Sacubitril/valsartan inhibits the proliferation of vascular smooth muscle cells through notch signaling and ERK1/2 pathway.

AIMS: To explore the role and mechanism of Notch signaling and ERK1/2 pathway in the inhibitory effect of sacubitril/valsartan on the proliferation of vascular smooth muscle cells (VSMCs). MAIN METHODS: Human aortic vascular smooth muscle cells (HA-VSMCs) were cultured in vitro. The proliferating VSMCs were divided into three groups as control group, Ang II group and Ang II + sacubitril/valsartan group. Cell proliferation and migration were detected by CCK8 and scratch test respectively. The mRNA and protein expression of PCNA, MMP-9, Notch1 and Jagged-1 were detected by qRT-PCR and Western blot respectively. The p-ERK1/2 expression was detected by Western blot. KEY FINDINGS: Compared with the control group, proliferation and migration of VSMCs and the expression of PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 was increased in Ang II group. Sacubitril/valsartan significantly reduced the proliferation and migration. Additionally, pretreatment with sacubitril/valsartan reduced the PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 expression.

Dienogest attenuates STAT3 activation in ovarian endometriotic cysts.

OBJECTIVE: Recent studies have suggested that endometriosis could be the result of excessive activation of signal transducer and activator of transcription 3 (STAT3), which is associated with the regulation of essential cellular mechanisms such as proliferation, invasion, and apoptosis. That finding implies that regulating STAT3 activation could play a key role in treating endometriosis. In the present study, we aimed to evaluate whether the anti-endometriotic effects of dienogest is mediated by the regulation of STAT3 activation. STUDY DESIGN: STAT3 activation was evaluated in normal endometrial and ovarian endometriotic tissues obtained from patients with/without preoperative dienogest treatment. A subsequent in vitro analysis with endometriotic cyst stromal cells (ECSCs) was used to confirm the direct influence of dienogest in STAT3 activation. RESULT: STAT3 activation is significantly higher in endometriotic tissues from non-treated patients than in normal endometrial tissues, and that difference is reversed by preoperative administration of dienogest. Similarly, the inhibitory effects of dienogest on STAT3 activation are demonstrated by in vitro results showing that dienogest treatment significantly inhibits IL-6-stimulated STAT3 activation in cultured ECSCs. That inhibition was accompanied by decreased expression of proliferative (PCNA), invasive (MMP-2), and anti-apoptotic (BCL-2) proteins. Furthermore, downregulating STAT3 activity with siRNA decreased PCNA, MMP-2, and BCL-2 expression in IL-6-treated ECSCs. CONCLUSION: Dienogest inhibits STAT3 activation in ECSCs, which affects their proliferation, invasiveness, and apoptosis.

Glucagon-Like Peptide Receptor Agonist Inhibits Angiotensin II-Induced Proliferation and Migration in Vascular Smooth Muscle Cells and Ameliorates Phosphate-Induced Vascular Smooth Muscle Cells Calcification.

BACKGRUOUND: Glucagon-like peptide-1 receptor agonist (GLP-1RA), which is a therapeutic agent for the treatment of type 2 diabetes mellitus, has a beneficial effect on the cardiovascular system. METHODS: To examine the protective effects of GLP-1RAs on proliferation and migration of vascular smooth muscle cells (VSMCs), A-10 cells exposed to angiotensin II (Ang II) were treated with either exendin-4, liraglutide, or dulaglutide. To examine the effects of GLP-1RAs on vascular calcification, cells exposed to high concentration of inorganic phosphate (Pi) were treated with exendin-4, liraglutide, or dulaglutide. RESULTS: Ang II increased proliferation and migration of VSMCs, gene expression levels of Ang II receptors AT1 and AT2, proliferation marker of proliferation Ki-67 (Mki-67), proliferating cell nuclear antigen (Pcna), and cyclin D1 (Ccnd1), and the protein expression levels of phospho-extracellular signal-regulated kinase (p-Erk), phospho-c-JUN N-terminal kinase (p-JNK), and phospho-phosphatidylinositol 3-kinase (p-Pi3k). Exendin-4, liraglutide, and dulaglutide significantly decreased the proliferation and migration of VSMCs, the gene expression levels of Pcna, and the protein expression levels of p-Erk and p-JNK in the Ang II-treated VSMCs. Erk inhibitor PD98059 and JNK inhibitor SP600125 decreased the protein expression levels of Pcna and Ccnd1 and proliferation of VSMCs. Inhibition of GLP-1R by siRNA reversed the reduction of the protein expression levels of p-Erk and p-JNK by exendin-4, liraglutide, and dulaglutide in the Ang II-treated VSMCs. Moreover, GLP-1 (9-36) amide also decreased the proliferation and migration of the Ang II-treated VSMCs. In addition, these GLP-1RAs decreased calcium deposition by inhibiting activating transcription factor 4 (Atf4) in Pi-treated VSMCs. CONCLUSION: These data show that GLP-1RAs ameliorate aberrant proliferation and migration in VSMCs through both GLP-1Rdependent and independent pathways and inhibit Pi-induced vascular calcification.

