A Study of the Protective Effect of Bushen Huoxue Prescription on Cerebral Microvascular Endothelia Based on Proteomics and Bioinformatics

Diabetic cognitive dysfunction is a serious complication of type 2 diabetes mellitus (T2DM), which can cause neurological and microvascular damage in the brain. At present, there is no effective treatment for this complication. Bushen Huoxue prescription (BSHX) is a newly formulated compound Chinese medicine containing 7 components. Previous research indicated that BSHX was neuroprotective against advanced glycosylation end product (AGE)-induced PC12 cell insult; however, the effect of BSHX on AGE-induced cerebral microvascular endothelia injury has not been studied. In the current research, we investigated the protective effects of BSHX on AGE-induced injury in bEnd.3 cells. Our findings revealed that BSHX could effectively protect bEnd.3 cells from apoptosis. Moreover, we analyzed the network regulation effect of BSHX on AGE-induced bEnd.3 cells injury at the proteomic level. The LC-MS/MS-based shotgun proteomics analysis showed BSHX negatively regulated multiple AGE-elicited proteins. Bioinformatics analysis revealed these differential proteins were involved in multiple processes, such as Foxo signaling pathway. Further molecular biology analysis confirmed that BSHX could downregulate the expression of FoxO1/3 protein and inhibit its nuclear transfer and inhibit the expression of downstream apoptotic protein Bim and the activation of caspase, so as to play a protective role in AGE-induced bEnd.3 injury. Taken together, these findings demonstrated the role of BSHX in the management of diabetic cerebral microangiopathy and provide some insights into the proteomics-guided pharmacological mechanism study of traditional Chinese Medicine.

Quercetin Attenuates Podocyte Apoptosis of Diabetic Nephropathy Through Targeting EGFR Signaling

Podocytes injury is one of the leading causes of proteinuria in patients with diabetic nephropathy (DN), and is accompanied by podocytes apoptosis and the reduction of podocyte markers such as synaptopodin and nephrin. Therefore, attenuation of podocyte apoptosis is considered as an effective strategy to prevent the proteinuria in DN. In this study, we evaluated the anti-podocyte-apoptosis effect of quercetin which is a flavonol compound possessing an important role in prevention and treatment of DN and verified the effect by using db/db mice and high glucose (HG)-induced mouse podocytes (MPs). The results show that administration of quercetin attenuated the level of podocyte apoptosis by decreasing the expression of pro-apoptotic protein Bax, cleaved caspase 3 and increasing the expression of anti-apoptotic protein Bcl-2 in the db/db mice and HG-induced MPs. Furthermore, epidermal growth factor receptor (EGFR) was predicted to be the potential physiological target of quercetin by network pharmacology. In vitro and vivo experiments confirmed that quercetin inhibited activation of the EGFR signaling pathway by decreasing phosphorylation of EGFR and ERK1/2. Taken together, this study demonstrates that quercetin attenuated podocyte apoptosis through inhibiting EGFR signaling pathway, which provided a novel approach for further research of the mechanism of quercetin in the treatment of DN.

Modulating effect of growth hormone on the functional state of cultured cells from hen preovulatory follicles

