), enhanced the ALP activity, and elevated the mineralization by PDLSCs, as shown by ARS staining. However, with the overexpression of circ_0003764, the osteogenic differentiation capacity of PDLSCs was significantly reduced. The CCK-8 and EdU results indicated that circ_0003764 could inhibit the proliferation of PDLSCs. CONCLUSION: Circ_0003764 is involved in the osteogenesis process and inhibits the osteogenic differentiation and proliferation of PDLSCs. CLINICAL RELEVANCE: This study indicates that circ_0003764 can serve as a diagnostic and therapeutic target in bone regeneration-related diseases treated by PDLSCs-based tissue engineering.

High-dose ascorbate exerts anti-tumor activities and improves inhibitory effect of carboplatin through the pro-oxidant function pathway in uterine serous carcinoma cell lines.

OBJECTIVE: Uterine serous carcinoma is a highly aggressive non-endometrioid subtype of endometrial cancer with poor survival rates overall, creating a strong need for new therapeutic strategies to improve outcomes. High-dose ascorbate (vitamin C) has been shown to inhibit cell proliferation and tumor growth in multiple preclinical models and has shown promising anti-tumor activity in combination with chemotherapy, with a favorable safety profile. We aimed to study the anti-tumor effects of ascorbate and its synergistic effect with carboplatin on uterine serous carcinoma cells. METHODS: Cell proliferation was evaluated by MTT and colony formation assays in ARK1, ARK2 and SPEC2 cells. Cellular stress, antioxidant ability, cleaved caspase 3 activity and adhesion were measured by ELISA assays. Cell cycle was detected by Cellometer. Invasion was measured using a wound healing assay. Changes in protein expression were determined by Western immunoblotting. RESULTS: High-dose ascorbate significantly inhibited cell proliferation, caused cell cycle arrest, induced cellular stress, and apoptosis, increased DNA damage, and suppressed cell invasion in ARK1 and SPEC2 cells. Treatment of both cells with 1 mM N-acetylcysteine reversed ascorbate-induced apoptosis and inhibition of cell proliferation. The combination of ascorbate and carboplatin produced significant synergistic effects in inhibiting cell proliferation and invasion, inducing cellular stress, causing DNA damage, and enhancing cleaved caspase 3 levels compared to each compound alone in both cells. CONCLUSIONS: Ascorbate has potent antitumor activity and acts synergistically with carboplatin through its pro-oxidant effects. Clinical trials of ascorbate combined with carboplatin as adjuvant treatment of uterine serous carcinoma are worth exploring.

The effect of selenium on the proliferation of bovine endometrial epithelial cells in a lipopolysaccharide-induced damage model.

BACKGROUND: Endometritis is a common bovine postpartum disease. Rapid endometrial repair is beneficial for forming natural defense barriers and lets cows enter the next breeding cycle as soon as possible. Selenium (Se) is an essential trace element closely related to growth and development in animals. This study aims to observe the effect of Se on the proliferation of bovine endometrial epithelial cells (BEECs) induced by lipopolysaccharide (LPS) and to elucidate the possible underlying mechanism. RESULTS: In this study, we developed a BEECs damage model using LPS. Flow cytometry, cell scratch test and EdU proliferation assay were used to evaluate the cell cycle, migration and proliferation. The mRNA transcriptions of growth factors were detected by quantitative reverse transcription-polymerase chain reaction. The activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/ß-catenin pathways were detected by Western blotting and immunofluorescence. The results showed that the cell viability and BCL-2/BAX protein ratio were significantly decreased, and the cell apoptosis rate was significantly increased in the LPS group. Compared with the LPS group, Se promoted cell cycle progression, increased cell migration and proliferation, and significantly increased the gene expressions of TGFB1, TGFB3 and VEGFA. Se decreased the BCL-2/BAX protein ratio, promoted ß-catenin translocation from the cytoplasm to the nucleus and activated the Wnt/ß-catenin and PI3K/AKT signaling pathways inhibited by LPS. CONCLUSIONS: In conclusion, Se can attenuate LPS-induced damage to BEECs and promote cell proliferation and migration in vitro by enhancing growth factors gene expression and activating the PI3K/AKT and Wnt/ß-catenin signaling pathways.