Repurposing AZD5438 and Dabrafenib for Cisplatin-Induced AKI.

SIGNIFICANCE STATEMENT: To combat both untoward effects of nephrotoxicity and ototoxicity in cisplatin-treated patients, two potential therapeutic oral anticancer drugs AZD5438 and dabrafenib, a phase-2 clinical trial protein kinase CDK2 inhibitor and an US Food and Drug Administration-approved drug BRAF inhibitor, respectively, were tested in an established mouse AKI model. Both drugs have previously been shown to protect significantly against cisplatin-induced hearing loss in mice. Each drug ameliorated cisplatin-induced increases in the serum biomarkers BUN, creatinine, and neutrophil gelatinase-associated lipocalin. Drugs also improved renal histopathology and inflammation, mitigated cell death by pyroptosis and necroptosis, and significantly enhanced overall survival of cisplatin-treated mice. BACKGROUND: Cisplatin is an effective chemotherapy agent for a wide variety of solid tumors, but its use is dose-limited by serious side effects, including AKI and hearing loss. There are no US Food and Drug Administration-approved drugs to treat both side effects. Recently, two anticancer oral drugs, AZD5438 and dabrafenib, were identified as protective against cisplatin-induced hearing loss in mice. We hypothesize that similar cell stress and death pathways are activated in kidney and inner ear cells when exposed to cisplatin and tested whether these drugs alleviate cisplatin-induced AKI. METHODS: The HK-2 cell line and adult FVB mice were used to measure the protection from cisplatin-induced cell death and AKI by these drugs. Serum markers of kidney injury, BUN, creatinine, and neutrophil gelatinase-associated lipocalin as well as histology of kidneys were analyzed. The levels of markers of kidney cell death, including necroptosis and pyroptosis, pERK, and proliferating cell nuclear antigen, were also examined by Western blotting and immunofluorescence. In addition, CDK2 knockout (KO) mice were used to confirm AZD5438 protective effect is through CDK2 inhibition. RESULTS: The drugs reduced cisplatin-induced cell death in the HK-2 cell line and attenuated cisplatin-induced AKI in mice. The drugs reduced serum kidney injury markers, inhibited cell death, and reduced the levels of pERK and proliferating cell nuclear antigen, all of which correlated with prolonged animal survival. CDK2 KO mice were resistant to cisplatin-induced AKI, and AZD5438 conferred no additional protection in the KO mice. CONCLUSIONS: Cisplatin-induced damage to the inner ear and kidneys shares similar cellular beneficial responses to AZD5438 and dabrafenib, highlighting the potential therapeutic use of these agents to treat both cisplatin-mediated kidney damage and hearing loss.

Effect of quercetin on granulosa cells development from hierarchical follicles in chicken.

1. The bioflavonoid quercetin is a biologically active component, but its functional regulation of granulosa cells (GCs) during chicken follicular development is little studied. To investigate the effect of quercetin on follicular development in laying hens, an in vitro study was conducted on granulosa cells from hierarchical follicles treated with quercetin.2. The effect of quercetin on cell activity, proliferation and apoptosis of granulosa cells was detected by CCK-8, EdU and apoptosis assays. The effect on progesterone secretion from granulosa cells was investigated by enzyme-linked immunosorbent assay (ELISA). Expression of proliferating cell nuclear antigen (PCNA) mRNA and oestrogen receptors (ERs), as well as the expression of steroid acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA during progesterone synthesis, were measured by real-time quantitative polymerase chain reaction (RT-qPCR). PCNA, StAR and CYP11A1 protein expression levels were detected using Western blotting (WB).3. The results showed that treatment with quercetin in granulosa cells significantly enhanced cell vitality and proliferation, reduced apoptosis and promoted the expression of gene and protein levels of PCNA. The levels of progesterone secretion increased significantly following quercetin treatment, as did the expression levels of StAR and CYP11A1 using the Western Blot (WB) method.4. The mRNA expression levels of ERα were significantly upregulated in the 100 ng/ml and 1000 ng/ml quercetin-treated groups, while there was no significant difference in expression levels of ERß mRNA.

