High-dose Vitamin C injection ameliorates against sepsis-induced myocardial injury by anti-apoptosis, anti-inflammatory and pro-autophagy through regulating MAPK, NF-κB and PI3K/AKT/mTOR signaling pathways in rats.

AIMS: This study aimed to evaluate the effects of VC on SIMI in rats. METHODS: In this study, the survival rate of high dose VC for SIMI was evaluated within 7 days. Rats were randomly assigned to three groups: Sham group, CLP group, and high dose VC (500 mg/kg i.v.) group. The animals in each group were treated with drugs for 1 day, 3 days or 5 days, respectively. Echocardiography, myocardial enzymes and HE were used to detect cardiac function. IL-1ß, IL-6, IL-10 and TNF-α) in serum were measured using ELISA kits. Western blot was used to detect proteins related to apoptosis, inflammation, autophagy, MAPK, NF-κB and PI3K/Akt/mTOR signaling pathways. RESULTS: High dose VC improved the survival rate of SIMI within 7 days. Echocardiography, HE staining and myocardial enzymes showed that high-dose VC relieved SIMI in rats in a time-dependent manner. And compared with CLP group, high-dose VC decreased the expressions of pro-apoptotic proteins, while increased the expression of anti-apoptotic protein. And compared with CLP group, high dose VC decreased phosphorylation levels of Erk1/2, P38, JNK, NF-κB and IKK α/ß in SIMI rats. High dose VC increased the expression of the protein Beclin-1 and LC3-II/LC3-I ratio, whereas decreased the expression of P62 in SIMI rats. Finally, high dose VC attenuated phosphorylation of PI3K, AKT and mTOR compared with the CLP group. SIGNIFICANCE: Our results showed that high dose VC has a good protective effect on SIMI after continuous treatment, which may be mediated by inhibiting apoptosis and inflammatory, and promoting autophagy through regulating MAPK, NF-κB and PI3K/AKT/mTOR pathway.

Shenmai Injection Alleviates Myocardial Ferroptosis via Activating the AKT1/mTOR Pathway in Rats with Acute Myocardial Infarction.

OBJECTIVE: Acute myocardial infarction (AMI) poses a serious burden on public health. Shenmai Injection (SMI) has been reported to have a cardioprotective effect and is used clinically attributed to its targeting of ferroptosis. This study aims to explore the underlying mechanisms of SMI in treating AMI through the application of network pharmacology analysis. METHODS: This study utilized network pharmacology to identify the bioactive ingredients and potential targets of SMI in treating AMI. A rat model of AMI was created by ligating the coronary arteries of rats, and a cell model was established by subjecting H9c2 cells to oxygen-glucose deprivation (OGD) to reveal the cardioprotective effects of SMI. Western blotting was employed to measure protein expressions, while hematoxylin-eosin staining was used to observe relevant pathological changes. Enzyme linked immunosorbent assay was conducted to measure the levels of biomarkers associated with cardiac injury and oxidative stress. RESULTS: A comprehensive analysis revealed a total of 225 putative targets of SMI in the context of AMI which exerted regulatory effects on numerous pathways and targeted multiple biological processes. AKT1 was identified as a core target mediating the effects of SMI on AMI by topological analysis. In vivo experiments revealed that SMI attenuated myocardial injury, oxidative stress, and ferroptosis in rats with AMI. Furthermore, SMI was found to enhance the expression levels of p-AKT1 and p-mTOR proteins in the myocardial tissues of rats afflicted with AMI. Similar findings were also observed in H9c2 cells subjected to OGD. Of particular interest, the suppression of OGD-induced iron accumulation, oxidative stress, and ferroptosis-associated proteins by SMI in H9c2 cells was reversed upon inhibition of the AKT1/mTOR pathway via MK2206. CONCLUSION: This study revealed that SMI exerts a protective effect against myocardial injury and ferroptosis caused by AMI via the activation of the AKT1/mTOR pathway.

The Modulation of Phospho-Extracellular Signal-Regulated Kinase and Phospho-Protein Kinase B Signaling Pathways plus Activity of Macrophage-Stimulating Protein Contribute to the Protective Effect of Stachydrine on Acetaminophen-Induced Liver Injury.