Corrigendum: Safe and effective delivery of supplemental iron to healthy adults: a two-phase, randomized, double-blind trial - the safe iron study.

[This corrects the article DOI: 10.3389/fnut.2023.1230061.].

Dietary Zn Deficiency Inhibits Cell Proliferation via the GPR39-Mediated Suppression of the PI3K/AKT/mTOR Signaling Pathway in the Jejunum of Broilers.

A prior investigation revealed that a lack of Zinc (Zn) could hinder intestinal cell proliferation in broiler chickens; however, the mechanisms responsible for this effect remain unclear. We aimed to investigate the possible mechanisms of dietary Zn deficiency in inhibiting the jejunal cell proliferation of broilers. For this study, a total of 112 chickens (21 days old) were randomly divided into two treatments (seven replicate cages per treatment, eight chickens per replicate cage): the control group (CON) and the Zn deficiency group. The duration of feeding was 21 d. Chickens in the control group were provided with a basal diet containing an extra addition of 40 mg Zn/kg in the form of Zn sulfate, whereas chickens in the Zn deficiency group were given the basal diet with no Zn supplementation. The results indicated that, in comparison to the CON, Zn deficiency increased (p < 0.05) the duodenal and jejunal crypt depth (CD) of broilers on d 28 and jejunal and ileal CD on d 35, and decreased (p < 0.05) the duodenal, jejunal, and ileal villus height/crypt depth (VH/CD) on d 28 and the jejunal VH, jejunal and ileal villus surface area, and VH/CD on d 35. Furthermore, Zn deficiency decreased (p < 0.0001) the number of proliferating cell nuclear antigen-positive cells and downregulated (p < 0.01) the mRNA or protein expression levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, phosphorylated serine-threonine kinase (AKT), phosphorylated mechanistic target of rapamycin (mTOR), G protein-coupled receptor 39 (GPR39), and extracellular-regulated protein kinase, but upregulated (p < 0.05) the mRNA or protein expression levels of P38 mitogen-activated protein kinase, c-jun N-terminal kinase (JNK) 1 and JNK2, and phosphorylated protein kinase C in the jejunum of the broilers on d 42. It was concluded that dietary Zn deficiency inhibited cell proliferation possibly via the GPR39-mediated suppression of the PI3K/AKT/mTOR signaling pathway in the jejunum of broilers.

Ginseng Glucosyl Oleanolate from Ginsenoside Ro, Exhibited Anti-Liver Cancer Activities via MAPKs and Gut Microbiota In Vitro/Vivo.

Ginseng is widely recognized for its diverse health benefits and serves as a functional food ingredient with global popularity. Ginsenosides with a broad range of pharmacological effects are the most crucial active ingredients in ginseng. This study aimed to derive ginseng glucosyl oleanolate (GGO) from ginsenoside Ro through enzymatic conversion and evaluate its impact on liver cancer in vitro and in vivo. GGO exhibited concentration-dependent HepG2 cell death and markedly inhibited cell proliferation via the MAPK signaling pathway. It also attenuated tumor growth in immunocompromised mice undergoing heterograft transplantation. Furthermore, GGO intervention caused a modulation of gut microbiota composition by specific bacterial populations, including Lactobacillus, Bacteroides, Clostridium, Enterococcus, etc., and ameliorated SCFA metabolism and colonic inflammation. These findings offer promising evidence for the potential use of GGO as a natural functional food ingredient in the prevention and treatment of cancer.

Shikonin suppresses rheumatoid arthritis by inducing apoptosis and autophagy via modulation of the AMPK/mTOR/ULK-1 signaling pathway.