Synergistic effects of electroacupuncture and bone marrow mesenchymal stem cell transplantation on rat intrauterine adhesion： an observation. / 电针联合骨髓间å è´¨å¹²ç»†èƒžç§»æ¤æ²»ç–—å¤§é¼ å®«è ”ç²˜è¿žçš„ååŒæ•ˆåº”è§‚å¯Ÿ.

OBJECTIVES: To investigate the effects of electroacupuncture(EA) combined with bone marrow mesen-chymal stem cells(BMSCs) transplantation on the endometrium of rats with intrauterine adhesions(IUA), so as to explore the possible mechanisms underlying their combined therapeutic effects. METHODS: Forty adult female SD rats were randomly divided into control, model, cell, and combined groups. The IUA rat model was established using a dual injury method of mechanical scratching and lipopolysaccharide infection. After successful modeling, on days 1, 3, and 7, rats in the model group received tail vein injection of phosphate buffered solution, while rats in the cell group received tail vein injection of BMSCs suspension for BMSCs transplantation；and rats in the combined group received BMSCs transplantation combined with EA treatment (2 Hz/15 Hz, 1-2 mA), targeting the "Guanyuan"(CV4), bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6) for 20 min daily for 3 consecutive estrous cycles. After intervention, uterine tissue was collected from 5 rats in each group. Histological analysis was performed using hematoxylin and eosin staining to evaluate endometrial thickness and glandular number. Masson staining was used to assess endometrial fibrosis area. Immunohistochemistry was performed to detect the positive expressions of vascular endothelial growth factor(VEGF), proliferating cell nuclear antigen(PCNA), and estrogen receptor(ER). Western blot analysis was conducted to determine the protein expressions of homeobox A10(HoxA10) and leukemia inhibitory factor(LIF), both key regulators of endometrial receptivity. The remaining 5 rats in each group were co-housed with male rats, and the uterine function recovery was evaluated by assessing the number of embryo implantations. RESULTS: Compared with the control group, the model group showed thinning endometrium(P<0.001), decreased glandular number(P<0.001), increased endometrial fibrosis area(P<0.001), reduced positive expressions of VEGF, PCNA, ER, expressions of HoxA10 and LIF, and decreased embryo implantation number (P<0.001) on the injured side of the uterus. Compared with the model group, the combined group showed a reversal of the aforementioned indicators(P<0.001, P<0.01)；the cell group exhibited thicker endometrium(P<0.001) and reduced endometrial fibrosis area(P<0.001). Compared with the cell group, the combined group showed increased endometrial thickness(P<0.01), elevated glandular number(P<0.05), significantly decreased endometrial fibrosis area(P<0.05), enhanced positive expressions of VEGF, PCNA and ER, expressions of HoxA10 and LIF in the endometrium, and a significant increase in embryo implantation number (P<0.001, P<0.05, P<0.01) on the injured side of the uterus, indicating better results than the cell group. CONCLUSIONS: The combination of EA and BMSCs synergistically promotes the repair of damaged endometrium, improves endometrial morphology, reduces fibrosis levels, enhances vascular regeneration and matrix cell proliferation, improves endometrial receptivity, which ultimately facilitates embryo implantation.

[Panax notoginseng saponins improve monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting ADAM10/Notch3 signaling pathway].

In this study, we investigated the effects of Panax notoginseng saponins (PNS) on pulmonary vascular remodeling and ADAM10/Notch3 pathway in pulmonary arterial hypertension (PAH). PAH rat model was established, and male Sprague Dawley (SD) rats were randomly divided into control group, monocrotaline (MCT) group and MCT+PNS group, with 10 rats in each group. Rats in the control group were intraperitoneally injected with equal volume of normal saline. Rats in the MCT group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with the same volume of normal saline every day. Rats in the MCT+PNS group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with 50 mg/kg PNS every day. The modeling time of each group lasted for 21 days. After the model was established, the mean pulmonary artery pressure (mPAP) was measured by right heart catheterization technique, the right ventricular hypertrophy index (RVHI) was calculated, the microscopic morphology and changes of pulmonary vascular wall were observed by HE and Masson staining, and the expressions of ADAM10, Notch3, Hes-1, P27, PCNA, Caspase-3 proteins and mRNA in pulmonary vascular tissue of rats were detected by Western blot and qPCR. The expression and localization of Notch3 and α-SMA were detected by immunofluorescence staining. The protein expression of ADAM10 was detected by immunohistochemical staining. The results showed that compared with the control group, mPAP, RVHI, pulmonary vessels and collagen fibers in the MCT group were significantly increased, the expressions of ADAM10, Notch3, Hes-1, and PCNA protein and mRNA were significantly increased, while the expressions of P27 and Caspase-3 protein and mRNA were decreased significantly. Compared with the MCT group, mPAP and RVHI were significantly decreased, pulmonary vessels were significantly improved and collagen fibers were significantly reduced, the expressions of protein and mRNA of ADAM10, Notch3, Hes-1, and PCNA were decreased in MCT+PNS group, but the expressions of protein and mRNA of P27 and Caspase-3 were increased slightly. The results of immunofluorescence showed that Notch3 and α-SMA staining could overlap, which proved that Notch3 was expressed in smooth muscle cells. The expression of Notch3 in the MCT group was increased significantly compared with that in the control group, while PNS intervention decreased the expression of Notch3. Immunohistochemical staining showed that compared with the control group, the amount of ADAM10 in the MCT group was increased significantly, and the expression of ADAM10 in the MCT+PNS group was decreased compared with the MCT group. These results indicate that PNS can improve the PAH induced by MCT in rats by inhibiting ADAM10/Notch3 signaling pathway.

