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.

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.

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.

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.

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.

Britannin inhibits cell proliferation, migration and glycolysis by downregulating KLF5 in lung cancer.

Lung cancer is a harmful type of malignancy and the leading cause of cancer-associated mortality. It is therefore imperative to develop novel drugs effective for treating this cancer. The Traditional Chinese Medicine compound Britannin has been previously reported to inhibit the development of certain cancers, such as pancreatic, breast and liver cancer. Moreover, Kruppel-like factor 5 (KLF5) has been identified an on oncogene in lung cancer. In the present study, the possible regulatory effects and underlying mechanism of Britannin in lung cancer were investigated. A549 and 16HBE cells were treated with different concentrations of Britannin. Subsequently, Cell counting kit-8, EdU staining and colony formation assays were used to detect the proliferative ability of these cells. Cell migration was detected by wound healing and Transwell assays, respectively. XF96 extracellular flux analyzer was used to analyze the extent of extracellular acidification and oxygen consumption rate in cells, whereas assay kits were used to detect glucose and lactic acid levels in the cell supernatant. The targeting effect between Britannin and the KLF5 protein was investigated using molecular docking technology. The protein expression levels of KLF5 in cells challenged with Britannin was detected by western blotting. Finally, overexpression of KLF5 in A549 cells was performed before cell proliferation, migration and the glycolysis rate were measured to explore the regulatory effects of Britannin. Britannin was found to inhibit the proliferation, migration and glycolysis of lung cancer cells, during which the protein expression levels of KLF5 were decreased. This suggests that Britannin regulated the expression of KLF5 in A549 cells. Overexpression of KLF5 reversed the inhibitory effects of Britannin on the proliferation, migration and glycolysis in lung cancer cells. In conclusion, these results suggest that Britannin can inhibit cell proliferation, migration and glycolysis by downregulating KLF5 expression in lung cancer cells.

Influence of Different Photobiomodulation Parameters on Multi-Potent Adipose Tissue Mesenchymal Cells In Vitro.

Objective: Investigating the effect of different parameters of photobiomodulation (PBM) with low-power laser on multi-potent mesenchymal stem cells (MSCs) derived from adipose tissue in terms of proliferation and cell death. Methods: MSCs were submitted to PBM applications with combinations of the following physical parameters: control group (no intervention), wavelengths of 660 and 830 nm; energy of 0.5, 2, and 4 J; and power of 40 and 100 mW. MSC analysis was performed using MetaXpress® software at 24, 48, and 72 h. Results: Irradiation promoted a significant increase in cell proliferation (p < 0.05), with 830 nm laser, 100 mW, with energy of 0.5, 2, and 4 J in relation to the control group at all times. PBM with 660 nm, power of 40 mW, and energy of 0.5, 2, and 4 J produced greater cell death at 24 h compared with the control group. At the time of 72 h, there was no significant difference concerning cell death. Conclusions: According to the results found, we can conclude that both wavelengths were effective; however, the 830 nm laser was more effective in terms of cell proliferation compared with the 660 nm laser. The 660 nm wavelength showed a significant increase in cell death when compared with the 830 nm laser.

GFAP-expressing cells in the adult hypothalamus can generate multiple neural cell lineages in vitro.

Adult neural stem cells (NSCs) located in the two canonical neurogenic niches, the subventricular zone (SVZ) and the subgranular zone (SGZ), express the glial fibrillary acidic protein (GFAP). Recently, proliferative activity has been described in the hypothalamus although the characterization of hypothalamic neural stem/progenitor cells (NSPCs) is still uncertain. We therefore investigated whether hypothalamic GFAP-positive cells, as in the SVZ and SGZ, also have neurogenic potential. We used a transgenic mouse line expressing green fluorescent protein (GFP) under the control of the GFAP promoter. GFAP-GFP expressing cells are localized in the ependymal layer as well as in the parenchyma of the mediobasal hypothalamus (MBH) and express Sox2, a marker for NSCs. Interestingly, no sexual dimorphism was observed in the numbers of GFP + and GFP-Sox2 + cells. After cells sorting, these cells were able to generate neurospheres in vitro and give rise to neurons, astrocytes and oligodendrocytes. Taken together, these results show that hypothalamic GFAP-expressing cells form a population of NSPCs.

