Functionalized siRNA-chitosan nanoformulations promote triple-negative breast cancer cell death via blocking the miRNA-21/AKT/ERK signaling axis: in-silico and in vitro studies.

Oncogenic microRNA (miRNA), especially miRNA-21 upregulation in triple-negative breast cancer (TNBC), suggests a new class of therapeutic targets. In this study, we aimed to create GE11 peptide-conjugated small interfering RNA-loaded chitosan nanoparticles (GE11-siRNA-CSNPs) for the targeting of EGFR overexpressed TNBC and selectively inhibit miRNA-21 expression. A variety of in-silico and in vitro cellular and molecular studies were conducted to investigate the binding affinities of specific targets used as well as the anticancer efficacies and mechanisms of GE11-siRNA-CSNPs in TNBC cells. An in-silico assessment reveals a distinct binding affinity of miRNA-21 with siRNA as well as between the extracellular domain of EGFR and synthesized peptides. Notably, the in vitro results showed that GE11-siRNA-CSNPs were revealed to have better cytotoxicity against TNBC cells. It significantly inhibits miRNA-21 expression, cell migration, and colony formation. The results also indicated that GE11-siRNA-CSNPs impeded cell cycle progression. It induces cell death by reducing the expression of the antiapoptotic gene Bcl-2 and increasing the expression of the proapoptotic genes Bax, Caspase 3, and Caspase 9. Additionally, the docking analysis and immunoblot investigations verified that GE1-siRNA-CSNPs, which specifically target TNBC cells and suppress miRNA-21, can prevent the effects of miRNA-21 on the proliferation of TNBC cells via controlling EGFR and subsequently inhibiting the PI3K/AKT and ERK1/2 signaling axis. The GE11-siRNA-CSNPs design, which specifically targets TNBC cells, offers a novel approach for the treatment of breast cancer with improved effectiveness. This study suggests that GE11-siRNA-CSNPs could be a promising candidate for further assessment as an additional strategy in the treatment of TNBC.

In vitro and in vivo anti-colorectal cancer effect of the newly synthesized sericin/propolis/fluorouracil nanoplatform through modulation of PI3K/AKT/mTOR pathway.

The present work aimed to assess the potential effect of sericin/propolis/fluorouracil nanoformula against colorectal cancer (CRC) (the fourth most common cause of cancer-related mortalities). A novel anti-cancerous formula of the synthesized sericin/propolis nanoparticles was developed and tested both in vitro (using Caco-2 cell line) and in vivo (in experimentally induced colorectal cancer animal models). The combination index of the prepared nanoformula proved that the combination between sericin/propolis nanoparticles and 5-fluorouracil demonstrated the highest synergistic effect (0.86), with dose reduction index (DRI) of the chemotherapeutic drug reaching 1.49. The mechanism of action of the prepared nanoformula revealed that it acts through the inhibition of the PI3K/AKT/mTOR signaling pathway and consequently inhibiting cancerous cells proliferation. Treatment and prophylactic studies of both sericin and propolis showed increased TBARS (Thiobarbituric Acid Reactive Substance) formation, downregulated BCL2 (B-cell lymphoma 2) and activated BAX, Caspase 9 and Caspase 3 expression. The prepared nanoformula decreased the ROS (Reactive Oxygen Species) production in vivo owing to PI3K/AKT/mTOR pathway inhibition and FOXO-1 (Forkhead Box O1) activation that resulted in autophagy/apoptosis processes stimulation. The potent anticancer effect of the prepared nanoformula was further emphasized through the in vivo histopathological studies of experimentally induced tumors. The newly formulated sericin/propolis/fluorouracil nanoparticles exhibited clear-cut cytotoxic effects toward tumor cells with provided evidence for the prophylactic effect.

AMPK-mediated autophagy is involved in the protective effect of canagliflozin in the vitamin D3 plus nicotine calcification model in rats.