Ketogenic diet inhibits neointimal hyperplasia by suppressing oxidative stress and inflammation.

OBJECTIVE: Neointimal hyperplasia is the primary mechanism underlying atherosclerosis and restenosis after percutaneous coronary intervention. Ketogenic diet (KD) exerts beneficial effects in various diseases, but whether it could serve as non-drug therapy for neointimal hyperplasia remains unknown. This study aimed to investigate the effect of KD on neointimal hyperplasia and the potential mechanisms. METHODS AND RESULTS: Carotid artery balloon-injury model was employed in adult Sprague-Dawley rats to induce neointimal hyperplasia. Then, animals were subjected to either standard rodent chow or KD. For in-vitro experiment, impacts of ß-hydroxybutyrate (ß-HB), the main mediator of KD effects, on platelet-derived growth factor BB (PDGF-BB) induced vascular smooth muscle cell (VSMC) migration and proliferation were determined. Balloon injury induced event intimal hyperplasia and upregulation of protein expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA), and these changes were significantly ameliorated by KD. In addition, ß-HB could markedly inhibit PDGF-BB induced VMSC migration and proliferation, as well as inhibiting expressions of PCNA and α-SMC. Furthermore, KD inhibited balloon-injury induced oxidative stress in carotid artery, indicated by reduced ROS level, malondialdehyde (MDA) and myeloperoxidase (MPO) activities, and increased superoxide dismutase (SOD) activity. We also found balloon-injury induced inflammation in carotid artery was suppressed by KD, indicated by decreased expressions of proinflammatory cytokines IL-1ß and TNF-α, and increased expression of anti-inflammatory cytokine IL-10. CONCLUSION: KD attenuates neointimal hyperplasia through suppressing oxidative stress and inflammation to inhibit VSMC proliferation and migration. KD may represent a promising non-drug therapy for neointimal hyperplasia associated diseases.

Effects of dietary folic acid supplementation on lactation performance and mammary epithelial cell development of dairy cows and its regulatory mechanism.

The experiment investigated the impacts of FA on the proliferation of bovine mammary gland epithelial cells (BMECs) and to investigate the underlying mechanisms. Supplementation of 10 µM FA elevated the mRNA expression of proliferating cell nuclear antigen (PCNA), cyclin A2 and cyclin D1, and protein expression of PCNA and Cyclin A1. The mRNA and protein expression of B-cell lymphoma-2 (BCL2) and the BCL2 to BCL2 associated X 4 (BAX4) ratio elevated, while that of BAX, Caspase-3 and Caspase-9 reduced by FA. Both Akt and mTOR signaling pathways were activated by FA. Moreover, the stimulation of BMECs proliferation, the alteration of proliferative genes and protein expression, the change of apoptotic genes and protein expression, and the activation of mTOR signaling pathway caused by FA were obstructed by Akt inhibitor. Suppression of mTOR with Rapamycin reversed the FA-modulated promotion of BMECs proliferation and change of proliferous genes and protein expression, with no impact on mRNA or proteins expression related to apoptosis and FA-activated Akt signaling pathway. Supplementation of rumen-protected FA in cow diets evaluated milk yields and serum insulin-like growth factor-1 and estradiol levels. The results implied that the proliferation of BMECs was stimulated by FA through the Akt-mTOR signaling pathway.

Novel studies on Isolation, purification and characterization of dibenzonitro compound from Glycosmis pentaphylla (Retz.) DC. and effect in downregulating neuronal cancers.