Somatotropic hormone (STH) is an important positive modulator of ovarian function in mammals. Local production of STH and the expression of the corresponding specific receptors were also detected in hen ovarian follicles, which indicates the participation of this hormone in the endocrine/paracrine control of folliculogenesis in birds. Nevertheless, the role of STH in the regulation of growth of avian follicles at the final stage of maturation is still not clear.Objective: To study in vitro the effect of STH on the proliferative activity and apoptotic changes of granulosa and theca cells from preovulatory follicles of domestic hens.Materials and methods. Young laying hens aged 34-35 weeks with a long clutch were used in the experiments. Granulosa and theca cells were isolated from the largest yellow follicle in the hierarchy (F1). The cells were cultured in a medium containing 10% fetal bovine serum until a monolayer was formed, and then for 24 h in the medium without serum in the absence (control) or in the presence of STH at various concentrations (1-100 ng/ml). The proliferative activity and apoptotic changes in the cells were assessed by immunocytochemical assay, based on the expression level of proliferating cell nuclear antigen PCNA and pro-apoptotic protein Bax, respectively.Results. The proportion of PCNA-positive granulosa cells increased 1.3-1.8 times (P<0.01-0.05) as compared to control with increasing the content of STH in the medium to 10-100 ng/ml. Furthermore, within this concentration range, the studied hormone reduced 1.2-1.6 times (P<0.05) the relative number of granulosa cells with the positive reaction to Bax. The sensitivity of theca cells to the growth-stimulating effect of STH was lower than that of granulosa cells. Such the effect of STH led to an increase in the proportion of PCNA-positive thecal cells by 1.2-1.3 times (P<0.05) and was detected only at concentrations of 25 and 100 ng/ml. Meanwhile, STH (25-100 ng/ml) increased 1.3 times (P<0.05) the level of Bax expression in theca cells.Conclusions. The results of the present study indicate the stimulating effect of STH in vitro on the proliferative activity of granulosa and theca cells from the most mature hen preovulatory follicle. In addition, STH is able to reduce the expression of the pro-apoptotic protein Bax in granulosa cells and increase this expression in thecal cells. Thus, the data obtained indicate the possible participation of STH in the regulation of growth and development of follicles at the final stage of maturation during the period of maximum egg-laying intensity in laying hens.

Insight into Analysis of Essential Oil from Anisosciadium lanatum Boiss.—Chemical Composition, Molecular Docking, and Mitigation of Hepg2 Cancer Cells through Apoptotic Markers

Essential oils have been used in various traditional healing systems since ancient times worldwide, due to their diverse biological activities. Several studies have demonstrated their plethora of biological activities—including anti-cancer activity—in a number of cell lines. Anisosciadium lanatum Boiss. is a perennial aromatic herb. Traditionally, it is an edible safe herb with few studies exploring its importance. The current study aims to investigate the chemical composition of essential oil isolated from Anisosciadium lanatum using GC-MS, as well as report its anti-cancer potential and its mechanistic effect on HepG2 liver cancer cell lines, and conduct molecular docking studies. To achieve this, the essential oil was isolated using a Clevenger apparatus and analyzed using GC-MS. The cell viability of HepG2 liver cancer and normal fibroblast NIH-3T3 cell lines was assessed by MTT cytotoxicity assay. The effects of the essential oil on cell migration and invasion were assessed using wound healing and matrigel assays, respectively. The effect of the essential oil on migration and apoptotic-regulating mRNA and proteins was quantified using quantitative real-time PCR and Western blot techniques, respectively. Finally, computational docking tools were used to analyze in silico binding of major constituents from the essential oil against apoptotic and migration markers. A total of 38 components were identified and quantified. The essential oil demonstrated regulation of cell proliferation and cell viability in HepG2 liver cancer cells at a sub-lethal dose of 10 to 25 μg/mL, and expressed reductions of migration and invasion. The treatment with essential oil indicated mitigation of cancer activity by aborting the mRNA of pro-apoptotic markers such as BCL-2, CASPASE-3, CYP-1A1, and NFκB. The algorithm-based binding studies demonstrated that eucalyptol, nerol, camphor, and linalool have potent binding towards the anti-apoptotic protein BCL-2. On the other hand, camphor and eucalyptol showed potent binding towards the pro-apoptotic protein CASPASE-3. These findings highlight the effectiveness of the essential oil isolated from Anisosciadium lanatum to drive alleviation of HepG2 cancer cell progression by modulating apoptotic markers. Our findings suggest that Anisosciadium lanatum could be used as a phytotherapeutic anti-cancer agent, acting through the regulation of apoptotic markers. More well-designed in vivo trials are needed in order to verify the obtained results.

Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopamine neurons and the deposition of misfolded proteins known as Lewy bodies (LBs), which contain α-synuclein (α-syn). The causes and molecular mechanisms of PD are not clearly understood to date. However, misfolded proteins, oxidative stress, and impaired autophagy are believed to play important roles in the pathogenesis of PD. Importantly, α-syn is considered a key player in the development of PD. The present study aimed to assess the role of Ellagic acid (EA), a polyphenol found in many fruits, on α-syn aggregation and toxicity. Using thioflavin and seeding polymerization assays, in addition to electron microscopy, we found that EA could dramatically reduce α-syn aggregation. Moreover, EA significantly mitigated the aggregated α-syn-induced toxicity in SH-SY5Y cells and thus enhanced their viability. Mechanistically, these cytoprotective effects of EA are mediated by the suppression of apoptotic proteins BAX and p53 and a concomitant increase in the anti-apoptotic protein, BCL-2. Interestingly, EA was able to activate autophagy in SH-SY5Y cells, as evidenced by normalized/enhanced expression of LC3-II, p62, and pAKT. Together, our findings suggest that EA may attenuate α-syn toxicity by preventing aggregation and improving viability by restoring autophagy and suppressing apoptosis.