BACKGROUND: The overproliferation of fibroblast-like synoviocytes (FLS) contributes to synovial hyperplasia, a pivotal pathological feature of rheumatoid arthritis (RA). Shikonin (SKN), the active compound from Lithospermum erythrorhizon, exerts anti-RA effects by diverse means. However, further research is needed to confirm SKN's in vitro and in vivo anti-proliferative functions and reveal the underlying specific molecular mechanisms. PURPOSE: This study revealed SKN's anti-proliferative effects by inducing both apoptosis and autophagic cell death in RA FLS and adjuvant-induced arthritis (AIA) rat synovium, with involvement of regulating the AMPK/mTOR/ULK-1 pathway. METHODS: SKN's influences on RA FLS were assessed for proliferation, apoptosis, and autophagy with immunofluorescence staining (Ki67, LC3B, P62), EdU incorporation assay, staining assays of Hoechst, Annexin V-FITC/PI, and JC-1, transmission electron microscopy, mCherry-GFP-LC3B puncta assay, and western blot. In AIA rats, SKN's anti-arthritic effects were assessed, and its impacts on synovial proliferation, apoptosis, and autophagy were studied using Ki67 immunohistochemistry, TUNEL, and western blot. The involvement of AMPK/mTOR/ULK-1 pathway was examined via western blot. RESULTS: SKN suppressed RA FLS proliferation with reduced cell viability and decreased Ki67-positive and EdU-positive cells. SKN promoted RA FLS apoptosis, as evidenced by apoptotic nuclear fragmentation, increased Annexin V-FITC/PI-stained cells, reduced mitochondrial potential, elevated Bax/Bcl-2 ratio, and increased cleaved-caspase 3 and cleaved-PARP protein levels. SKN also enhanced RA FLS autophagy, featuring increased LC3B, reduced P62, autophagosome formation, and activated autophagic flux. Autophagy inhibition by 3-MA attenuated SKN's anti-proliferative roles, implying that SKN-induced autophagy contributes to cell death. In vivo, SKN mitigated the severity of rat AIA while also reducing Ki67 expression, inducing apoptosis, and enhancing autophagy within AIA rat synovium. Mechanistically, SKN modulated the AMPK/mTOR/ULK-1 pathway in RA FLS and AIA rat synovium, as shown by elevated P-AMPK and P-ULK-1 expression and decreased P-mTOR expression. This regulation was supported by the reversal of SKN's in vitro and in vivo effects upon co-administration with the AMPK inhibitor compound C. CONCLUSION: SKN exerted in vitro and in vivo anti-proliferative properties by inducing apoptosis and autophagic cell death via modulating the AMPK/mTOR/ULK-1 pathway. Our study revealed novel molecular mechanisms underlying SKN's anti-RA effects.

Specneuzhenide improves bleomycin-induced pulmonary fibrosis in mice via AMPK-dependent reduction of PD-L1.

BACKGROUND: Pulmonary fibrosis (PF) is an escalating global health issue, characterized by rising rates of morbidity and mortality annually. Consequently, further investigation of potential damage mechanisms and potential preventive strategies for PF are warranted. Specnuezhenide (SPN), a prominent secoiridoid compound derived from Ligustrum lucidum Ait, exhibits anti-inflammatory and anti-oxidative capacities, indicating the potential therapeutic actions on PF. However, the underlying mechanisms of SPN on PF remain unclear. PURPOSE: This work was aimed at investigating the protective actions of SPN on PF and the potential mechanism. METHODS: In vivo, mice were administrated with bleomycin (BLM) to establish PF model. PF mice were treated with SPN (45/90 mg/kg) by gavage. In vitro, we employed TGF-ß1 (10 ng/mL)-induced MLE-12 and PLFs cells, which then were treated with SPN (5, 10, 20 µM). DARTS assay, biofilm interference experiment and molecular docking were performed to investigate the molecular target of SPN. RESULTS: In vivo, we found SPN treatment improved survival rate, alleviated pathological changes through reducing BLM-induced extracellular matrix (ECM) deposition, as well as BLM-induced epithelial-mesenchymal transition (EMT). In vitro, SPN inhibited EMT and lung fibroblast transdifferentiation. Mechanistically, SPN activated the AMPK protein to decrease the abnormally high level of PD-L1. Furthermore, the compound C, known as an AMPK inhibitor, exhibited a significant hindrance to the inhibition of SPN on TGF-ß1-caused fibroblast transdifferentiation and proliferation. This outcome could be attributed to the fact that compound C could eliminate the inhibitory effects of SPN on PD-L1 expression. Interestingly, DARTS assay, biofilm interference experiment and molecular docking results all indicated that SPN could bind to AMPK, which suggested that SPN might be a potential agonist targeting AMPK protein. CONCLUSION: Altogether, the results in our work illustrated that SPN promoted AMPK-dependent reduction of PD-L1 protein, contributing to the inhibition of fibrosis progression. Thus, SPN may represent a potential AMPK agonist for PF treatment.