Stachydrine, a prominent bioactive alkaloid derived from Leonurus heterophyllus, is a significant herb in traditional medicine. It has been noted for its anti-inflammatory and antioxidant characteristics. Consequently, we conducted a study of its hepatoprotective effect and the fundamental mechanisms involved in acetaminophen (APAP)-induced liver injury, utilizing a mouse model. Mice were intraperitoneally administered a hepatotoxic dose of APAP (300 mg/kg). Thirty minutes after APAP administration, mice were treated with different concentrations of stachydrine (0, 2.5, 5, and 10 mg/kg). Animals were sacrificed 16 h after APAP injection for serum and liver tissue assays. APAP overdose significantly elevated the serum alanine transferase levels, hepatic pro-inflammatory cytokines, malondialdehyde activity, phospho-extracellular signal-regulated kinase (ERK), phospho-protein kinase B (AKT), and macrophage-stimulating protein expression. Stachydrine treatment significantly decreased these parameters in mice with APAP-induced liver damage. Our results suggest that stachydrine may be a promising beneficial target in the prevention of APAP-induced liver damage through attenuation of the inflammatory response, inhibition of the ERK and AKT pathways, and expression of macrophage-stimulating proteins.

San Pian decoction can treat nitroglycerin-induced migraine in rats by inhibiting the PI3K/AKT and MAPK signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: San Pian decoction (SPD), a traditional Chinese medicine preparation composed of eight herbs, has been reported to alleviate migraine. However, its active ingredients and the potential mechanism of action remains unclear. The purpose of this study was to comprehensively analyze SPD for the treatment of chronic migraine based on pharmacological direction and to identify the active ingredients and pharmacological mechanism of SPD in the treatment of migraine. MATERIALS AND METHODS: The active components in SPD were identified by AB SCIEX quadrupole time-of-flight mass spectrometer, and the prediction targets and pharmacological networks related to migraine were constructed. The mechanism of SPD in treating migraine was studied through network pharmacology, which was further verified using pharmacological experiments. RESULTS: A total of 489 targets of 26 compounds were identified. Based on Venn analysis, we found 117 intersection targets between SPD and migraine, that is, these targets were related to the treatment of migraine. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the treatment of migraine using SPD was related to the PI3K/AKT and MAPK signaling pathways. The effect of SPD on migraine was verified by measuring the levels of the inflammatory factors, nitric oxide (NO), interleukin (IL-6), endothelin (ET),5-hydroxytryptamine(5-HT), indoleamine 2,3-dioxygenas (IDO), tumor necrosis factor (TNF-α) and calcitonin gene-related peptide (CGRP). Lastly, real-time polymerase chain reaction and western blotting were used to verify gene and protein expression in the PI3K/AKT and MAPK signaling pathways. Expression of the genes P38, JNK, ERK, PI3K and AKT, and the protein expression of p-P38, p-JNK, p-ERK, p-AKT and p-PI3K were significantly downregulated. Our findings indicated that SPD could prevent inflammation by regulating the inflammatory cytokines and key genes and proteins in the PI3K/AKT and MAPK signaling pathways to treat migraine. CONCLUSION: Our findings reveal that SPD could treat nitroglycerin-induced migraine by regulating p-AKT, p-pI3k, p-p38, p-ERK, p-JNK, IL-6, and TNF-α inflammatory factors in the PI3K/AKT and MAPK signaling pathways.

Taraxasterol attenuates melanoma progression via inactivation of reactive oxygen species-mediated PI3K/Akt signaling pathway.

Background: Taraxasterol (TX), a pentacyclic triterpene, is one of the main active constituents isolated from Taraxacum officinale. A growing number of studies have reported that TX exhibits a wide range of biological activities such as anti-oxidative, anti-inflammatory, and neuro-protective effects. Recently, TX has been demonstrated to be a potential drug candidate for treatment of some types of cancers. However, the specific role of TX in melanoma remains unclear.Purpose: In this study, we aimed at exploration of the effect of TX on melanoma cell viability, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT) as well as the underlying mechanisms.Research design: A375 and SK-MEL-28 cells were treated with various concentrations of TX for different times. Cell viability was measured using CCK-8 assay. Cell apoptosis was determined by flow cytometry. Transwell assays were performed to measure cell migration and invasion. The expression of E-cadherin, α-catenin, N-cadherin, vimentin, p-PI3K, PI3K, p-Akt and Akt was detected using western blot.Results: The study showed that TX induced A375 and SK-MEL-28 cell apoptosis. Furthermore, exposure to TX inhibited A375 and SK-MEL-28 cell migration and invasion. Besides, the EMT process was reversed in A375 and SK-MEL-28 cells after TX treatment. We also observed that TX reduced the protein expression of p-PI3K and p-Akt; thus, inhibiting activity of the PI3K/Akt pathway in A375 and SK-MEL-28 cells. In addition, TX treatment increased the levels of reactive oxygen species (ROS) in A375 and SK-MEL-28 cells, and treatment with the ROS scavenger NAC significantly rescued TX-induced down-regulation of p-PI3K and p-Akt in A375 and SK-MEL-28 cells.Conclusions: In conclusion, our study demonstrated that TX induced ROS accumulation followed by inactivation of the PI3K/Akt pathway and subsequently attenuated melanoma progression, suggesting that TX may be a potential candidate for treatment of melanoma.