To isolate Letrozole from Glycosmis pentaphylla (Retz.) DC. and to determine its effect on regulating the proliferation, cell cycle distribution, apoptosis and key mechanisms in human neuroblastoma cell lines. Letrozole was isolated through column chromatographic technique and its effect was checked on human neuroblastoma cell lines, IMR 32. The effects of Letrozole on cell viability were measured by MTT assay, and the cell cycle distribution was determined by flow cytometry. The expression changes in mRNA of proliferating cell nuclear antigen (PCNA), cyclin D1 and Bcl-xL were taken from real-time PCR analysis and the protein levels were detected by Western blotting. The results of the present study showed that Letrozole, isolated from leaves of G. pentaphylla could cause significant inhibitory effect on proliferation of IMR 32 cells in a dose dependent manner. Cell arrest was obtained at S phase with the treatment of Letrozole. Apart from this, the expression of PCNA, cyclin D1 and Bcl-xL were decreased both at mRNA and protein levels for the same treatment. Letrozole can inhibit proliferation, induce cell arrest and cause apoptosis in IMR 32 cell lines. The decreased expression of PCNA, cyclin D1 and Bcl-xL induced by Letrozole contributes to the above effects in vitro. This is the first report on the isolation of Letrozole from G. pentaphylla.

The impact of busulfan on the testicular structure in prepubertal rats: A histological, ultrastructural and immunohistochemical study.

Busulfan is a widely used cancer chemotherapeutic agent. Temporary or permanent sterility in male patients is one of the most common side effects of this drug. The present study was performed to evaluate the changes in the microscopic structure of the testes of prepubertal rats, as well as the changes in PCNA and caspase-3 immune expression, at different durations after busulfan administration. The rats were 5 weeks old and were divided into two main groups. Control group and busulfan treated group. Busulfan treated group received a single dose of busulfan (40 mg/kg), then animals were subdivided to three subgroups; IIa, IIb, IIc which were sacrificed after four, ten and twenty weeks, respectively, from the beginning of the experiment. Light and electron microscopic studies were done. Serum testosterone level and relative testes weight were assessed. Immunohistochemical staining for anti-proliferating cell nuclear antigen (PCNA) and anti-caspase-3 antigen was also done. Morphometric and statistical studies were carried out. Group II revealed histological and ultrastructural degenerative changes including congested blood vessels and degenerated spermatogenic epithelium, Sertoli cells, and Leydig cells. These changes were more evident after 10 weeks of busulfan administration and were accompanied by absence of mature sperms in the lumen of seminiferous tubules. These changes were associated with a significant reduction in relative testes weight, testosterone level, germinal epithelial height and seminiferous tubule diameter. Moreover, PCNA and caspase-3 immune expression was significantly altered in busulfan treated group. Mild improvement in testicular structure was observed 20 weeks after busulfan treatment.

BPA induces placental trophoblast proliferation inhibition and fetal growth restriction by inhibiting the expression of SRB1.

Bisphenol-A (BPA) is a common environmental toxicant that is known to be associated with fetal growth restriction (FGR). However, the mechanisms of how BPA induce FGR is poorly characterized. We conducted proteomics to identify the abnormal expression of SRB1 in female placental tissues with high BPA-induced FGR and further verified its decreased expression in human placenta and BeWo cells. Next, the effect of BPA on fetal development was further confirmed in pregnant C57BL/6 mice. The expression of SRB1 was consistently downregulated in human FGR placentas, BPA-exposed trophoblasts and mouse placentas. In addition, we found that SRB1 interacted with PCNA, and BPA exposure indirectly reduced the expression of PCNA and further inhibited placental proliferation. In vitro studies showed that BPA exposure reduced the expression of CDK1, CDK2, cyclin B and phosphorylated Rb in placental trophoblast cells, indicating cell cycle arrest after exposure to BPA. In addition, the expression of γ-H2AX and phosphorylated ATM was upregulated in BPA-exposed trophoblasts, indicating increased DNA damage. Our results indicate that BPA-induced FGR is achieved by reducing the expression of SRB1, inhibiting placental proliferation and increasing DNA damage. Our findings not only explain the mechanism of BPA-associated developmental toxicity but also shed light upon developing novel therapeutic targets.

Tan IIA mitigates vascular smooth muscle cell proliferation and migration induced by ox-LDL through the miR-137/TRPC3 axis.