Vascular calcification (VC) is a major risk factor for cardiovascular events. A mutual interplay between inflammation, oxidative stress, apoptosis, and autophagy is implicated in its development. Herein, we aimed to evaluate the potential protective effects of canagliflozin in a vitamin D3 plus nicotine (VDN) model of VC, and to explore potential mechanisms. VC was induced by VDN in adult male Wistar rats on day one. Then, rats were randomly assigned into three groups to receive canagliflozin (10 mg or 20 mg/kg/day) or its vehicle for 4 weeks. Age-matched normal rats served as a control group. After euthanization, aorta and kidneys were harvested for biochemical and histopathological evaluation of calcification. Aortic markers of oxidative stress, alkaline phosphatase (ALP) activity, runt-related transcription factor (Runx2) and bone morphogenic protein-2 (BMP-2) levels were determined. Additionally, the protein expression of autophagic markers, LC3 and p62, and adenosine monophosphate activated protein kinase (AMPK) were also assessed in aortic homogenates. Canagliflozin dose-dependently improved renal function, enhanced the antioxidant capacity of aortic tissues and reduced calcium deposition in rat aortas and kidneys. Both doses of canagliflozin attenuated ALP and osteogenic markers while augmented the expression of autophagic markers and AMPK. Histopathological examination of aortas and kidneys by H&E and Von Kossa stain further support the beneficial effect of canagliflozin. Canagliflozin could alleviate VDN-induced vascular calcification, in a dose dependent manner, via its antioxidant effect and modulation of autophagy. Further studies are needed to verify whether this effect is a member or a class effect.

Engineered Microencapsulated Lactoferrin Nanoconjugates for Oral Targeted Treatment of Colon Cancer.

Despite current progress in the development of targeted therapies for cancer treatment, there is a lack in convenient therapeutics for colorectal cancer (CRC). Lactoferrin nanoparticles (Lf NPs) are a promising drug delivery system in cancer therapy. However, numerous obstacles impede their oral delivery, including instability against stomach enzymes and premature uptake during passage through the small intestine. Microencapsulation of Lf NPs offer a great solution for these obstacles. It can protect Lf NPs and their drug payloads from degradation in the upper gastrointestinal tract (GIT), reduce burst drug release, and improve the release profile of the encapsulated NPs triggered by stimuli in the colon. Here, we developed nanoparticle-in-microparticle delivery systems (NIMDs) for the oral delivery of docetaxel (DTX) and atorvastatin (ATR). The NPs were obtained by dual conjugation of DTX and ATR into the Lf backbone, which was further microencapsulated into calcium-crosslinked microparticles using polysaccharide-protein hybrid copolymers. The NIMDs showed no detectable drug release in the upper GIT compared to NPs. Furthermore, sustained release of the NPs from the NIMDs in rat cecal content was observed. Moreover, the in vivo study demonstrated the superiority of the NIMDs over NPs in CRC treatment by suppressing p-AKT, p-ERK1/2, and NF-κB. This study provides the proof of concept for using NIMDs to enhance the effect of protein NPs in CRC treatment.

HR LC-MS/MS metabolomic profiling of Yucca aloifolia fruit and the potential neuroprotective effect on rotenone-induced Parkinson's disease in rats.

Yucca aloifolia L. fruit (Yucca or Spanish bayonet, family Asparagaceae) is recognized for its purplish red color reflecting its anthocyanin content, which has a powerful antioxidant activity. This study aimed to investigate yucca (YA) fruit extract's protective effect on Parkinson's disease (PD). In vitro study, the anti-inflammatory activity of yucca fruit extracts was explored by measuring tumor necrosis factor receptor 2 (TNF-R2) and nuclear factor kappa B (NF-KB) to choose the most effective extract. Afterward, a detailed in vivo investigation of the protective effect of the most active extract on rotenone-induced PD was performed on male albino Wister rats. First, the safety of the extract in two different doses (50 and 100 mg/kg in 0.9% saline orally) was confirmed by a toxicological study. The rats were divided into four groups: 1) normal control (NC); 2) rotenone group; and third and fourth groups received 50 and 100 mg/kg yucca extract, respectively. The neurobehavioral and locomotor activities of the rats were tested by rotarod, open field, and forced swim tests. Striatal dopamine, renal and liver functions, and oxidative stress markers were assessed. Western blot analysis of brain tissue samples was performed for p-AMPK, Wnt3a, and ß-catenin. Histopathological examination of striatal tissue samples was performed by light and electron microscopy (EM). The metabolites of the active extract were characterized using high-resolution LC-MS/MS, and the results showed the prevalence of anthocyanins, saponins, phenolics, and choline. Biochemical and histopathological tests revealed a dose-dependent improvement with oral Yucca extract. The current study suggests a possible neuroprotective effect of the acidified 50% ethanol extract (YA-C) of the edible Yucca fruit, making it a promising therapeutic target for PD.

Therapeutic effect of dithiophenolato chitosan nanocomposites against carbon tetrachloride-induced hepatotoxicity in rats.

