Electro-acupuncture modulated miR-214 expression to prevent chondrocyte apoptosis and reduce pain by targeting BAX and TRPV4 in osteoarthritis rats

Osteoarthritis (OA) is a highly prevalent joint disorder characterized by progressive degeneration of articular cartilage, subchondral bone remodeling, osteophyte formation, synovial inflammation, and meniscal damage. Although the etiology of OA is multifactorial, pro-inflammatory processes appear to play a key role in disease pathogenesis. Previous studies indicate that electroacupuncture (EA) exerts chondroprotective, anti-inflammatory, and analgesic effects in preclinical models of OA, but the mechanisms underlying these potential therapeutic benefits remain incompletely defined. This study aimed to investigate the effects of EA on OA development in a rat model, as well as to explore associated molecular mechanisms modulated by EA treatment. Forty rats were divided into OA, EA, antagomiR-214, and control groups. Following intra-articular injection of monosodium iodoacetate to induce OA, EA and antagomiR-214 groups received daily EA stimulation at acupoints around the knee joint for 21 days. Functional pain behaviors and chondrocyte apoptosis were assessed as outcome measures. The expression of microRNA-214 (miR-214) and its downstream targets involved in apoptosis and nociception, BAX and TRPV4, were examined. Results demonstrated that EA treatment upregulated miR-214 expression in OA knee cartilage. By suppressing pro-apoptotic BAX and pro-nociceptive TRPV4, this EA-induced miR-214 upregulation ameliorated articular pain and prevented chondrocyte apoptosis. These findings suggested that miR-214 plays a key role mediating EA's therapeutic effects in OA pathophysiology, and represents a promising OA treatment target for modulation by acupuncture.

Phillygenin Inhibits TGF-ß1-induced Hepatic Stellate Cell Activation and Inflammation: Regulation of the Bax/Bcl-2 and Wnt/ß-catenin Pathways.

Hepatic fibrosis (HF), a precursor to cirrhosis and hepatocellular carcinoma, is caused by abnormal proliferation of connective tissue and excessive accumulation of extracellular matrix in the liver. Notably, activation of hepatic stellate cells (HSCs) is a key link in the development of HF. Phillygenin (PHI, C21H24O6) is a lignan component extracted from the traditional Chinese medicine Forsythiae Fructus, which has various pharmacological activities such as anti-inflammatory, antioxidant and anti-tumour effects. However, whether PHI can directly inhibit HSC activation and ameliorate the mechanism of action of HF has not been fully elucidated. Therefore, the aim of the present study was to investigate the in vitro anti-HF effects of PHI and the underlying molecular mechanisms. Transforming growth factor-ß1 (TGF-ß1)-activated mouse HSCs (mHSCs) and human HSCs (LX-2 cells) were used as an in vitro model of HF and treated with different concentrations of PHI for 24 h. Subsequently, cell morphological changes were observed under the microscope, cell viability was analyzed by MTT assay, cell cycle and apoptosis were detected by flow cytometry, and the mechanism of anti-fibrotic effect of PHI was explored by immunofluorescence, ELISA, RT-qPCR and western blot. The results showed that PHI suppressed the proliferation of TGF-ß1-activated mHSCs and LX-2 cells, arrested the cell cycle at the G0/G1 phase, decreased the levels of α-SMA, Collagen I, TIMP1 and MMP2 genes and proteins, and promoted apoptosis in activated mHSCs and LX-2 cells. Besides, PHI reduced the expression of inflammatory factors in activated mHSCs and LX-2 cells, suggesting a potential anti-inflammatory effect. Mechanically, PHI inhibited TGF-ß1-induced HSC activation and inflammation, at least in part through modulation of the Bax/Bcl-2 and Wnt/ß-catenin pathways. Overall, PHI has significant anti-HF effects and may be a promising agent for the treatment of HF.