Calotropis procera extract inhibits prostate cancer through regulation of autophagy.

Current treatment options available for prostate cancer (PCa) patients have many adverse side effects and hence, new alternative therapies need to be explored. Anticancer potential of various phytochemicals derived from Calotropis procera has been studied in many cancers but no study has investigated the effect of leaf extract of C. procera on PCa cells. Hence, we investigated the effect of C. procera leaf extract (CPE) on cellular properties of androgen-independent PC-3 and androgen-sensitive 22Rv1 cells. A hydroalcoholic extract of C. procera was prepared and MTT assay was performed to study the effect of CPE on viability of PCa cells. The effect of CPE on cell division ability, migration capability and reactive oxygen species (ROS) production was studied using colony formation assay, wound-healing assay and 2',7'-dichlorodihydrofluorescein diacetate assay, respectively. Caspase activity assay and LDH assay were performed to study the involvement of apoptosis and necrosis in CPE-mediated cell death. Protein levels of cell cycle, antioxidant, autophagy and apoptosis markers were measured by western blot. The composition of CPE was identified using untargeted LC-MS analysis. Results showed that CPE decreased the viability of both the PCa cells, PC-3 and 22Rv1, in a dose- and time-dependent manner. Also, CPE significantly inhibited the colony-forming ability, migration and endogenous ROS production in both the cell lines. Furthermore, CPE significantly decreased NF-κB protein levels and increased the protein levels of the cell cycle inhibitor p27. A significant increase in expression of autophagy markers was observed in CPE-treated PC-3 cells while autophagy markers were downregulated in 22Rv1 cells after CPE exposure. Hence, it can be concluded that CPE inhibits PCa cell viability possibly by regulating the autophagy pathway and/or altering the ROS levels. Thus, CPE can be explored as a possible alternative therapeutic agent for PCa.

Phospholipase D Mediates Glutamine-Induced mTORC1 Activation to Promote Porcine Intestinal Epithelial Cell Proliferation.

BACKGROUND: The intestinal epithelium is one of the fastest self-renewal tissues in the body, and glutamine plays a crucial role in providing carbon and nitrogen for biosynthesis. In intestinal homeostasis, phosphorylation-mediated signaling networks that cause altered cell proliferation, differentiation, and metabolic regulation have been observed. However, our understanding of how glutamine affects protein phosphorylation in the intestinal epithelium is limited, and identifying the essential signaling pathways involved in regulating intestinal epithelial cell growth is particularly challenging. OBJECTIVES: This study aimed to identify the essential proteins and signaling pathways involved in glutamine's promotion of porcine intestinal epithelial cell proliferation. METHODS: Phosphoproteomics was applied to describe the protein phosphorylation landscape under glutamine treatment. Kinase-substrate enrichment analysis was subjected to predict kinase activity and validated by qRT-PCR and Western blotting. Cell Counting Kit-8, glutamine rescue experiment, chloroquine treatment, and 5-fluoro-2-indolyl deschlorohalopemide inhibition assay revealed the possible underlying mechanism of glutamine promoting porcine intestinal epithelial cell proliferation. RESULTS: In this study, glutamine starvation was found to significantly suppress the proliferation of intestinal epithelial cells and change phosphoproteomic profiles with 575 downregulated sites and 321 upregulated sites. Interestingly, phosphorylation of eukaryotic initiation factor 4E-binding protein 1 at position Threonine70 was decreased, which is a crucial downstream of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Further studies showed that glutamine supplementation rescued cell proliferation and mTORC1 activity, dependent on lysosomal function and phospholipase D activation. CONCLUSION: In conclusion, glutamine activates mTORC1 signaling dependent on phospholipase D and a functional lysosome to promote intestinal epithelial cell proliferation. This discovery provides new insight into regulating the homeostasis of the intestinal epithelium, particularly in pig production.