Our previous study showed that dithiophenolate (DTP) and its chitosan nanoparticles (DTP-CSNPs) have abilities to bind with DNA helixes. So in this study, their lethal doses (LD50) and therapeutic roles against rat liver injuries induced by carbon tetrachloride (CCl4) were evaluated. The study focused on the determination of the markers of oxidative stress (OS) and apoptosis and compare the results with those of cisplatin treatment. The results revealed that LD50 values of DTP and DTP-CSNPs are 2187.5 and 1462.5 mg/kg, respectively. Treatment with DPT and DPT-CSNPs after CCl4 administration reduced liver injuries, induced by CCl4, and improved liver functions and architecture through the reduction of OS and apoptosis. Where the oxidant marker was decreased with elevations of antioxidant markers. Also, there was an elevation in Bcl-2 value, with decreases in caspase-8, Bax, and Bax/Bcl-2 ratio. DPT-CSNPs treatment gave preferable results than those treated with DPT. Moreover, DTP and DPT-CSNPs treatment gave better results than cisplatin treatment. The administration of healthy rats with low doses of DTP and DTP-CSNPs for 14 days had no effect. Otherwise, the study on HepG2 cell line showed that DTP and DPT-CSNPs inhibited cell growth by arresting cells in the G2/M phase and inducing cell death. In conclusion, DTP and DTP-CSNPs have antiapoptotic and anti-oxidative stress toward hepatotoxicity induced by CCl4. Moreover, DTP and DTP-CSNPs have anticancer activity against the HepG2 cell line. Generally, DTP-CSNPs are more effective than DTP. So, they can be used in the pharmacological fields, especially DTP-CSNPs.

Effective amelioration of hepatic inflammation and insulin response in high fat diet-fed rats via regulating AKT/mTOR signaling: Role of Lepidium sativum seed extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Obesity-induced insulin resistance and chronic inflammation appears to be the most frequent cause of diabetes and its related metabolic complications; in this way a new therapeutic approaches are needed to prevent the chronic obesity and insulin resistance. Lepidium sativum has been extensively used in traditional alternative medicine for cough, skin disease, liver disorder, diuretic, gastrointestinal problems, hair loss treatment, milk secretion during lactation as well as antioxidant, antihypertensive, anti-inflammatory, and antidiabetic activities. The hypoglycemic and hypolipidemic effect of Lepidium sativum have been observed by previous studies, but the underlying molecular mechanisms are unclear. AIM OF THE STUDY: In this study, we investigated the beneficial effect of Lepidium sativum ethanol and aqueous seed extracts on obesity, oxidative, inflammatory, and insulin sensitivity changes in the liver tissue of high fat diet (HFD)-fed rats. The bioactive constituents responsible for these activities have been identified for both extracts using HPLC and GC-MS. MATERIALS AND METHODS: Rats were fed HFD for 10 weeks. The obese rats were treated orally with the Lepidium sativum ethanol extracts (LSEE) at dose 200 and 400 mg/kg body weight (BW) and Lepidium sativum aqueous extracts (LSAE) at dose 200 mg/kg BW daily for 8 weeks. RESULTS: The findings of the present study pointed out a significant increase in the hepatic transaminases, lipid profile, leptin, and hepatic oxidative stress with decreased antioxidant capacity of HFD-fed rats. Consistent with this depiction; we determined the up-regulation of liver inflammatory markers with a significant down-regulation of insulin signaling components phospho-insulin receptor (p-IR), p-AKT, p-mammalian target of rapamycin (p-mTOR), and p-p70S6K after consumption of HFD for 10 weeks that indicates a deterioration of insulin sensitivity. Interestingly, the phytochemical screening of LSEE and LSAE exhibited positive results for phenolic, flavonoid, lipid, and some bioactive components as well as the in vitro antioxidant activity of both extracts clearly demonstrated their high antioxidant activities. Notably, LSEE and LSAE displayed a wide range of biological features including anti-obesity, anti-inflammatory, and antioxidant properties. Both extracts significantly decreased high glucose, leptin, lipid profile, liver enzymes levels, and body weight. We also found that LSEE and LSAE significantly alleviated lipid peroxidation and restored the antioxidant enzymes to normal levels. In parallel, the intracellular phosphorylation of classical markers of insulin signaling cascade p-IR/p-AKT/p-mTOR/p-p70S6K was up-regulated in the hepatic tissues of LSEE and LSAE-treated groups. CONCLUSION: This study provides evidence that LSEE and LSAE might be one promising dietary supplementation that could safely and effectively prevent the early metabolic alterations and weight gain caused by HFD further regulate the activation of insulin signaling pathway beside their powerful antioxidant and low-toxicity properties.