A new labdane diterpenoid from Scoparia dulcis improving pancreatic function against islets cell apoptotic by Bax/Bcl-2/Caspase-3 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Scoparia dulcis has been identified as a significant ethnopharmacological substance in the Li, Zhuang, and Dai ethnic groups of China. Traditional medicine use S. dulcis to treat numerous illnesses, most notably diabetes. The considerable antidiabetic properties of this herbal remedy have been established by several clinical investigations and animal experiments. The islet is the intended target of S. dulcis, although the cause of its activity and mechanism for diabetes treatment is unclear. The diterpenoids from S. dulcis have been shown in the literature to have significant hypoglycemic efficacy and to protect islet cells in vitro. Diterpenoids may be the components of this herbal remedy that preserve islets, but further research is needed. AIM OF THE STUDY: This study was projected to investigate the new diterpenoid scoparicol E from S. dulcis and examined its islet-protective effect and the potential mechanism both in vitro and in vivo. METHODS: The structure of the novel diterpenoid scoparicol E was clarified by employing a wide range of spectroscopic methods. Using CCK-8 tests, cytotoxicity and antiapoptotic activity of scoparicol E were detected. Serum biochemical analysis and pathologic examination were performed to study the protective effect of scoparicol E against islet damage. The specific mechanism of action of scoparicol E was investigated through the mitochondrial membrane potential, Annexin V-FITC flow cytometry, and western blotting. RESULTS: Scoparicol E reduced MLD-STZ-induced hyperglycemia in mice and increased insulin and islet apoptosis. Scoparicol E effectively suppressed the Bax/Bcl-2/Caspase-3 pathway, according to the in vivo western blot investigation. Scoparicol E showed significant antiapoptotic action in vitro. We also showed that scoparicol E might prevent islet cells from dying by inhibiting the Bax/Bcl-2/Caspase-3 pathway. The Annexin V-FITC flow cytometry results revealed that MIN6 cell apoptosis was considerably decreased following scoparicol E intervention, showing anti-islet cell apoptosis action. Furthermore, the Caspase-3-mediated apoptosis pathway depends on cytochrome c and the potential of the mitochondrial membrane. Scoparicol E prevented the release of cytochrome c, restored the mitochondrial membrane potential, and prevented MIN6 cell apoptosis. CONCLUSION: We demonstrated the new diterpenoid scoparicol E could protect islet cells apoptosis by modulating the Bax/Bcl-2/Caspase-3 pathway.

Selenium protection from DNA damage and regulation of apoptosis signaling following cyclophosphamide induced cardiotoxicity in rats.

We investigated the mechanism of the cardioprotective effect of selenium (Se) against cyclophosphamide (CPA) induced cardiotoxicity in rats. We divided 24 female Wistar albino rats into four groups. The control group was injected intraperitoneally (i.p.) with normal saline. The CPA group was injected i.p. with 200 mg/kg CPA. The Se group was injected i.p. with 1 mg/kg Se. The CPA + Se group was injected i.p. with 200 mg/kg CPA and 1 mg/kg Se. Rats were euthanized 24 h after injection and heart tissues were harvested. Histopathological examination revealed reduced severity of myocardial lesions in the CPA + Se group compared to CPA induced cardiotoxicity of the CPA group; this finding was confirmed by increased immunoreactivity of cardiac troponin-I (cTn-I) in the CPA + Se group compared to decreased cTn-I immunoreactivity in the CPA group. Administration of CPA increased the immunoreactivity of phosphorylated histone-2AX (γH2AX). Se reduced the CPA induced increase in γH2AX immunoreactivity. Se administration reversed the CPA induced increase of Bax and decrease of Bcl2 gene expressions. Our findings suggest that Se is cardioprotective by reducing DNA damage and regulating the genes responsible for apoptosis caused by CPA in rats.

Protective effect of a standardized Allium jesdianum extract in an Alzheimer's disease induced rat model.

Alzheimer's disease (AD) is a complex disorder with multiple underlying mechanisms. Existing treatment options mostly address symptom management and are associated with numerous side effects. Therefore, exploring alternative therapeutic agents derived from medicinal plants, which contain various bioactive compounds with diverse pharmacological effects, holds promise for AD treatment. This study aims to assess the protective effects of the hydroalcoholic extract of Allium jesdianum on cognitive dysfunction, mitochondrial and cellular parameters, as well as genetic parameters in an intracerebroventricular Streptozotocin (icv-STZ) induced rat model of AD. Male Wistar rats were injected with a single dose of STZ (3 mg/kg, icv) to establish a sporadic AD model. A. jesdianum extract (100, 200, and 400 mg/kg/day) and donepezil (5 mg/kg/day) were orally administered for 14 days following model induction. Cognitive function was evaluated using the radial arm water maze test. Mitochondrial toxicity parameters in various brain regions (whole brain, frontal cortex, hippocampus, and cerebellum) were assessed. Gene expression analysis of miR-330, miR-132, Bax, and Bcl-2 in isolated rat brain neurons was performed using RT-qPCR. A. jesdianum extract significantly attenuated cognitive dysfunction and mitigated mitochondrial toxicity induced by icv-STZ administration. Following STZ injection, there was upregulation of Bax gene expression and downregulation of miR-330, miR-132, and Bcl-2 gene expression. Treatment with A. jesdianum extract resulted in the reversal of the expression of these microRNAs and genes, indicating its potential for improving AD and reducing neuronal apoptosis. This study demonstrates the neuroprotective capabilities of A. jesdianum against STZ-induced oxidative stress and cognitive impairment in rats, highlighting its therapeutic potential in the management of AD.

Protective effect of vitamin B6 against doxorubicin-induced cardiotoxicity by modulating NHE1 expression.