Synergistic effect of Aloe vera flower and Aloe gel on cutaneous wound healing targeting MFAP4 and its associated signaling pathway: In-vitro study.

ETHNOPHARMACOLOGICAL RELEVANCE: Aloe vera (L.) Burm. f. (Liliaceae family) is a well-known traditional medicinal plant, that has been used to treat a variety of illnesses, for decades ranging from cancer to skin disorders including wounds. It has been included in the traditional and herbal healthcare systems of many cultures around the world, as well as the pharmacopeia of different countries. Several in vitro and in vivo studies have also confirmed its potential antioxidant, anti-inflammatory, and wound-healing activities, etc. in the consistency of its historical and traditional uses. However, most studies to date are based on the A. vera gel and latex including its wound-healing effects. Very few studies have been focused on its flower, and rarely with its effects on cutaneous wound healing and its molecular mechanism. AIM OF THE STUDY: To the best of our knowledge, this is the first study to report on the synergistic effect of the A. vera flower (AVF) and Aloe gel (PAG) on cutaneous wound-healing, as well as revealing its molecular mechanism targeting microfibril-associated glycoprotein 4 (MFAP4) and its associated signaling pathway. METHODS: To investigate the synergistic effect of A. vera flower and Aloe gel in cutaneous wound healing, cell viability, and cell migration, as well proliferation assay was performed. This was followed by quantitative real-time polymerase chain reaction and Western blot analyses in wounded conditions to check the effects of this mixture on protein and mRNA levels in normal human dermal fibroblast (NHDF) cells. Moreover, small interfering RNA (siRNA) -mediated knockdown of MFAP4 in NHDF cells was performed followed by migration assay and cell cycle analysis, to confirm its role in cutaneous wound healing. Additionally, HaCaT cells were included in this study to evaluate its migratory and anti-inflammatory effects. RESULTS: Based on our obtained results, the PAG and AVF mixture synergistically induced the proliferation, migration, and especially ECM formation of NHDF cells by enhancing the expression of MFAP4. Other extracellular components associated with MFAP4 signaling pathway, such as fibrillin, collagen, elastin, TGF ß, and α-SMA, also increased at both the protein and mRNA levels. Subsequently, this mixture initiated the phosphorylation of the extracellular signal-regulated kinase (ERK) and AKT signaling pathways, and the S-phase of the cell cycle was also slightly modified. Also, the mixture induced the migration of HaCaT cells along with the suppression of inflammatory cytokines. Moreover, the siRNA-mediated knockdown highlighted the crucial role of MFAP4 in cutaneous wound healing in NHDF cells. CONCLUSION: This study showed that the mixture of PAG and AVF has significant wound healing effects targeting MFAP4 and its associated signaling pathway. Additionally, MFAP4 was recognized as a new potential biomarker of wound healing, which can be confirmed by further in vivo studies.

Norcantharidin counteracts acquired everolimus resistance in renal cell carcinoma by dual inhibition of mammalian target of rapamycin complex 1 and complex 2 pathways in Vitro.