Tissue engineered periosteum: Fabrication of a gelatin basedtrilayer composite scaffold with biomimetic properties for enhanced bone healing.

The periosteum, a vascularized tissue membrane, is essential in bone regeneration following fractures and bone loss due to some other reasons, yet there exist several research gaps concerning its regeneration. These gaps encompass reduced cellular proliferation and bioactivity, potential toxicity, heightened stiffness of scaffold materials, unfavorable porosity, expensive materials and procedures, and suboptimal survivability or inappropriate degradation rates of the implanted materials. This research used an interdisciplinary approach by forming a new material fabricated through electrospinning for the proposed application as a layer-by-layer tissue-engineered periosteum (TEP). TEP comprises poly(ε-caprolactone) (PCL), PCL/gelatin/magnesium-doped zinc oxide (vascular layer), and gelatin/bioactive glass/COD liver oil (osteoconductive layer). These materials were selected for their diverse properties, when integrated into the scaffold formation, successfully mimic the characteristics of native periosteum. Scanning electron microscopy (SEM) was employed to confirm the trilayer structure of the scaffold and determine the average fiber diameter. In-vitro degradation and swelling studies demonstrated a uniform degradation rate that matches the typical recovery time of periosteum. The scaffold exhibited excellent mechanical properties comparable to natural periosteum. Furthermore, the sustained release kinetics of COD liver oil were observed in the trilayer scaffold. Cell culture results indicated that the three-dimensional topography of the scaffold promoted cell growth, proliferation, and attachment, confirming its non-toxicity, biocompatibility, and bioactivity. This study suggests that the fabricated scaffold holds promise as a potential artificial periosteum for treating periostitis and bone fractures.

Vincazalidine A, a unique bisindole alkaloid from Catharanthus roseus.

A new dimeric indole alkaloid, vincazalidine A consisting of an aspidosperma type and a modified iboga type with 1-azatricyclo ring system consisting of one azepane and two piperidine rings coupled with an oxazolidine ring was isolated from Catharanthus roseus, and the structure including absolute stereochemistry was elucidated on the basis of spectroscopic data as well as DP4 statistical analysis. Vincazalidine A induced G2 arrest and subsequent apoptosis in human lung carcinoma cell line, A549 cells.

Iridoid Glycosides and Coumarin Glycoside Derivatives from the Roots of Nymphoides peltata and Their In Vitro Wound Healing Properties.

Nymphoides peltata has been used as a medicinal herb in traditional medicines to treat strangury, polyuria, and swelling. The phytochemical investigation of the MeOH extract of N. peltata roots led to the isolation of three iridoid glycosides and three coumarin glycoside derivatives, which were characterized as menthiafolin (1), threoninosecologanin (2), callicoside C (3), and scopolin (4), as well as two undescribed peltatamarins A (5) and B (6). The chemical structures of the undescribed compounds were determined by analyzing their 1 dimensional (D) and 2D nuclear magnetic resonance (NMR) spectra and using high-resolution (HR)-electrospray ionization mass spectroscopy (ESI-MS), along with the chemical reaction of acid hydrolysis. The wound healing activities of the isolated compounds 1-6 were evaluated using a HaCaT cell scratch test. Among the isolates, scopolin (4) and peltatamarin A (5) promoted HaCaT cell migration over scratch wounds, and compound 5 was the most effective. Furthermore, compound 5 significantly promoted cell migration without adversely affecting cell proliferation, even when treated at a high dose (100 µM). Our results demonstrate that peltatamarin A (5), isolated from N. peltata roots, has the potential for wound healing effects.

Schisandrin B Alleviates LPS Induced Mitochondrial Damage in C28I2 Cells.