Doxorubicin (DOX) has been used to treat various types of cancer, but its application is limited due to its heart toxicity as well as other drawbacks. Chronic inhibition of Na+ /H+ exchanger (NHE1) reduces heart failure and reduces the production of reactive oxygen species (ROS); vitamin B6 (VitB6 ) has been demonstrated to have a crucial role in antioxidant mechanism. So, this study was designed to explore the effect of VitB6 supplement on the DOX-induced cardiotoxicity and to imply whether NHE1 is involved. Ultrasonic cardiogram analysis revealed that VitB6 supplement could alleviate DOX-induced cardiotoxicity; hematoxylin and eosin (HE) and Masson's staining further confirmed this effect. Furthermore, VitB6 supplement exhibited significant antioxidative stress and antiapoptosis effect, which was evidenced by decreased serum malondialdehyde (MDA) content and increased serum superoxide dismutase (SOD) content, and decreased Bcl-2-associated X protein/B-cell lymphoma-2 ratio, respectively. Collectively, VitB6 supplement may exert antioxidative and antiapoptosis effects to improve cardiac function by decreasing NHE1 expression and improve DOX-induced cardiotoxicity.

LCMS/MS Phytochemical Profiling, Molecular, Pathological, and Immune-Histochemical Studies on the Anticancer Properties of Annona muricata.

Annona muricate is a tropical plant that is well-known for its edible fruit of therapeutic interest. LCMS/MS analyses were applied to identify phytoconstituents of the ethanolic extract of the whole fruits and the aqueous extract of the edible fruit part, in addition to the investigation of their anticancer properties against Ehrlich ascites carcinoma (EAC) in male albino mice. LCMS/MS analyses resulted in the identification of 388 components, representing a wide array of classes of compounds, including acetogenins as the major constituents, alkaloids, flavonoids, and phenolics. Among them, four compounds were tentatively characterized as new compounds (1-4), including an acid derivative, protocatechuic-coumaroyl-quinic acid (1), and three flavonoid derivatives, dihydromyricetin galloyl hexoside (2), apigenin gallate (3), and dihydromyricetin hexouronic acid hexoside (4). Induction with EAC cells resulted in abnormalities in the gene expression of pro-apoptotic genes (Bax and caspase-3) and anti-apoptotic gene (Bcl-2) in the tumor mass. Moreover, microscopic, histopathological, and immune-histochemical examinations of the tumor mass and liver tissues exhibited extensive growth of malignant Ehrlich carcinoma cells and marked hydropic degeneration of hepatocytes and infiltration by tumor cells to liver tissue with marked inflammatory reaction. These abnormalities were markedly ameliorated aftertreatment of EAC mice with A. muricata extracts.

Investigating the role of Zibai ointment on apoptosis-related factors Bcl-2 and Bax in wound healing after anal fistula surgery.

OBJECTIVE: In this study, we investigated the impact of Zibai ointment on wound healing by analyzing the expression levels of two key apoptosis-related factors-B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax), in patients following surgery for anal fistula. METHODS: We included 90 patients with anal fistulas who were treated in the People's Hospital Affiliated to Fujian University of Traditional Chinese Medicine. Patients were randomly assigned to receive treatment with Zibai ointment (n = 45) or petroleum jelly (n = 45). The levels of apoptosis-related factors Bcl-2 and Bax were evaluated using enzyme-linked immunosorbent assay (ELISA), while cell apoptosis was assessed using Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. RESULTS: The results of ELISA showed that on Day 21 after the surgery, the levels of Bcl-2 and Bax in the Zibai ointment group were significantly different compared to the petroleum jelly group, with values of (60.11 ± 1.31) ng/mL and (7.05 ± 0.01) versus (83.79 ± 1.74) ng/mL and (6.00 ± 0.05) ng/mL, respectively (p < .05). Furthermore, light microscopy revealed a large number of apoptotic cells within the field of vision 14 days postsurgery in the Zibai ointment group, and the healing time in the Zibai ointment group was significantly different from that in the petroleum jelly group (p < .05). CONCLUSION: We found that Zibai ointment effectively promoted wound healing in patients following anal fistula surgery, possibly by regulating Bcl-2 and Bax apoptosis-related factors.

Nyctanthes arbor-tristis alkaloids activates p53 independent cell death receptor and necroptosis pathways in HepG2 cells.