Everolimus, an oral mammalian target of rapamycin complex 1 (mTORC1) inhibitor, presents a therapeutic option in metastatic renal cell carcinoma (RCC) patients who were intolerant to, or previously failed, immune- and vascular endothelial growth factor-targeted therapies. However, the onset of drug resistance limits its clinical use. One possible mechanism underpinning the resistance is that inhibiting mTORC1 by everolimus results in mTORC2-dependent activation of v-Akt murine thymoma viral oncogene (AKT) and upregulation of hypoxia-inducible transcription factors (HIF). Norcantharidin (NCTD) is a demethylated derivative of cantharidin with antitumor properties which is an active ingredient of the traditional Chinese medicine Mylabris. In this study, everolimus-resistant RCC cells (786-O-R) obtained by chronic everolimus treatment revealed higher level of HIF2α and over-activated mTORC2 pathway and NCTD inhibits cell proliferation in both everolimus-resistant and -sensitive RCC cells by arresting cell cycle in G0/G1 phase and reducing cell cycle-related proteins of C-Myc and cyclin D. Furthermore, NCTD shows synergistic anticancer effects combined with everolimus in everolimus-resistant 786-O-R cells. Mechanically, NCTD repressed both mTORC1 and mTORC2 signaling pathways as well as downstream molecular signaling pathways, such as p-4EBP1, p-AKT, HIF1α and HIF2α. Our findings provide sound evidence that combination of NCTD and everolimus is a potential therapeutic strategy for treating RCC and overcoming everolimus resistance by dual inhibition of mTORC1 and mTORC2.

Jolkinolide B sensitizes bladder cancer to mTOR inhibitors via dual inhibition of Akt signaling and autophagy.

The monotherapy of mTOR inhibitors (mTORi) in cancer clinical practice has achieved limited success due to the concomitant activation of compensatory pathways, such as Akt signaling and cytoprotective autophagy. Thus, the combination of mTORi and the inhibitors of these pro-survival pathways has been considered a promising therapeutic strategy. Herein, we report the synergistic effects of a natural anti-cancer agent Jolkinolide B (JB) and mTORi (temsirolimus, rapamycin, and everolimus) for the effective treatment of bladder cancer. A mechanistic study revealed that JB induced a dual inhibition of Akt feedback activation and cytoprotective autophagy, potentiating the anti-proliferative efficacy of mTORi in both PTEN-deficient and cisplatin-resistant bladder cancer cells. Meanwhile, mTORi augmented the pro-apoptotic and pro-paraptotic effects of JB by reinforcing JB-activated endoplasmic reticulum stress and MAPK pathways. These synergistic mechanisms were related to cellular reactive oxygen species accumulation. Our study suggests that dual inhibition of Akt feedback activation and cytoprotective autophagy is an effective strategy in mTORi-based therapy, and JB + mTORi combination associated with multiple anti-cancer mechanisms and good tolerance in mouse models may serve as a promising treatment for bladder cancer.

Activating BK channels ameliorates vascular smooth muscle calcification through Akt signaling.

Vascular calcification (VC) is characterized by pathological depositions of calcium and phosphate in the arteries and veins via an active cell-regulated process, in which vascular smooth muscle cells (VSMCs) transform into osteoblast/chondrocyte-like cells as in bone formation. VC is associated with significant morbidity and mortality in chronic kidney disease (CKD) and cardiovascular disease, but the underlying mechanisms remain unclear. In this study we investigated the role of large-conductance calcium-activated potassium (BK) channels in 3 experimental VC models. VC was induced in vascular smooth muscle cells (VSMCs) by ß-glycerophosphate (ß-GP), or in rats by subtotal nephrectomy, or in mice by high-dosage vitamin D3. We showed that the expression of BK channels in the artery of CKD rats with VC and in ß-GP-treated VSMCs was significantly decreased, which was functionally confirmed by patch-clamp recording. In ß-GP-treated VSMCs, BK channel opener NS1619 (20 µM) significantly alleviated VC by decreasing calcium content and alkaline phosphatase activity. Furthermore, NS1619 decreased mRNA expression of ostoegenic genes OCN and OPN, as well as Runx2 (a key transcription factor involved in preosteoblast to osteoblast differentiation), and increased the expression of α-SMA protein, whereas BK channel inhibitor paxilline (10 µM) caused the opposite effects. In primary cultured VSMCs from BK-/- mice, BK deficiency aggravated calcification as did BK channel inhibitor in normal VSMCs. Moreover, calcification was more severe in thoracic aorta rings of BK-/- mice than in those of wild-type littermates. Administration of BK channel activator BMS191011 (10 mg· kg-1 ·d-1) in high-dosage vitamin D3-treated mice significantly ameliorated calcification. Finally, co-treatment with Akt inhibitor MK2206 (1 µM) or FoxO1 inhibitor AS1842856 (3 µM) in calcified VSMCs abrogated the effects of BK channel opener NS1619. Taken together, activation of BK channels ameliorates VC via Akt/FoxO1 signaling pathways. Strategies to activate BK channels and/or enhance BK channel expression may offer therapeutic avenues to control VC.

Network pharmacology and experimental investigation of Rhizoma polygonati extract targeted kinase with herbzyme activity for potent drug delivery.