Polysaccharide of Atractylodes macrocephala Koidz alleviates LPS-induced proliferation, differentiation inhibition and excessive apoptosis in chicken embryonic myogenic cells.

BACKGROUND: Lipopolysaccharide (LPS) can induce systemic inflammation and affect the growth and development of poultry. As a kind of traditional Chinese medicine, polysaccharide of Atractylodes macrocephala Koidz (PAMK) can effectively improve the growth performance of animals and improve the immunity of animal bodies. OBJECTIVES: The purpose of this study was to investigate the effects of PAMK on LPS-induced inflammatory response, proliferation, differentiation and apoptosis of chicken embryonic myogenic cells. METHODS: We used chicken embryonic myogenic cells as a model by detecting EdU/MYHC immunofluorescence, the expression of inflammation, proliferation, differentiation-related genes and proteins and the number of apoptotic cells in the condition of adding LPS, PAMK, belnacasan (an inhibitor of Caspase1) or their combinations. RESULTS: The results showed that LPS stimulation increased the expression of inflammatory factors, inhibited proliferation and differentiation, and excessive apoptosis in chicken embryonic myogenic cells, and PAMK alleviated these adverse effects induced by LPS. After the addition of belnacasan (inhibitor of Caspase1), apoptosis in myogenic cells was inhibited, and therefore, the number of apoptotic cells and the expression of pro-apoptotic genes Caspase1 and Caspase3 were increased. In addition, belnacasan inhibited the increased expression of inflammatory factors, inhibited proliferation, differentiation and excessive apoptosis in chicken embryonic myogenic cells induced by LPS. CONCLUSIONS: This study provides a theoretical basis for further exploring the mechanism of action of PAMK and exogenous LPS on chicken embryonic myogenic cells and lays the foundation for the development and application of green feed additives in animal husbandry industry.

Pterostilbene suppresses gastric cancer proliferation and metastasis by inhibiting oncogenic JAK2/STAT3 signaling: In vitro and in vivo therapeutic intervention.