Nyctanthes arbor-tristis is a traditional medicinal plant with potential anti-cancer properties. In this study, crude and alkaloid extracts were prepared from different parts of the plant, and their cytotoxicity was evaluated on four different cancer cell lines. The alkaloid extracts from the leaf and fruit showed promising results, with the HepG2 cell line exhibiting significant cytotoxicity. The promising extracts were further studied for their apoptotic potential using various methods, including DNA fragmentation, TUNEL, Caspase-3 activity, Giemsa, and Hoechst staining. Our results indicated that the fruit extract had the highest apoptotic potential, with clear nuclear condensation, fragmentation, and apoptotic bodies observed. We also investigated the alteration of the Bax/Bcl-2 ratio both at the mRNA and protein levels. Our results showed a significant upregulation of the Bax gene and downregulation of the Bcl-2 gene for the fruit alkaloid extract. This indicates that the phenomenon of cell death expression might be following a p53-independent extrinsic pathway and Bax-activated caspase-independent AIF-mediated necroptosis in the HepG2 cancer cell line. Overall, our findings suggest that Nyctanthes arbor-tristis has potential as a therapeutic option for cancer treatment. The alkaloid extracts from the leaf and fruit may hold promise as a source of bioactive compounds for further development into anti-cancer agents. Further studies are needed to explore the underlying mechanisms of their cytotoxic and apoptotic effects and to evaluate their safety and efficacy in animal models and clinical trials.

Modified Erchen decoction ameliorates cognitive dysfunction in vascular dementia rats via inhibiting JAK2/STAT3 and JNK/BAX signaling pathways.

BACKGROUND: Vascular dementia (VaD) is one of the most common clinical syndromes of progressive neurocognitive dysfunction with uncertain mechanisms. Modified Erchen decoction (MECD), developed from "Erchen decoction (ECD)" recorded in "Taiping Huimin Heji Jufang", showed a good effect in the treatment of VaD. However, its therapeutic mechanism is still unclear. PURPOSE: This study aimed to elucidate the multi-target mechanisms of MECD against VaD in vivo and in vitro. METHODS: VaD model was established by two-vessel obstruction (2-VO) in Sprague-Dawley rats. Six groups, including the control, 2-VO operation, MECD treatment (2.5, 5.0 and 10.0 g kg-1 d-1), donepezil hydrochloride (positive control, 0.45 g kg-1 d-1) were designed in the whole experiment. After oral administration for 4 weeks, the effects of MECD were verified by behavioral experiments, histological observation, and biochemical index analysis. The chemical profiling of MECD was performed by UHPLC-Orbitrap Fusion-HRMS, and a "compound-target-pathway" multivariate network was constructed to validate and elucidate its pharmacological mechanisms. RESULTS: Compared with 2-VO group, MECD treatment significantly alleviated anxiety and improved spatial memory in VaD rats according to the open field test (OFT) and Y-maze test. A significant increase in neuron number was observed from hematoxylin and eosin (H&E) stained images in cornu ammonis 1 (CA1) of the hippocampal region after MECD treatment. On the one hand, MECD reduced the plasma levels of triglyceride (TG), low-density lipoprotein (LDL), malondialdehyde (MDA), and amyloid-beta 42 (Aß42), and inhibited mRNA expression of interleukin-1 beta (Il-1ß) and Il-6 in the hippocampus. On the other hand, superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) were significantly increased after treatment with MECD. Moreover, MECD reduced the mRNA expression and protein expression of janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), c-Jun N-terminal kinase (JNK), and BCL2-associated X (BAX) in the brain of 2-VO rats. Furthermore, 71 compounds were identified from the extract of MECD. Among them, liquiritin and isochlorogenic acid C gave inhibiting effects on the mRNA expression of Jnk. In addition, liquiritin and hesperetin were conformed with the inhibition of Jak2 transcription level in vitro experiments. CONCLUSION: MECD has demonstrated a significant amelioration effect on cognitive dysfunction in VaD rats via JAK2/STAT3 and JNK/BAX signaling pathways, which represents an innovative insight into the "activate blood and eliminate phlegm" theory.

Effects of Au@Ag core-shell nanostructure with alginate coating on male reproductive system in mice.

Despite the widespread use of silver nanoparticles (NPs), these NPs can accumulate and have toxic effects on various organs. However, the effects of silver nanostructures (Ag-NS) with alginate coating on the male reproductive system have not been studied. Therefore, this study aimed to investigate the impacts of this NS on sperm function and testicular structure. After the synthesis and characterization of Ag-NS, the animals were divided into five groups (n = 8), including one control group, two sham groups (received 1.5 mg/kg/day alginate solution for 14 and 35 days), and two treatment groups (received Ag-NS at the same dose and time). Following injections, sperm parameters, apoptosis, and autophagy were analyzed by the TUNEL assay and measurement of the mRNA expression of Bax, Bcl-2, caspase-3, LC3, and Beclin-1. Fertilization rate was assessed by in vitro fertilization (IVF), and testicular structure was analyzed using the TUNEL assay and hematoxylin and eosin (H&E) staining. The results showed that the NS was rod-shaped, had a size of about 60 nm, and could reduce sperm function and fertility. Gene expression results demonstrated an increase in the apoptotic markers and a decrease in autophagy markers, indicating apoptotic cell death. Moreover, Ag-NS invaded testicular tissues, especially in the chronic phase (35 days), resulting in tissue alteration and epithelium disintegration. The results suggest that sperm parameters and fertility were affected. In addition, NS has negative influences on testicular tissues, causing infertility in men exposed to these NS.