Rhizoma polygonati (Huangjing, RP) has been used for a long history with many chemical components in inducing anti-cancer, anti-aging, anti-diabetes, anti-fatigue, and more prevention of diseases or acts as nutrition sources in food. Here we investigated RP extract combination with kinase inhibitors in anti-cell growth and blockade in pathways targeting kinases. Experimental investigation and network pharmacology analysis were applied to test the potent kinase-mediated signaling. Herbzyme activity was determined by substrate with optical density measurement. Extract of processed RP inhibits cell growth in a much greater manner than alone when applied in combination with inhibitors of mTOR or EGFR. Moreover, processing methods of RP from Mount Tai (RP-Mount Tai) play essential roles in herbzyme activity of phosphatase suggesting the interface is also essential, in addition to the chemical component. The network pharmacology analysis showed the chemical component and target networks involving AKT and mTOR, which is consistent with experimental validation. Finally, EGFR inhibitor could be associated with nano-extract of RP-Mount Tai but not significantly affects the phosphatase herbzyme activity in vitro. Thus the processed extract of RP-Mount Tai may play a dual role in the inhibition of cell proliferation signaling by both chemical component and nanoscale herbzyme of phosphatase activity to inhibit kinases including mTOR/AKT in potent drug delivery of kinase inhibitors.

Study on the Mechanism of Prunella Vulgaris L on Diabetes Mellitus Complicated with Hypertension Based on Network Pharmacology and Molecular Docking Analyses.

The role of traditional Chinese medicine Prunella vulagaris L in the treatment of tumors and inflammation has been widely confirmed. We found that some signaling pathways of Prunella vulgaris L action can also regulate diabetes and hypertension, so we decided to study the active ingredients, potential targets and signaling pathway of Prunrlla vulgaris L, and explore the "multi-target, multi-pathway" molecular mechanism of Prunella vulgaris L on diabetes mellitus complicated with hypertension(DH). Methods. Based on TCMSP(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform) and CNKI(China National Knowledge Infrastructure), the components and action targets related to Prunella vulgaris L were screened. The OMIM(Online Mendelian Inheritance in Man) and GeneCards (The human gene database) were used to search for targets related to DH. The "gene - drug - disease" relationship map was drawn by Cytoscape_v3.7.2 plug-in. The target was amplified by the STRING platform, and the "protein - protein" interaction relationship (PPI) network of the interacting target was obtained by the STRING online analysis platform and the Cytoscape_v3.7.2 plug-in. Finally, GO enrichment analysis and KEGG pathway enrichment analysis were conducted on David and Metascape platform to study the co-acting targets. Results. 11 active components, 41 key targets and 16 significant signaling pathways were identified from Prunella vulgaris L. The main active components of Prunella vulgaris L against DH were quercetin and kaumferol, etc, and potential action targets were IL-6 and INS, etc and signaling pathways were AGE-RAGE signaling pathway, TNF signaling pathway, MAPK signaling pathway, PI3K-AKT signaling pathway, etc. It involves in biological processes such as cell proliferation, apoptosis and inflammatory response. Conclusions. The main molecular mechanism of Prunella vulgaris L against DH is that sterols and flavonoids play an active role by affecting TNF signaling pathway, AGE-RAGE signaling pathway, MAPK pathway, PI3K-Akt pathway related targets such as IL-6 and INS.

Berberine exerts its antineoplastic effects by reversing the Warburg effect via downregulation of the Akt/mTOR/GLUT1 signaling pathway.