Tanshinone IIA (Tan IIA) has an important role in treatment of cardiovascular diseases, including atherosclerosis. The vascular smooth muscle cells (VSMCs) are a major part of the atherosclerotic plaque. However, the biological functions of Tan IIA in regulating VSMCs function remain mostly unclear. This research aimed at identifying the explicit molecular mechanism that Tan IIA regulates oxidized low-density lipoprotein (ox-LDL)-mediated VSMC proliferation and migration. VSMCs challenged by ox-LDL were adopted as cellular model of atherosclerosis, and suffered from Tan IIA treatment. After that, cells proliferation, apoptosis or migration were measured. The expression levels of microRNA (miR)-137, transient receptor potential cation channel subfamily C member 3 (TRPC3) and proliferating cell nuclear antigen (PCNA) were measured. The targeting relationship between miR-137 and TRPC3 was determined. It was found that Tan IIA blunted VSMC proliferation, PCNA expression and migration mediated by ox-LDL. Tan IIA promoted miR-137 level, and miR-137 knockdown reversed the influences of Tan IIA on VSMC proliferation, PCNA expression and migration in the presence of ox-LDL. TRPC3 was verified to be targeted by miR-137. Moreover, TRPC3 silencing exacerbated the influences of Tan IIA on VSMC proliferation, apoptosis and migration, and it mitigated the inhibitive effects of miR-137 knockdown on function of Tan IIA. We confirmed for the first time that Tan IIA constrained ox-LDL-stimulated VSMC proliferation and migration via regulating the miR-137/TRPC3 axis, which provided a theoretical basis for the research and promotion of Tan IIA as a therapeutic drug.

Vitamin D Receptor Regulates Autophagy to Inhibit Apoptosis and Promote Proliferation in Hepatocyte Injury.

BACKGROUND: Oxidative stress is an important mechanism in liver ischemia/reperfusion (I/R) injury. Hepatocyte apoptosis and proliferation occur in parallel with liver I/R injury, and the degree of apoptosis and proliferation determines the effects on hepatocytes. Vitamin D receptor (VDR) can lessen liver I/R injury, but previous studies focused mostly on inflammation and immunity. METHODS: H2O2 was used to induce hepatocyte injury. Before treatment with H2O2, Hep-3B cells were pretreated with paricalcitol (PC) and siRNA-VDR. Rapamycin and chloroquine were also applied in the study. RESULTS: The number of apoptotic cells was measured with an annexin V (AV) -fluorescein isothiocyanate apoptosis detection kit. Expression of proteins was measured by western blotting. As compared with the H2O2+Hep-3B group, levels of AV/PI, cleaved caspase-3, and p62 were lower, and expression levels of Bcl-2, proliferating cell nuclear antigen, and VDR were higher, in the PC+H2O2+Hep-3B group. When the VDR gene was silenced by siRNA-VDR in the siRNA-VDR+H2O2+Hep-3B group, expressions of AV/PI, cleaved caspase-3, and p62 were upregulated, and expressions of Bcl-2, proliferating cell nuclear antigen, and VDR were downregulated, as compared with values for the siRNA-NC+H2O2+Hep-3B group. Treatment with rapamycin or chloroquine partially reversed the effect of PC and siRNA-VDR on apoptosis and proliferation. CONCLUSIONS: VDR mediates hepatocyte apoptosis and proliferation through autophagy.

Metformin and thymoquinone co-treatment enhance 5-fluorouracil cytotoxicity by suppressing the PI3K/mTOR/HIF1α pathway and increasing oxidative stress in colon cancer cells.

This study investigated the chemotherapeutic effects of 5-fluorouracil (5-FU), metformin (Met), and/or thymoquinone (TQ) single/dual/triple therapies in the HT29, SW480 and SW620 colon cancer (CRC) cell lines. Cell cycle/apoptosis were measured by flow cytometry. The gene and protein expression of apoptosis (PCNA/survivin/BAX/Cytochrome-C/Caspase-3) and cell cycle (CCND1/CCND3/p21/p27) molecules, the PI3K/mTOR/HIF1α oncogenic pathway, and glycolysis regulatory enzymes were measured by quantitative-PCR and Western blot. Markers of oxidative stress were also measured by colorimetric assays. Although all treatments induced anti-cancer effects related to cell cycle arrest and apoptosis, the triple therapy showed the highest pro-apoptotic actions that coincided with the lowest expression of CCND1/CCND3/PCNA/survivin and the maximal increases in p21/p27/BAX/Cytochrome-C/Caspase-3 in all cell lines. The triple therapy also revealed the best suppression of the PI3K/mTOR/HIF1α pathway by increasing its endogenous inhibitors (PTEN/AMPKα) in all cell lines. Moreover, the lowest expression of lactate dehydrogenase and pyruvate dehydrogenase kinase-1 with the highest expression of pyruvate dehydrogenase were seen with the triple therapy, which also showed the highest increases in oxidative stress markers (ROS/RNS/MDA/protein carbonyl groups) alongside the lowest antioxidant levels (GSH/CAT) in all cell lines. In conclusion, this is the first study to reveal enhanced anti-cancer effects for metformin/thymoquinone in CRC that were superior to all monotherapies and the other dual therapies. However, the triple therapy approach showed the best tumoricidal actions related to cell cycle arrest and apoptosis in all cell lines, possibly by enhancing oxidative glycolysis and augmenting oxidative stress through stronger modulation of the PI3K/mTOR/HIF1α oncogenic network.