BACKGROUND: Gastric cancer (GC) represents a significant health burden with dire prognostic implications upon metastasis and recurrence. Pterostilbene (PTE) has been proven to have a strong ability to inhibit proliferation and metastasis in other cancers, while whether PTE exhibits anti-GC activity and its potential mechanism remain unclear. PURPOSE: To explore the efficacy and potential mechanism of PTE in treating GC. METHODS: We employed a comprehensive set of assays, including CCK-8, EdU staining, colony formation, flow cytometry, cell migration, and invasion assays, to detect the effect of PTE on the biological function of GC cells in vitro. The xenograft tumor model was established to evaluate the in vivo anti-GC activity of PTE. Network pharmacology was employed to predict PTE's potential targets and pathways within GC. Subsequently, Western blotting, immunofluorescence, and immunohistochemistry were utilized to analyze protein levels related to the cell cycle, EMT, and the JAK2/STAT3 pathway. RESULTS: Our study demonstrated strong inhibitory effects of PTE on GC cells both in vitro and in vivo. In vitro, PTE significantly induced cell cycle arrest at G0/G1 and S phases and suppressed proliferation, migration, and invasion of GC cells. In vivo, PTE led to a dose-dependent reduction in tumor volume and weight. Importantly, PTE exhibited notable safety, leaving mouse weight, liver function, and kidney function unaffected. The involvement of the JAK2/STAT3 pathway in PTE's anti-GC effect was predicted utilizing network pharmacology. PTE suppressed JAK2 kinase activity by binding to the JH1 kinase structural domain and inhibited the downstream STAT3 signaling pathway. Western blotting confirmed PTE's inhibition of the JAK2/STAT3 pathway and EMT-associated protein levels. The anti-GC effect was partially reversed upon STAT3 activation, validating the pivotal role of the JAK2/STAT3 signaling pathway in PTE's activity. CONCLUSION: Our investigation validates the potent inhibitory effects of PTE on the proliferation and metastasis of GC cells. Importantly, we present novel evidence implicating the JAK2/STAT3 pathway as the key mechanism through which PTE exerts its anti-GC activity. These findings not only establish the basis for considering PTE as a promising lead compound for GC therapeutics but also contribute significantly to our comprehension of the intricate molecular mechanisms underlying its exceptional anti-cancer properties.

Solanum lycopersicum (Tomato)-Derived Nanovesicles Accelerate Wound Healing by Eliciting the Migration of Keratinocytes and Fibroblasts.

Plant-derived nanovesicles have been considered interesting in medicine for their breakthrough biological effects, including those relevant to wound healing. However, tomato-derived nanovesicles (TDNVs) have not been studied for their effects on wound closure yet. TDNVs were isolated from Solanum lycopersicum (var. Piccadilly) ripe tomatoes by ultracentrifugation. Extract (collected during the isolation procedure) and NVs (pellet) were characterized by transmission electron microscopy and laser Doppler electrophoresis. Wound healing in the presence of Extract or NVs was analyzed by a scratch assay with monocultures of human keratinocytes (HUKE) or NIH-3T3 mouse fibroblasts. Cell proliferation and migration were studied by MTT and agarose spot assay, respectively. The vesicles in the Extract and NV samples were nanosized with a similar mean diameter of 115 nm and 130 nm, respectively. Both Extract and NVs had already accelerated wound closure of injured HUKE and NIH-3T3 monocultures by 6 h post-injury. Although neither sample exerted a cytotoxic effect on HUKE and NIH-3T3 fibroblasts, they did not augment cell proliferation. NVs and the Extract increased cell migration of both cell types. NVs from tomatoes may accelerate wound healing by increasing keratinocyte and fibroblast migration. These results indicate the potential therapeutic usefulness of TDNVs in the treatment of chronic or hard-to-heal ulcers.