Euphorbium total triterpenes improve Freund's complete adjuvant-induced arthritis through PI3K/AKT/Bax and NF-κB/NLRP3 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbium is the resinous secretion of Euphorbia resinifera Berg. According to the record, Euphorbium was first used by Roman doctors to treat the emperor's joint pain. In China, it is applied in folk medicine to treat damp-cold or mucous diseases, such as arthralgia and ascites, etc. This herb is used for rheumatoid arthritis and skin tumors in the folklore of northeastern Brazil. Triterpenes are mainly characteristic constituents of Euphorbium, and possibly possess anti-rheumatoid arthritis. AIM OF THE STUDY: To explore the preventive effect of Euphorbium total triterpenes (TTE) on Freund's complete adjuvant (FCA) induced arthritis in rats and its mechanism. MATERIAL AND METHODS: TTE was extracted and isolated from Euphorbium, and its components were analyzed by HPLC. The safety of TTE was evaluated by an acute toxicity test in mice. Arthritis was induced in rats by injecting 0.2 mL FCA into the right hind paw toe, except for the control group, which was given the same volume of physiological saline. Tripterygium Glycosides (TG, 7.5 mg/kg) and TTE (32, 64 and 128 mg/kg) were administered by gavage for 30 days. Body weights, paw swelling, and arthritic scores were measured during the experiment process. After 30 days, blood and joints were harvested to determine various indicators of arthritis. RESULTS: The contents of euphol and euphorbol in TTE were 47.03% and 18.77% respectively, and the maximal feasible dose of TTE in mice is 12 g/kg. The experimental results showed that arthritis indicators in rats deteriorated after FCA inducement compared with the control group. After treatment with TTE, the swelling degree and histopathological change of the hind paws in rats were significantly improved as well as arthritic score; the serum TNF-α, CRP, IL-1ß, IL-6, IL-18 and RF levels in rats were significantly reduced; The expression of PI3K, AKT, P-AKT, Bcl-2, NF-κB, NLRP3 and Pro-caspase-1 protein in joint tissue were down-regulated, and the expression of Bax protein was up-regulated. CONCLUSION: The results suggested that TTE possessed anti-arthritis effects, and its mechanism may be related to its anti-inflammatory and immunomodulatory properties, as well as regulation of PI3K/AKT/Bax and NF-κB/NLRP3 signaling pathway.

Mechanism of Ethyl Acetate Extract of Gentianopsis paludosa in Treatment of Ulcerative Colitis Based on Bcl-2/Bax Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of ethyl acetate extract of Tibetan medicine dampness bud Gentianopsis paludosa in the prevention and treatment of recurrent ulcerative colitis （UC） in rats with dampness-heat in large intestine syndrome based on the apoptotic pathway mediated by the B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax）. MethodUsing the disease-syndrome combination method， a recurrent UC model of dampness-heat in large intestine syndrome was constructed in rats. Seventy SPF-grade male SD rats were randomly divided into control group， model group， high-， medium-， and low-dose ethyl acetate of G.paludosa groups （150， 75， 37.5 mg·kg-1）， and mesalazine group （135 mg·kg-1）. The rats were orally administered with respective drugs for 14 days. The general conditions of the rats were recorded， and colon length and mucosal damage were observed. The colon wet weight index and organ coefficients of the liver， spleen， and thymus were calculated. Enzyme-linked immunosorbent assay （ELISA） was used to measure the levels of interleukin-6 （IL-6） and interleukin-1β （IL-1β） in the serum of each group. Hematoxylin-eosin （HE） staining was performed to observe pathological changes in the colon. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） was used to detect apoptosis in colonic epithelial cells. Western blot was used to measure the expression levels of Bcl-2， Bax， Caspase-3， Caspase-9， Zona Occludens-1 （ZO-1）， Claudin3， and Occludin in colonic tissue. Immunohistochemistry （IHC） was used to observe the expression of Bax and Caspase-3 in colonic epithelial cells. ResultCompared with the control group， the model group showed significant increases in the disease activity index （DAI） score， colonic mucosal damage index （CMDI）， intestinal epithelial apoptosis， liver and spleen indexes， and levels of inflammatory factors IL-1β and IL-6 in the serum （P<0.01）， decreased expression of intestinal mucosal protective proteins ZO-1， Claudin3， and Occluding （P<0.01）， increased expression of pro-apoptotic proteins Bax， Caspase-3， and Caspase-9 （P<0.01）， and decreased expression of anti-apoptotic protein Bcl-2 （P<0.01）. Compared with the model group， the high-， medium-， and low-dose ethyl acetate of G.paludosa groups all significantly improved the general condition of the rats， reduced colonic lesions， decreased intestinal epithelial cell apoptosis， reduced liver and spleen indexes， upregulated the expression of ZO-1， Claudin3， Occludin， and Bcl-2 proteins， and downregulated the expression of Bax， Caspase-3， and Caspase-9 proteins， with the high- and medium-dose ethyl acetate of G.paludosa groups showing the superior effects （P<0.05， P<0.01）. ConclusionEthyl acetate of G.paludosa can alleviate colonic mucosal damage and exert a therapeutic effect on UC by regulating the Bcl-2/Bax signaling pathway.