Identification and Validation of Novel Microtubule Suppressors with an Imidazopyridine Scaffold through Structure Based Virtual Screening and Docking.

Targeting the colchicine binding site of alpha/beta tubulin microtubules can lead to suppression of microtubule dynamics, cell cycle arrest and apoptosis. Therefore, development of microtubule (MT) inhibitors is considered a promising route to anticancer agents. Our approach to identify novel scaffolds as MT inhibitors depends on a 3D-structure based pharmacophore approach and docking using three programmes MOE, Autodock and BUDE (Bristol University Docking Engine) to screen a library of virtual compounds. From this work we identified the compound 7-(3-Hydroxy-4-methoxy-phenyl)-3-(3-trifluoromethyl-phenyl)-6,7-dihydro-3H-imidazo[4,5-b] pyridin-5-ol (6) as a novel inhibitor scaffold. This compound inhibited several types of cancer cell proliferation at low micromolar concentrations with low toxicity. Compound 6 caused cell cycle arrest in the G2/M phase and blocked tubulin polymerization at low micromolar concentration (IC50 = 6 micromolar, inducing apoptosis via activation of caspase 9, increasing the level of the pro-apoptotic protein Bax and decreasing the level of the anti-apoptotic protein Bcl2. In summary, our approach identified a lead compound with potential antimitotic and antiproliferative activity.

The Influence of BCL6 on the WNT Pathway in Glioblastoma Therapy Resistance

<p>Glioblastoma is a devastating disease with a median survival of 18 months from diagnosis and a 5 years survival rate of only 10%. The gold standard of treatment for glioblastoma is surgical resection followed by chemotherapeutic treatment with Temozolomide, a DNA alkylating agent, and irradiation around the remaining tumour margins. These treatments are both designed to create DNA damage to the cancerous cells, causing the cell cycle to halt, and result in apoptosis. This treatment does extend patients life for a few months, however glioblastoma cells quickly become resistant to therapy, and disease is always fatal. The anti-apoptotic protein BCL6, confers resistance to apoptosis in response to DNA damage and has been shown to be upregulated in Glioblastoma in response to DNA damaging chemotherapy and irradiation. This upregulation has been hypothesised to increase resistance to these therapies. By minimizing resistance to the standard therapies, the outlook for sufferers of glioblastoma could be greatly improved. Dysregulation of the WNT pathway has also been shown to be very important in carcinogenesis of glioblastoma and is responsible for the diffuse nature of the tumour which makes total resection nearly impossible. An RNA-seq screen was carried out on a glioblastoma cell line in which BCL6 was inhibited using the small molecule inhibitor FX1. This resulted in a change in expression of a large number of WNT related genes. This indicates that there is a link between BCL6 and the WNT pathway. Changes in expression in the WNT genes DKK1, WNT5a and WNT5b were validated. Experiments were carried out to investigate the effects of chemotherapy and BCL6 inhibition on both the canonical and non-canonical WNT pathways. It was found that BCL6 has an influence of the level of activity of the canonical WNT pathway. It also influences migration, the cell cycle, and clonogenicity. Understanding this link between WNT and BCL6 could be crucial in finding an effective treatment for glioblastoma.</p>