Qinzhuliangxue mixture ameliorates psoriasis by restraining apoptosis in psoriasis via downregulating the MDA-5 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoriasis is characterized by hyperkeratosis that produces the classic silvery scales, and the pathogenesis of psoriasis involves abnormal proliferation of keratinocytes. Emerging evidence supports that apoptosis regulates keratinocyte proliferation and formation of stratum corneum, which maintains the homeostasis of the skin. Qinzhuliangxue mixture (QZLX) is a representative formula for the treatment of psoriasis, which was earliest recorded in the classic Chinese medicine book Xia's Surgery. In our previous clinical studies, QZLX demonstrated 83.33% efficacy with few side effects in the treatment of psoriasis. Furthermore, our published basic research has also proved that the QZLX mixture effectively inhibits the hyperproliferation of keratinocytes, thus exerting therapeutic effects on psoriasis. However, whether QZLX mixture can regulate keratinocytes apoptosis requires further clarification. OBJECTIVE OF THE STUDY: To investigate the mechanism of QZLX in the treatment of psoriasis from the perspective of keratinocyte apoptosis. MATERIALS AND METHODS: First, psoriasis-like mice with imiquimod (IMQ)-induced were given QZLX intragastric administration and Psoriasis Area Severity Index (PASI) scores were recored for 11 consecutive days to appraise the efficacy. Then, tissue samples were collected for transcriptome analysis. The DEseq2 method detected significantly differentially expressed genes (DEGs), Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway databases were used to analyze the functions and pathway enrichment of DEGs. After that, the therapeutic mechanisms of QZLX in intervening with psoriasis were explored using TUNEL, immunohistochemical staining, and western blotting. RESULTS: QZLX ameliorated the symptoms and pathological characteristics of IMQ-induced psoriasis in mice. The epidermal cell hyperplasia in the skin was inhibited, in accordance with the suppressed expression of PCNA and Ki67 after treatment. Transcriptome sequencing showed that melanoma differentiation associated gene-5 (MDA-5) was downregulated. GO and KEGG enrichment analysis of the signaling pathways indicated that the differentially expressed genes were significantly enriched in apoptosis pathways. Besides, QZLX treatment decreased the apoptosis of keratinocyte as shown by reduced TUNEL-positive cells. As MDA-5 protein levels decreased, so did the expression of the downstream protein Caspase-8, which indicates that the apoptotic pathway was triggered. Furthermore, QZLX therapy might also help to balance the apoptotic Bcl-2 family expression. CONCLUSION: QZLX restrains the apoptosis of keratinocyte in psoriasis-like mice by downregulating the MDA-5 pathway. The restoration of the balance between cell apoptosis and proliferation in the skin may lead to considerable psoriasis relief. Our study reveals the possible molecular processes behind the effects of QZLX therapy on the skin lesions of psoriasis, and lends support to its clinical efficacy.

Zishen Qingre Lishi Huayu recipe promotes proliferation and inhibits apoptosis of GCs of PCOS via KLF4-C/EBPß pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zishen Qingre Lishi Huayu recipe (ZQLHR) is a herbal recipe created on the basis on the theory of traditional Chinese medicine and clinical practice, and is mainly used in the treatment of polycystic ovary syndrome (PCOS). However, the underlying mechanism for this fact has not been clearly elucidated. AIM OF THE STUDY: To verify whether ZQLHR regulates granulosa cells (GCs) proliferation and apoptosis through the Krüppel-like factor 4 (KLF4) - CCATT enhancer-binding proteinß (C/EBPß) pathway, and to provide in vitro molecular mechanism supporting for the effects of ZQLHR to enhance follicular development and treat patients with PCOS. MATERIALS AND METHODS: Based on previous experiments, we performed the following experiments. Firstly, we treated KGN cells (a steroidogenic human granulosa-like tumor cell line) for 48 h using different concentrations of ZQLHR in order to observe apoptosis in each group. Secondly, the mRNA and protein expression levels of KLF4 and C/EBPß in KGN cells after administrated with ZQLHR were examined by quantitative real-time PCR(q-PCR) and Western blot assay. Thirdly, after knocking down KLF4 and C/EBPß using siRNAs, the relationship between KLF4 and C/EBPß in KGN cells was detected. Further, cell counting kit-8 assay, colony formation assay and flow cytometry were used to verify whether ZQLHR promotes proliferation and facilitates apoptosis in KGN cells through the KLF4-C/EBPß pathway. Finally, q-PCR and Western blot were used to test whether ZQLHR mediated proliferation and apoptosis-related factors such as B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (BAX), proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 to affect the proliferation and apoptosis of KGN cells through the KLF4-C/EBPß pathway. CONCLUSIONS: ZQLHR, containing 0.2% by volume, administered to KGN cells resulted in the lowest rate of apoptosis. The expression levels of KLF4 and C/EBPß were increased in KGN cells following ZQLHR treatment. Additionally, ZQLHR promoted proliferation and inhibited apoptosis of KGN cells by modulating proliferation and apoptosis-related factors via the KLF4-C/EBPß pathway. Furthermore, we confirmed that KLF4 and C/EBPß regulate each other in KGN cells. These findings indicate that ZQLHR enhances the proliferation of GCs and suppresses their apoptosis, which constitutes a therapeutic mechanism for treating patients with PCOS.