[Xanthoceras sorbifolium Bunge flower extract inhibits benign prostatic hyperplasia in rats].

OBJECTIVE: To assess the inhibitory effect of the extract of Xanthoceras sorbifolium Bunge flower against benign prostatic hyperplasia (BPH) and explore its possible mechanism. METHODS: MTT assay was used to examine the effect of the extract of Xanthoceras sorbifolium Bunge flower on proliferation of benign prostatic hyperplasia cells (BPH-1), and cell apoptosis and cell cycle changes following the treatment were analyzed using annexin V/PI double staining and flow cytometry. The protein expression levels of Bcl-2, Bax, caspase-3, PI3K and AKT in the treated cells were detected using Western blotting. A rat model of BPH established by subcutaneous injection of testosterone propionate was treated with the flower extract for 28 days, and pathological changes in the prostate tissue were observed with HE staining. The protein expression levels of Bcl-2, Bax, caspase3 and PI3K/AKT in the prostate tissue were detected with Western blotting. RESULTS: Within the concentration range of 125-1000 µg/mL, the flower extract of Xanthoceras sorbifolium Bunge significantly inhibited the proliferation of BPH-1 cells and caused obvious cell cycle arrest at G0/G1 phase; the apoptotic rate of the cells was positively correlated with the concentration of the flower extract (P < 0.05). Bcl-2, p-PI3K and p-AKT expression levels were significantly down-regulated and Bax and caspase-3 expression levels were significantly increased in the cells after treatment with the flowers extract (P < 0.05). In the rat models of BPH, the rats treated with the flowers extract at moderate and high doses showed obviously decreased expressions of p-AKT and Bcl-2 and an increased expression of Bax in the prostate tissue; a significantly lowered p-AKT expression was observed in the prostate tissue of rats receiving the low-dose treatment (P < 0.05). CONCLUSION: The flower extract of Xanthoceras sorbifolium Bunge has a inhibitory effect on BPH both in vitro and in rats, suggesting its potential value in the development of medicinal plant preparations for treatment of BPH.

Ferula gummosa gum exerts cytotoxic effects against human malignant glioblastoma multiforme in vitro.

Background and purpose: Ferula gummosa (F. gummosa), a potent medicinal herb, has been shown to possess anticancer activities in vitro. The present examination evaluated the cytotoxic and apoptogenic impacts of F. gummosa gum on the U87 glioblastoma cells. Experimental approach: MTT assay to determine the cell viability, flow cytometry by annexin V/FITC-PI to apoptosis evaluation, reactive oxygen species (ROS) assay, and quantitative RT-PCR were performed. Findings / Results: The results revealed that F. gummosa inhibited the growth of U87 cells in a concentration- and time-dependent manner with IC50 values of 115, 82, and 52 µg/mL obtained for 24, 48, and 72 h post-treatment, respectively. It was also identified that ROS levels significantly decreased following 4, 12, and 24 h after treatment. The outcomes of flow cytometry analysis suggested that F. gummosa induced a sub-G1 peak which translated to apoptosis in a concentration-dependent manner. Further examination revealed that F. gummosa upregulated Bax/Bcl-2 ratio and p53 genes at mRNA levels. Conclusion and implications: Collectively, these findings indicate that sub-G1 apoptosis and its related genes may participate in the cytotoxicity of F. gummosa gum in U87 cells.

Ethyl Acetate Extract from Artemisia argyi Prevents Liver Damage in ConA-Induced Immunological Liver Injury Mice via Bax/Bcl-2 and TLR4/MyD88/NF-κB Signaling Pathways.