Osteoarthritis is a common joint disease characterized by damage to the joint cartilage that occurs throughout the entire joint tissue. This damage primarily manifests as pain in the affected area. In clinical practice, medication is commonly used to relieve pain, but the treatment's effectiveness is poor and recurrent attacks are likely. Schisandrin B is the most abundant biphenylcyclohexene lignan found in the traditional Chinese medicine Schisandra chinensis, and it possesses various pharmacological effects. This study aims to investigate the protective effect of Schisandrin B on mitochondrial damage in osteoarthritis (C28I2 cells) under an inflammatory environment induced by LPS. Cell proliferation and activity, scratch tests, and LDH release tests are utilized to assess cell growth and migration ability. The immunofluorescence assay was used to detect the expression levels of proliferation and apoptosis proteins. The Western Blot assay was used to detect the expression levels of mitochondrial fusion and division proteins. The JC-1 assay was used to detect changes in mitochondrial membrane potential. The mitochondrial fluorescence probe assay was used to detect mitochondrial activity. Through research, it was found that Schisandrin B promotes the proliferation, growth, and migration of C28I2 cells, reduces apoptosis of C28I2 cells, balances mitochondrial fusion and division, stabilizes mitochondrial membrane potential, and promotes mitochondrial activity in an LPS induced inflammatory environment.

Synthesis of salicylic acid from wintergreen oil by green chemistry overcomes its cytotoxicity in keratinocyte cells and teratogenicity in zebrafish embryos.

Salicylic acid topical is used to treat variety of skin conditions. However, salicylic acid in these products is generated through industrial synthesis and has been shown to negatively impact fetal development and cause congenital abnormalities. We hypothesized that teratogenic effects reported in salicylic acid can be prevented by naturally synthesizing salicylic acid from wintergreen oil using green chemistry method. For this purpose, we investigated the effects of natural salicylic acid (NSA) synthesized from wintergreen oil using green chemistry and synthetic salicylic acid (SSA) on keratinocyte cell (HaCaT) proliferation and zebrafish embryo development. NSA structures were analyzed by 1H NMR, 13C NMR, and GC/MS methods. Percentage inhibition against HaCaT cell was determined by MTS assay. xCelligence system was used for cellular activities. Zebrafish embryos were exposed to NSA and SSA for 72 h post-fertilization. Lipid peroxidation, nitric oxide, sialic acid, glutathione-S-transferase, catalase, and superoxide dismutase were evaluated using biochemical methods. Expressions of nqO1, gfap, bdnf, vtg, egr, cyp1a, and igf2 were determined by RT-PCR as developmental indicators. MTS and RT-cell analysis showed increased cell viability by NSA, whereas SSA decreased cell viability. NSA beneficially affected zebrafish embryo development while SSA exerted deleterious effects through oxidant-antioxidant status, inflammation, and development. Results of our study showed for the first time that synthesis of salicylic acid from wintergreen oil by green chemistry overcomes its cytotoxicity in keratinocyte cells and teratogenicity in zebrafish embryos. This finding is important for drug research on safe topical applications during pregnancy, when preventing exposure to drug and chemical-derived teratogens is vital.

Proliferation of mammalian cells with Chlorococcum littorale algal compounds without serum support.

In recent years, serum-free medium for mammalian cell cultivation has attracted a lot of attention, considering the high cost of production and environmental load involved in developing the conventional animal sera. The use of alternative growth-promoting products in mammalian cell cultivation such as extracts from microalgae has proven to be quite beneficial and environmental-friendly. This research aims to cultivate mammalian cells with growth-promoting factors derived from Chlorococcum littorale. We have established a simple extraction using the ultrasonication method and applied the extract in place of serum on mammalian C2C12 cell lines, 3T3 cell lines, and CHO cell lines to compare and analyze the effectiveness of the extract. Cell passage was conducted in a suspended culture condition with the addition of the extract. The results indicate that the extract from microalgae shows a high proliferation rate in all cell lines without fetal bovine serum. Moreover, it is eco-friendly and has huge potential to replace the traditional cell culture system. It could be applied in the fields of regenerative medicine, gene/cell therapies, as well as cultured meat production.