Therapeutic effects of kartogenin on temporomandibular joint injury by activating the TGF-ß/SMAD pathway in rats.

Patients with temporomandibular dysfunction (TMD) usually suffer from pathology or malpositioning of the temporomandibular joint (TMJ) disk, leading to the degenerative lesion of condyles. Kartogenin can promote the repair of damaged cartilage. This study aimed to explore whether intra-articular injection of kartogenin could alleviate the TMJ injury induced by type II collagenase. We measured the head withdrawal threshold and found that kartogenin alleviated the pain around TMD induced by type II collagenase. We observed the morphology of the condylar surface and found that kartogenin protected the integration of the condylar surface. We analyzed the density of the subchondral bone and found that kartogenin minimized the damage of TMJ injury to the subchondral bone. We next explored the histological changes and found that kartogenin increased the thickness of the proliferative layer and more collagen formation in the superficial layer. Then, to further ensure whether kartogenin promotes cell proliferation in the condyle, we performed immunohistochemistry of proliferating cell nuclear antigen (PCNA). The ratio of PCNA-positive cells was significantly increased in the kartogenin group. Next, immunofluorescence of TGF-ß1 and SMAD3 was performed to reveal that kartogenin activated the TGF-ß/SMAD pathway in the proliferative layer. In conclusion, kartogenin may have a therapeutic effect on TMJ injury by promoting cell proliferation in cartilage and subchondral bone. Kartogenin may be promising as an intra-articular injection agent to treat TMD.

Inflammation reduction potential of nanostructured lipid carriers encapsulated with rat's bone marrow cells' lysate.

Bone marrow-derived mesenchymal stromal cells (BMSCs) have been used for treating inflammatory disorders. Due to the large size of BMSCs compared to nanoparticles, BMSCs cannot be loaded into the nanoparticles. It is hypothesized that BMSCs lysate loading into the nanocarriers will effectively deliver cellular contents and regulatory elements of BMSCs at the injury site. This study aimed to investigate nanostructured lipid carriers (NLC) loading with BMSCs lysate through basic characterization and morphological analysis. Moreover, this study was mainly designed to investigate the role of NLC loaded BMSCs lysate in reducing inflammation via in-vitro and in-vivoassays. The in-vitro study involves cell viability assays, p53, annexin V and VEGF expression through ELISA and immunocytochemistry, real-time BAX, caspase-3, IL-6, IL-8, TOP2A, PCNA, and Ki-67 gene expression analysis. Additionally, to evaluate in-vivo anti-inflammatory activity, the carrageenan-induced rat paw oedema model was used. In-vitro results showed that NLC loaded BMSCs lysate increased cell viability, decreased apoptosis and pro-inflammatory genes expression and up-regulated angiogenesis and proliferation in H2O2 pre-stimulated cells. Findings of the in-vivo assay also indicated a reduction in rat's paw oedema volume in NLC-loaded BMSCs lysate, and downregulation of BAX, Caspase-3, IL-6, and IL-8 was observed. Enhanced expressions of TOP2A, PCNA, and Ki-67 were obtained. Concluding the results of this study, NLC-loaded BMSCs lysate could reduce inflammation and possibly regenerate damaged tissue mainly via increasing cell viability, angiogenesis and proliferation, and reducing apoptosis and pro-inflammatory cytokines.

Tetrandrine inhibits migration and invasion of BGC-823 and MKN-45 cells by regulating PI3K/AKT/mTOR signaling pathway.

Tetrandrine (Tet), a traditional Chinese herbal medicine extract, exhibits anti-cancer effect on many types of cancer. Nonetheless, the action mechanism of Tet in gastric cancer (GC) is still largely unclear. In the current study, proliferation, invasion, and migration of the BGC-823 and MKN-45 cells were effectively suppressed by Tet treatment in a dose-dependent manner. Moreover, Tet suppressed expression of the proliferation-associated protein PCNA, the interstitial cell phenotype N-cadherin, and the extracellular matrix-associated MMP-2 and MMP-9 in BGC-823 and MKN-45 cells in a dose-dependent manner. PI3K/AKT/mTOR, a cancer promoting signaling, was inactivated by Tet in a dose-dependent manner in BGC-823 and MKN-45 cells. Furthermore, our results demonstrated that the inhibition of Tet to PCNA, N-cadherin, MMP-2, and MMP-9 expression was partly rescuedby AKT inhibitor or mTOR inhibitor. In animal experiments, tumor growth was inhibited by Tet administration in a dose-dependent manner. In conclusion, the current data indicated that Tet had a critical effect on inhibiting BGC-823 and MKN-45 cells proliferation, migration, invasion, and tumor growth via regulating PI3K/AKT/mTOR signaling pathway, suggesting that Tet might be a potential treatment for GC.