BACKGROUND: Immunological liver injury (ILI) is a common liver disease and lacks potent drugs for treatment. Artemisia argyi Lévl. et Vant. (A. argyi), a medicinal and edible homologous plant usually used in diet therapy to cure various liver diseases, provides a great option for the prevention of ILI. PURPOSE: To investigate the effect that ethyl acetate extract of A. argyi (AaEA) on Concanavalin A (ConA)-induced ILI and the mechanism of regulating Bax/Bcl-2 and TLR4/MyD88/NF-κB signaling pathways. METHODS: The chemical components of AaEA were studied by LC-MS. In animal experiments, the positive control group was administrated diammonium glycyrrhizinate (DIG, 100 mg/kg), while different doses of AaEA groups (AaEA-H, AaEA-M, AaEA-L) were pretreated with AaEA 2.00, 1.00, and 0.50 g/kg, respectively, by intragastric for seven days, once every day. Then, ConA (12.00 mg/kg) was used through tail intravenous injection to establish the ILI model. The blood samples and livers were collected to test the degree of liver dysfunction, inflammation, oxidative stress, histopathological changes, and cell apoptosis. Real-time PCR and Western blotting analysis were used to explain the mechanism of regulating Bax/Bcl-2 and TLR4/MyD88/NF-κB signaling pathways. RESULTS: The way in which AaEA prevents liver damage in immunological liver injury (ILI) mice caused by ConA was investigated for the first time. Pretreatment with AaEA reduced the expression of ALT, AST, and inflammatory factors (TNF-α and IFN-γ). Meanwhile, AaEA also reduced MDA levels but upregulated the contents of IL-4, SOD, and GSH-px, alleviating oxidative stress induced by ILI. Western blotting and real-time PCR analysis demonstrated that AaEA could regulate the expression level and relative mRNA expression of key proteins on Bax/Bcl-2 and TLR4/MyD88/NF-κB signaling pathways. Finally, 504 components from AaEA were identified by LC-MS analysis, mainly including flavones, phenolic acids, and terpenoids with anti-inflammatory and liver protective activities, which highlights the potential of AaEA for diet treatment of ILI. CONCLUSION: AaEA can work against ConA-induced ILI in mice by regulating Bax/Bcl-2 and TLR4/MyD88/NF-κB signaling pathways, which has the potential to be a great strategy for the prevention of ILI.

Elevated Bax/Bcl-2 Ratio: A Cytotoxic Mode of Action of Kermanian Propolis Against an Acute Lymphoblastic Leukemia Cell Line, NALM-6.

Currently, alternative cancer remedies, especially herbal-derived medicines, have attracted great interest. Propolis, a honeybee-produced naturopathic formulation, is an available, affordable, and safe example of such remedies with different content according to its geographic location. Findings regarding the protective properties of this resinous substance across numerous pathological conditions are promising. Although the anti-tumor effects of propolis from different origins have been explored to some degree, yet there is no study on the effects of Kermanian propolis in the treatment of hematologic malignancies. Accordingly, the objective of the present experiment was to divulge the anti-tumor potential of this bioactive substance both as monotherapy and in combination with doxorubicin against an acute lymphoblastic leukemia cell line (NALM-6).The viability of cells treated with Kermanian propolis (5-500 µg/mL) and doxorubicin (5-100 µg/mL) was analyzed during 72 h. Based on the MTT results, the best incubation time, IC50 concentrations, and finally the cytotoxicity of the combination therapy were ascertained. Next, the apoptotic rate and expression of apoptosis-related genes (Bcl-2 and Bax) were assessed in mono and combination therapies using flow cytometry and real-time PCR assays, respectively. Kermanian propolis and doxorubicin have impressive tumor-suppressing activity in a dose-dependent manner (IC50 concentrations: 100 and 40 µg/mL respectively). The best incubation time was considered 48 h. For the combination approach, 50 and 10 µg/mL were determined as optimum concentrations of the compounds. The selected concentrations induced notable apoptosis in the studied cells through significant (P < 0.01) upregulation of Bax/Bcl-2 level. The present study clearly suggests that Kermanian propolis, as an adjunct treatment option, has a promising apoptosis-induced cell death potential in the NALM-6 cell line.

Neuroprotective Effects of Alpinia oxyphylla Miq against Mitochondria-Related Apoptosis by the Interactions between Upregulated p38 MAPK Signaling and Downregulated JNK Signaling in the Subacute Phase of Cerebral Ischemia-Reperfusion in Rats.