Oxidative stress and expression of insulin signaling proteins in the brain of diabetic rats: Role of Nigella sativa oil and antidiabetic drugs.

BACKGROUND AND OBJECTIVES: Insulin resistance of the brain is a specific form of type2-diabetes mellitus (T2DM) and the active insulin-signaling pathway plays a neuroprotective role against damaging conditions and Alzheimer's progression. The present study identifies the mediated emerging effects of the Nigella sativa oil (NSO) on the memory enhancing process, its anti-oxidative, acetylcholinestrase (AChE) inhibition, anti-brain insulin resistance and anti-amyloidogenic activities. In addition, the possible role of some anti-diabetic drugs in the neuro-protection processes and their effect in combination with NSO and/or the insulin receptor inhibitor IOMe-AG538 were investigated. METHODS: T2DM-induced rats were orally and daily administrated 2.0 ml NSO, 100 mg metformin (MT), 0.8 mg glimepiride (GI) and different combinations (100 mg MT & 2.0 ml NSO, 0.8 mg GI & 2.0 ml NSO and 2.0 ml NSO & intraperitoneal injection of 1/100 LD50 of IOMe-AG538) per kg body weight for 21 days. RESULTS: A significant increase in the brain lipid peroxidation and decrease in the antioxidant status with peripheral and central production of pro-inflammatory mediators were observed in diabetes-induced rats. The brain AChE was activated and associated with diminished brain glucose level and cholinergic function. In addition, the brain insulin resistance and the attenuated insulin signaling pathway (p-IRS/ p-AKT/p-GSK-3ß) were accompanied by an augmentation in GSK-3ß level, which in turn may contribute in the extensive alterations of Tau phosphorylation along with changes in PP2A level. Furthermore, neuronal loss and elevation in Aß-42 plaque formation were observed due to a low IDE formation and an increased expression of p53, BACE1 and APP with diminished ADAM10, SIRT1 and BDNF levels. The expression profile of AD-related miRNAs in sera and brain tissues displayed its neuro-protection role. The treatment of diabetes-induced rats with NSO and the anti-diabetic drugs alone and/or in combination have the potential to suppress the oxidative stress, the pro-inflammatory mediators and amyloidogenic pathway. Moreover, it lowers the insulin receptor inhibitory effect of IOMe-AG538 and modifies the insulin-signaling pathway. Therefore, it prevents the neurotoxicity, amyloid plaque formation and Tau hyper-phosphorylation and restores AD-related miRNA normal levels. CONCLUSION: These data suggest that NSO or its combined treatments with anti-diabetic drugs have a possible benefit as disease modifying agents for the insulin resistance in the brain through enhancing brain insulin signaling pathway.

Modulation of AP-endonuclease1 levels associated with hepatic cirrhosis in rat model treated with human umbilical cord blood mononuclear stem cells.

Oxidative stress in liver cells may contribute to the etiology of hepatic diseases, as in liver cirrhosis. AP-Endonuclease1 (APE1/Ref-1) is essential for cell protection toward oxidative stress by acting as a transcriptional regulator of pro-survival genes and as a redox sensitive protein. The aim of this study was to critically analyze the various parameters governing the success of human umbilical cord blood mononuclear stem cell-based (MNCs) therapy without the use of an immunosuppressant and to investigate for the first time the expression of APE1 during thioacetamide (TAA)-induced cirrhosis and MNCs therapy in a rat model. Umbilical cord blood samples from full-term deliveries were collected. Lethal fulminant hepatic cirrhosis in rats was induced by intraperitoneal injection of thio-acetamide. MNCs were then intrahepatically transplanted. We measured APE1 expression at mRNA and protein levels, mRNA expression of TGF-ß, α-SMA, STAP, CTGF, MMP-9 and TIMP-1 in a follow up study. Histopathological and immunohistochemical analyses were performed 10 weeks after intrahepatic injection of the cells. Transdifferentiated cells could be efficiently stained with antihuman hepatocytes. Interestingly, human hepatocyte-specific markers, human albumin, cytokeratin-18 and cytokeratin-19 mRNAs were detected in rat liver after 10 days of MNCs infusion. MNC transplanted by intrahepatic route, could engraft recipient liver, differentiated into functional hepatocytes, and rescued liver failure. Moreover up regulation of APE1 expression confirmed by marked immunohistochemical staining may be involved in MNCs-induced hepatocytes regeneration suggesting that maintaining high level of APE1 has protective effect as pro-survival signal.