Glucose transporter 1 (GLUT1) plays a primary role in the glucose metabolism of cancer cells. However, to the best of our knowledge, there are currently no anticancer drugs that inhibit GLUT1 function. The present study aimed to investigate the antineoplastic activity of berberine (BBR), the main active ingredient in numerous Traditional Chinese medicinal herbs, on HepG2 and MCF7 cells. The results of Cell Counting Kit­8 assay, colony formation assay and flow cytometry revealed that BBR effectively inhibited the proliferation of tumor cells, and induced G2/M cell cycle arrest and apoptosis. Notably, the results of luminescence ATP detection assay and glucose uptake assay showed that BBR also significantly inhibited ATP synthesis and markedly decreased the glucose uptake ability, which suggested that the antitumor effect of BBR may occur via reversal of the Warburg effect. In addition, the results of reverse transcription­quantitative PCR, western blotting and immunofluorescence staining indicated that BBR downregulated the protein expression levels of GLUT1, maintained the cytoplasmic internalization of GLUT1 and suppressed the Akt/mTOR signaling pathway in both HepG2 and MCF7 cell lines. Augmentation of Akt phosphorylation levels by the Akt activator, SC79, abolished the BBR­induced decrease in ATP synthesis, glucose uptake, GLUT1 expression and cell proliferation, and reversed the proapoptotic effect of BBR. These findings indicated that the antineoplastic effect of BBR may involve the reversal of the Warburg effect by downregulating the Akt/mTOR/GLUT1 signaling pathway. Furthermore, the results of the co­immunoprecipitation assay demonstrated that BBR increased the interaction between ubiquitin conjugating enzyme E2 I (Ubc9) and GLUT1, which suggested that Ubc9 may mediate the proteasomal degradation of GLUT1. On the other hand, BBR decreased the interaction between Gα­interacting protein­interacting protein at the C­terminus (GIPC) and GLUT1, which suggested that the retention of GLUT1 in the cytoplasm may be achieved by inhibiting the interaction between GLUT1 and GIPC, thereby suppressing the glucose transporter function of GLUT1. The results of the present study provided a theoretical basis for the application of the Traditional Chinese medicine component, BBR, for cancer treatment.

Identifying the molecular targets and mechanisms of xuebijing injection for the treatment of COVID-19 via network parmacology and molecular docking.

Xuebijing Injection have been found to improve the clinical symptoms of COVID-19 and alleviate disease severity, but the mechanisms are currently unclear. This study aimed to investigate the potential molecular targets and mechanisms of the Xuebijing injection in treating COVID-19 via network pharmacology and molecular docking analysis. The main active ingredients and therapeutic targets of the Xuebijing injection, and the pathogenic targets of COVID-19 were screened using the TCMSP, UniProt, and GeneCard databases. According to the 'Drug-Ingredients-Targets-Disease' network built by STRING and Cytoscape, AKT1 was identified as the core target, and baicalein, luteolin, and quercetin were identified as the active ingredients of the Xuebijing injection in connection with AKT1. R language was used for enrichment analysis that predict the mechanisms by which the Xuebijing injection may inhibit lipopolysaccharide-mediated inflammatory response, modulate NOS activity, and regulate the TNF signal pathway by affecting the role of AKT1. Based on the results of network pharmacology, a molecular docking was performed with AKT1 and the three active ingredients, the results indicated that all three active ingredients could stably bind with AKT1. These findings identify potential molecular mechanisms by which Xuebijing Injection inhibit COVID-19 by acting on AKT1.

Methyl-Donors Can Induce Apoptosis and Attenuate Both the Akt and the Erk1/2 Mediated Proliferation Pathways in Breast and Lung Cancer Cell Lines.

Dietary methyl-donors play important roles in physiological processes catalyzed by B vitamins as coenzymes, and are used for complementary support in oncotherapy. Our hypothesis was that methyl-donors can not only assist in tolerating cancer treatment but may also directly interfere with tumor growth and proliferation. Therefore, we investigated the proposed cancer inhibitory effects of methyl-donors (in a mixture of L-methionine, choline chloride, folic acid, and vitamin B12) on MCF7 and T47D breast cancer as well as A549 and H1650 lung cancer cell lines. Indeed, methyl-donor treatment significantly reduced the proliferation in all cell lines, possibly through the downregulation of MAPK/ERK and AKT signaling. These were accompanied by the upregulation of the pro-apoptotic Bak and Bax, both in MCF7 and H1650 cells, at reduced anti-apoptotic Mcl-1 and Bcl-2 levels in MCF7 and H1650 cells, respectively. The treatment-induced downregulation of p-p53(Thr55) was likely to contribute to protecting the nuclear localization and apoptosis inducing functions of p53. The presented features are known to improve the sensitivity of cancer therapy. Therefore, these data support the hypothesis, i.e., that methyl-donors may promote apoptotic signaling by protecting p53 functions through downregulating both the MAPK/ERK and the AKT pathways both in breast and lung adenocarcinoma cell lines. Our results can emphasize the importance and benefits of the appropriate dietary supports in cancer treatments. However, further studies are required to confirm these effects without any adverse outcome in clinical settings.

Phyllanthin prevents diethylnitrosamine (DEN) induced liver carcinogenesis in rats and induces apoptotic cell death in HepG2 cells.