Photobiomodulation effects of pulsed and continuous wave near-infrared laser on the proliferation and migration of human gingival fibroblasts: An in vitro study.

There are limited data on comparison of pulsed and continuous wave in photobiomodulation therapy (PBM). This study aimed to investigate the effect of PBM with 980 nm laser in pulsed and continuous wave on the proliferation and migration of human gingival fibroblasts (HGF) cells. Cultured HGF were divided into three main groups: (1) irradiated in pulsed mode (frequencies of 50 and 25 KHz; energy densities of 3 and 5 J/cm2 ), (2) irradiated in continuous mode (energy densities of 3.2 and 5.2 J/cm2 ), and (3) no irradiation as control group. HGF proliferation rate was measured by MTT assay at 24, 48, and 72 h post irradiation. In addition, HGF migration rate was measured by scratch test at 24 h post PBM. At 24 h, the group received continuous irradiation at 5.2 J/cm2 showed significantly higher proliferation compared with the control group (p = 0.012). At 48 and 72 h, the groups received continuous, and 50 Hz pulsed irradiation at energy densities of 5.2 and 5 J/cm2 respectively, had significantly higher HGF proliferation rates compared to the control (p < 0.05). Only the continuous irradiations were effective in significant increase of the cell migration. In conclusion, continuous PBM at energy density of 5.2 J/cm2 showed promising effect on HGF proliferation and migration.

Isovincathicine from Catharanthus roseus induces apoptosis in A549 cells.

A dimeric indole alkaloid, isovincathicine consisting of an aspidosperma type and modified iboga with C-7-C-20 connection type skeletons was first isolated from Catharanthus roseus, and the structure including stereochemistry was elucidated on the basis of spectroscopic data as well as DP4 statistical analysis. Isovincathicine inhibited cell proliferation in A549 cells. We investigated the detailed mode of action of isovincathicine-induced inhibitory effects on cell proliferation in A549 cells. Flow cytometric analysis showed that isovincathicine-treated cells accumulated in the G2 phase after 24 h, and the percentage of cells showing cell death increased after 48 h. Western blotting also showed increased expression of BimEL, an apoptosis-related protein, and decreased expression of Mcl-1 and Bcl-xL. Isovincathicine was suggested to induce apoptosis in A549 cells by a mechanism is similar to that of vinblastine.

Establishment and application of high throughput screening cell model for nutrient regulation of embryonic development.

Embryo development exerts far-reaching influence on pregnancy outcome, postnatal development and lifelong health. Thereafter, to select functional nutrients to improve embryo development is of great importance. Herein, a stable porcine trophectoderm cell line expressing a luciferase reporter gene driven by a 1,009 bp PCNA gene promoter was constructed through lentiviral transduction and G418 selection. A high throughput screening assay was subsequently developed using the stable reporter cell line to screen a library of 225 nutrients. Seven nutrients with a minimum Z-score of 2.0 were initially identified to be capable of enhancing embryonic development. Among these nutrients, resveratrol, apigenin, and retinol palmitate were furtherly confirmed the beneficial effects for embryo development. Resveratrol significantly increased the expression of key genes involved in pTr cell proliferation and the number of S-phase cells. Resveratrol was furtherly confirmed to promote the expression of key genes in trophoblast development and increase embryo adhesion rate in vitro. Similarly, dietary 0.05% resveratrol supplementation significantly increased the number of embryo attachment and serum level of P4 and E2 in rats. Resveratrol could also improve maternal antioxidant levels and reduce intracellular ROS. Collectively, a high throughput screening cell model for nutrient regulation of embryonic development was established, which can be used to highly effectively select the potential candidates for embryo development. These findings have great implications for exploring optimal functional nutrients to improve embryo development, ultimately beneficial for pregnancy outcome, offspring postnatal development and lifelong health for human beings and mammalian animals.