Effects of Holliday Junction-Recognition Protein-Mediated C-Jun N-Terminal Kinase/ Signal Transducer and Activator of Transcription 3 Signaling Pathway on Cell Proliferation, Cell Cycle and Cell Apoptosis in Bladder Urothelial Carcinoma.

The Holliday Junction-Recognition Protein (HJURP) was upregulated in several tumors, which was associated with poor outcome. This study investigated the effects of the HJURP-mediated c-Jun N-terminal kinase (JNK)/ signal transducer and activator of transcription 3 (STAT3) pathway on bladder urothelial carcinoma (BLUC). Online databases were used to analyze HJURP expression in BLUC and the correlation of HJURP to JNK1 [mitogen-activated protein kinase 8 (MAPK8)], JNK2 (MAPK9), STAT3, marker of proliferation Ki-67 (MKI67), proliferating cell nuclear antigen (PCNA), cyclin dependent kinase 2 (CDK2), CDK4 and CDK6. HJURP expression was detected in BLUC cells and human normal primary bladder epithelial cells (BdECs). BLUC cells were treated with HJURP lentivirus activation /shRNA lentivirus particles or JNK inhibitor SP600125. HJURP was upregulated in BLUC tissues and correlated with poor prognosis of patients (all P < 0.05). HJURP in tumor positively correlated with MAPK8 (R = 0.30), MAPK9 (R = 0.30), STAT3 (R = 0.15), MKI67 (R = 0.60), PCNA (R = 0.46), CDK2 (R = 0.39), CDK4 (R = 0.24) and CDK6 (R = 0.21). The JNK inhibitor SP600125 decreased p-JNK/JNK and p-STAT3/STAT3 in BLUC cells, which was reversed by HJURP overexpression (P < 0.05). The HJURP-mediated JNK/STAT3 pathway promoted BLUC cell proliferation and inhibited cell apoptosis (P < 0.05). HJURP reversed the arrested G0/G1 phase of BLUC cells by SP600125. HJURP acted as an oncogene to regulate BLUC cell proliferation, apoptosis and the cell cycle by mediating the JNK/STAT3 pathway. Therefore, HJURP targeting might be an attractive novel therapeutic target for early diagnosis and treatment in BLUC.

Determining the effects of in ovo administration of monosodium glutamate on the embryonic development of brain in chickens.

Monosodium glutamate (MSG) is a popular flavor enhancer largely used in the food industry. Although numerous studies have reported the neurotoxic effects of MSG on humans and animals, there is limited information about how it affects embryonic brain development. Thus, this study aimed to determine the effects of in ovo administered MSG on embryonic brain development in chickens. For this purpose, 410 fertilized chicken eggs were divided into 5 groups as control, distilled water, 0.12, 0.6 and 1.2 mg/g egg MSG, and injections were performed via the egg yolk. On days 15, 18, and 21 of the incubation period, brain tissue samples were taken from all embryos and chicks. The mortality rates of MSG-treated groups were significantly higher than those of the control and distilled water groups. The MSG-treated groups showed embryonic growth retardation and various structural abnormalities such as abdominal hernia, unilateral anophthalmia, hemorrhage, brain malformation, and the curling of legs and fingers. The relative embryo and body weights of the MSG-treated groups were significantly lower than those of the control group on incubation days 18 and 21. Histopathological evaluations revealed that MSG caused histopathological changes such as necrosis, neuronophagia, and gliosis in brain on incubation days 15, 18, and 21. There was a significant increase in the number of necrotic neurons in the MSG-treated groups compared to the control and distilled water groups in the hyperpallium, optic tectum and hippocampus regions. Proliferating cell nuclear antigen (PCNA) positive cells in brain were found in the hyperpallium, optic tectum, and hippocampus regions; there were more PCNA(+) immunoreactive cells in MSG-treated groups than in control and distilled water groups. In conclusion, it was determined that in ovo MSG administered could adversely affect embryonic growth and development in addition to causing necrosis in the neurons in the developing brain.