Liver cancer is a critical clinical condition with augmented malignancy, rapid progression, and poor prognosis. Liver cancer often initiates as fibrosis, develops as cirrhosis, and results in cancer. For centuries, medicinal plants have been incorporated in various liver-associated complications, and recently, research has recognized that many bioactive compounds from medicinal plants may interact with targets related to liver disorders. Phyllanthin from the Phyllanthus species is one such compound extensively used by folklore practitioners for various health benefits. However, most practices continue to be unrecognized scientifically. Hence, in this work, we investigated the protective role of phyllanthin on diethylnitrosamine (DEN) induced liver carcinoma in Wistar Albino rats and the anti-tumor potential on human hepatocellular carcinoma (HCC) HepG2 cells. The DEN-challenged liver cancer in experimental rats caused increased liver weight, 8-OHD, hepatic tissue injury marker, lipid peroxidation, and tumor markers levels. Remarkably, phyllanthin counteracted the DEN effect by ameliorating all the liver function enzymes, oxidative DNA damage, and tumor-specific markers by enhanced anti-oxidant capacity and induced caspase-dependent apoptosis through the mTOR/ PI3K signaling pathway. MTT assay demonstrated that phyllanthin inhibited the HepG2 cell growth in a dose-dependent manner. Fascinatingly, phyllanthin did not demonstrate any substantial effect on the normal cell line, HL7702. In addition, HepG2 cells were found in the late apoptotic stage upon treatment with phyllanthin as depicted by acridine orange/ethidium bromide staining. Overall, this work offers scientific justification that phyllanthin can be claimed to be a safe candidate with potential chemotherapeutic activity against HCC.

Metformin Prevents Follicular Atresia in Aging Laying Chickens through Activation of PI3K/AKT and Calcium Signaling Pathways.

Increased follicular atresia occurs with aging and results in reduced fecundity in laying chickens. Therefore, relieving follicular atresia of aging poultry is a crucial measure to maintain sustained high laying performance. As an antiaging agent, metformin was reported to play important roles in preventing aging in diverse animals. In this study, the physiological state of the prehierarchical follicles in the peak-laying hens (D280) and aged hens (D580) was compared, followed with exploration for the possible capacity of metformin in delaying atresia of the prehierarchical follicles in the aged D580 hens. Results showed that the capacity of yolk deposition within follicles declined with aging, and the point of endoplasmic reticulum- (ER-) mitochondrion contact decreased in the ultrastructure of the follicular cells. Meanwhile, the expression of apoptosis signaling genes was increased in the atretic small white follicles. Subsequently, the H2O2-induced follicular atresia model was established to evaluate the enhancing capacity of metformin on yolk deposition and inhibition of apoptosis in the atretic small white follicles. Metformin inhibited apoptosis through regulating cooperation of the mitochondrion-associated ER membranes and the insulin (PI3K/AKT) signaling pathway. Furthermore, metformin regulated calcium ion homeostasis to relieve ER-stress and inhibited release of mitochondrion apoptosis factors (BAD and caspase). Additionally, metformin activated PI3K/AKT that suppressed activation of BAD (downstream of the insulin signaling pathway) in the atretic follicles. Further, serum estrogen level and liver estrogen receptor-α expression were increased after dietary metformin supplementation in D580 hens. These results indicated that administration of dietary metformin activated the PI3K/AKT and calcium signaling pathway and enhanced yolk deposition to prevent chicken follicular atresia.

Network pharmacology and molecular docking analyses on Lianhua Qingwen capsule indicate Akt1 is a potential target to treat and prevent COVID-19.

OBJECTIVES: Coronavirus disease 2019 (COVID-19) is rapidly spreading worldwide. Lianhua Qingwen capsule (LQC) has shown therapeutic effects in patients with COVID-19. This study is aimed to discover its molecular mechanism and provide potential drug targets. MATERIALS AND METHODS: An LQC target and COVID-19-related gene set was established using the Traditional Chinese Medicine Systems Pharmacology database and seven disease-gene databases. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network were performed to discover the potential mechanism. Molecular docking was performed to visualize the patterns of interactions between the effective molecule and targeted protein. RESULTS: A gene set of 65 genes was generated. We then constructed a compound-target network that contained 234 nodes of active compounds and 916 edges of compound-target pairs. The GO and KEGG indicated that LQC can act by regulating immune response, apoptosis and virus infection. PPI network and subnetworks identified nine hub genes. The molecular docking was conducted on the most significant gene Akt1, which is involved in lung injury, lung fibrogenesis and virus infection. Six active compounds of LQC can enter the active pocket of Akt1, namely beta-carotene, kaempferol, luteolin, naringenin, quercetin and wogonin, thereby exerting potential therapeutic effects in COVID-19. CONCLUSIONS: The network pharmacological strategy integrates molecular docking to unravel the molecular mechanism of LQC. Akt1 is a promising drug target to reduce tissue damage and help eliminate virus infection.