Apoptosis in the penumbra region is the major cell death mechanism occurring during ischemia-reperfusion injury's early phase. Here, we evaluated how the Alpinia oxyphylla Miq (AOM) affects mitochondria-related apoptosis 3 days after transient middle cerebral artery occlusion (MCAo) and examined the mechanisms underlying the regulation of MAPK-mediated mitochondria-related apoptotic signaling in the peri-infarct cortex in rats. The rats were administered the AOM extract intraperitoneally at doses of 0.2[Formula: see text]g/kg (AOM-0.2[Formula: see text]g), 0.4[Formula: see text]g/kg (AOM-0.4[Formula: see text]g), or 0.8[Formula: see text]g/kg (AOM-0.8[Formula: see text]g) at MCAo initiation. The AOM-0.4[Formula: see text]g and AOM-0.8[Formula: see text]g significantly ameliorated apoptotic cell death and considerably downregulated cytochrome c (cyto c) and cleaved caspase-3 immunoreactivity 3 days after reperfusion. Simultaneously, they significantly downregulated cytosolic p-JNK/JNK, cathepsin B/actin, cyto c/actin, Smac/DIABLO/actin, cleaved caspase-3/actin, and AIF/actin and mitochondrial p53/HSP60 and Bax/HSP60 fractions but upregulated cytosolic p-p38 MAPK/p38 MAPK, p-p90RSK/actin, p-Bad/Bad, p-CREB/actin, and XIAP/actin and cytosolic and mitochondrial Bcl-2/Bax and Bcl-xL/Bax fractions in the peri-infarct cortex. Pretreatment with SB203580 - a p38 MAPK inhibitor - completely abrogated the effects of AOM-0.8[Formula: see text]g on the aforementioned protein expression, whereas treatment with SP600125 - a JNK inhibitor - exerted protective effects similar to those of AOM-0.8[Formula: see text]g. Treatment with 0.4 or 0.8[Formula: see text]g/kg AOM has neuroprotective effects against mitochondria-related apoptosis by suppressing cyto c, Smac/DIABLO, and AIF release from the mitochondria to cytosol. The anti-mitochondria related apoptotic effects of the AOM extract are attributable to the interactions between upregulated p38 MAPK/p90RSK-mediated p-Bad and CREB signaling and downregulated JNK/cathepsin B-mediated Bax and p53 signaling in the peri-infarct cortex 3 days after transient MCAo.

Realgar (As4S4), a traditional Chinese medicine, induces acute promyelocytic leukemia cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway in vitro.

Acute promyelocytic leukemia (APL) is a specific subtype of acute myelogenous leukemia (AML) characterized by the proliferation of abnormal promyelocytes. Realgar, a Chinese medicine containing arsenic, can be taken orally. Traditional Chinese medicine physicians have employed realgar to treat APL for over a thousand years. Therefore, realgar may be a promising candidate for the treatment of APL. Nevertheless, the underlying mechanism behind realgar therapy is largely unclear. The present study aimed to investigate the effect of realgar on cell death in the APL cell line (NB4) in vitro and to elucidate the underlying mechanism. In this study, after APL cells were treated with different concentrations of realgar, the cell survival rate, apoptotic assay, morphological changes, ATP levels and cell cycle arrest were assessed. The expression of Bcl-2, Bax, Cytochrome C (Cyt-C) and apoptosis-inducing factor (AIF) at the mRNA and protein levels were also measured by immunofluorescence, quantitative PCR (qPCR) and Western blotting. We found that realgar could significantly inhibit APL cell proliferation and cell death in a time- and dose-dependent manner. Realgar effectively decreased the ATP levels in APL cells. Realgar also induced APL cell cycle arrest at the S and G2/M phases. Following realgar treatment, the mRNA and protein levels of Bcl-2 were significantly downregulated, whereas the levels of Bax, Cyt-C, and AIF were significantly upregulated. In summary, realgar can induce APL cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway, suggesting that realgar may be an effective therapeutic for APL.

A new approach on the regulation of NF-κB and Bax protein signaling pathway activation by royal jelly in fluoride-induced pancreas damage in rats.

Forty-two healthy adult male rats (Wistar albino, n = 42, 8 weeks old, starting weights 200-250 g) employed in this study were subdivided into six groups randomly with seven rats per group as follows: (i) Control group: received standard diet; (ii) RJ group: received standard diet supplemented with royal jelly; (iii) F50 group: received standard diet supplemented with fluoride (50 mg/kg BW); (iv) F100 group: received standard diet supplemented with fluoride (100 mg/kg BW); (v) F50 +RJ group: received standard diet supplemented with fluoride (50 mg/kg BW) and royal jelly; (iv) F100 +RJ group: received standard diet supplemented with fluoride (100 mg/kg BW) and royal jelly. The study continued for a total of eight weeks. Western blot analysis was conducted to determine the post-translational expression levels of NF-κB, Bax, Bcl-2, TNF-α, Caspase-3 and Caspase-6 proteins in pancreas tissue. The pancreatic tissue was subjected to histopathological evaluation. Furthermore, MDA, GSH and CAT activities were examined by spectrophotometric analyzes. Our findings demonstrate that, compared to the control and RJ groups, Bcl-2 protein expression was augmented and, conversely, Caspase-6, Caspase-3 and Bax protein levels were decreased upon fluoride treatment. A statistically significant increase in TNF-α and NF-κB protein expressions was observed in the groups with fluoride-induced damage compared to the control and RJ groups. The MDA levels were increased in all fluoride-treated rats compared to those in the control and RJ groups, whereas the CAT and GSH activities were reduced in all rats with fluoride- induced damage. Although there was not a great difference between the groups regarding histopathological findings, there was a tendency to decrease in the rate of damage upon royal jelly treatment.