Piperlongumine inhibits head and neck squamous cell carcinoma proliferation by docking to Akt.

Piperlongumine (PL) is a biologically active alkaloid isolated from the long pepper roots and widely used as a traditional medicine in Ayurvedic medicine. However, the mechanism of PL's effect on head and neck squamous cell carcinoma (HNSCC) is not well understood. We performed cell experiments to confirm PL's inhibitory effect on HNSCC and employing cisplatin as positive control. Next, we conducted bioinformatics to predict PL's potential targets and verified by western blotting. Molecular docking, Biacore experiment and kinase activity assays were applied to elucidate the mechanism by which PL inhibited target activity. In vivo efficacy was verified by xenotransplantation and immunohistochemistry. PL inhibited proliferation, promoted late apoptosis, arrested cell cycle and inhibited DNA replication of the HEp-2 and FaDu cell lines. Employing bioinformatics, we found that PL's target was Akt and PL attenuated Akt phosphorylation. We found from molecular docking, Biacore experiment and kinase activity assay that PL inhibited Akt activation by docking to Akt to restrain its activity. In addition, PL significantly inhibited the growth of xenograft tumors by down regulating the expression of p-Akt in vivo. This study provides new insights into the molecular functions of PL and indicate its potential as a therapeutic agent for HNSCC.

The Chinese Herb Codonopsis pilosula Isolate Isorhapontigenin protects against oxidative stress injury by inhibiting the activation of PI3K/Akt signaling pathway.

We investigated the effects of the Chinese herb Codonopsis pilosula isolate isorhapontigenin on antioxidant factor and the PI3K/Serine/Akt signaling pathway in Parkinson's disease. This research was, therefore, carried out to explore a possible protective mechanism of isorhamnetin in Parkinson's disease. The results support that isorhapontigenin could effectively inhibit isorhapontigenin restored myeloperoxidase + induced reduction of antioxidant levels. Also, 1-Methyl-4-phenylpyridine up-regulated the expression of phosphorylated-Akt, phosphorylated-PI3K, and phosphorylated mammalian target of rapamycin, while isorhapontigenin inhibited the expression of phosphorylated-Akt, phosphorylated-PI3K, and phosphorylated- mammalian target of rapamycin. Furthermore, LY294002 improved the antioxidant effect of isorhapontigenin in PC12 cells, and insulin-like growth factor 1 inhibited the antioxidant effect of isorhapontigenin in PC12 cells. Our results support the finding that isorhamnetin enhanced the antioxidant effect induced by 1-Methyl-4-phenylpyridine in PC12 cells by suppressing the activation of the PI3K/Akt signaling pathway.

Superior hypoglycemic activity of mulberry lacking monosaccharides is accompanied by better activation of the PI3K/Akt and AMPK signaling pathways.

Mulberry has been used as a functional food to treat type 2 diabetes mellitus (T2DM). However, it contains relatively high levels of fructose and glucose, which are not suitable for excess consumption by diabetic patients. In this study we used microbial fermentation to remove fructose and glucose from mulberry fruit, and then determined the effects on glycemia, the phosphatidylinositol 3-hydroxykinase/Akt (PI3K/Akt) and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways and their downstream effectors in T2DM mice. After 5 weeks of administration, fermented mulberry (FM) significantly reduced fasting blood glucose, and also improved insulin sensitivity and glucose tolerance, more effectively than unfermented mulberry (MP). Moreover, compared with MP, FM had a more marked effect on the protein expression of intermediates in the PI3K/Akt and AMPK signaling pathways and their effectors: insulin receptor, phosphorylated Akt (Ser 308), phosphorylated glycogen synthase kinase-3ß (Ser 9), glycogen synthetase, phosphorylated forkhead transcription factor 1 (Ser 256), pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1, 6-bisphosphatase, glucose-6-phosphatase, lipoprotein lipase, and phosphorylated AMPK (Thr 172), glucose transporter 4 and pyruvate kinase. These findings indicate that mulberry fruit modified to remove fructose and glucose may be more promising than whole mulberry as a treatment for diabetes.