The Influence of BCL6 on the WNT Pathway in Glioblastoma Therapy Resistance

<p>Glioblastoma is a devastating disease with a median survival of 18 months from diagnosis and a 5 years survival rate of only 10%. The gold standard of treatment for glioblastoma is surgical resection followed by chemotherapeutic treatment with Temozolomide, a DNA alkylating agent, and irradiation around the remaining tumour margins. These treatments are both designed to create DNA damage to the cancerous cells, causing the cell cycle to halt, and result in apoptosis. This treatment does extend patients life for a few months, however glioblastoma cells quickly become resistant to therapy, and disease is always fatal. The anti-apoptotic protein BCL6, confers resistance to apoptosis in response to DNA damage and has been shown to be upregulated in Glioblastoma in response to DNA damaging chemotherapy and irradiation. This upregulation has been hypothesised to increase resistance to these therapies. By minimizing resistance to the standard therapies, the outlook for sufferers of glioblastoma could be greatly improved. Dysregulation of the WNT pathway has also been shown to be very important in carcinogenesis of glioblastoma and is responsible for the diffuse nature of the tumour which makes total resection nearly impossible. An RNA-seq screen was carried out on a glioblastoma cell line in which BCL6 was inhibited using the small molecule inhibitor FX1. This resulted in a change in expression of a large number of WNT related genes. This indicates that there is a link between BCL6 and the WNT pathway. Changes in expression in the WNT genes DKK1, WNT5a and WNT5b were validated. Experiments were carried out to investigate the effects of chemotherapy and BCL6 inhibition on both the canonical and non-canonical WNT pathways. It was found that BCL6 has an influence of the level of activity of the canonical WNT pathway. It also influences migration, the cell cycle, and clonogenicity. Understanding this link between WNT and BCL6 could be crucial in finding an effective treatment for glioblastoma.</p>

Bioactivity-guided Isolation of TRAIL-Resistance-Overcoming Activity Compounds From the Leaves of Murraya exotica

Fractionation of the leaf extract from Murraya exotica led to the successful isolation of 12 compounds (1-12) with TRAIL-resistance-overcoming activity. Xanthinosin (1), 11α, 13-dihydroxanthinin (2), 11β, 13-dihydroxanthinosin (3), 4α, 11α, 13-trihydroxanthuminol (4), desacetylxanthanol (5), and lasidiol p-methoxybenzoate (6) were sesquiterpenes isolated from this plant for the first time, and 3 was isolated from natural sources for the first time. Among them, compounds 1 and 5 showed strong TRAIL-resistance-overcoming activity, but their mechanisms have already been revealed. Furthermore, dihydroxanthinin (2), 1, 5-dicaffeoylquinic acid (7), and (-) loliolide (8), which belong to different phytochemical groups, were investigated for their effects on increasing apoptosis induction to overcome TRAIL resistance using Western blot analysis. The results demonstrated that 2, 7, and 8 promoted TRAIL-induced apoptosis by increasing the expression of several proapoptotic markers, including cleaved caspases −3 and −8, and suppressing anti-apoptotic protein Bcl-2.

Studies on the effect of Celastrus orbiculatus (Celastraceae) extract on chemosensitivity of liver cancer cells via Wnt/β-catenin pathway

Purpose: To examine the efficacy of Celastrus orbiculatus extract (COE) on the chemosensitivity of liver cancer (LC) cells and its mechanism of action.Methods: Hep G2/ADM cells in the logarithmic growth phase were assigned to a control group (no treatment for cell culture medium only) and a study group (120 μg/ml COE added to the culture medium). After 48 h of incubation, the biological responses were compared. The study group wasdivided into groups A and B, while control group was divided into groups C and D, with 1 μmol/L XAV939 added in groups A and C. Cell proliferation, cell invasion, cell apoptosis rate, and apoptosis protein in the four groups were evaluated.Results: The study group showed significantly lower values in terms of cell proliferation and cell invasiveness (p < 0.05) and a higher apoptotic rate than the control group (p < 0.05)). The study group also demonstrated an elevated pro-apoptotic protein Bax level and a declined anti-apoptotic protein Bcl-2 level. In contrast to group B, the proliferation and invasiveness of Hep G2/ADM cells in group A treated with the inhibitor, XAV939, were significantly lower (p < 0.05), while the apoptotic rate exhibited a significant increase (p < 0.05). There was a rise in the level of pro-apoptotic protein, Bax, and a fall in the anti-apoptotic protein Bcl-2 level in group A. Lower levels of β-catenin, c-Myc, and cyclin D1 protein were observed in the study group compared with the control group (p < 0.05). Compared with other groups, the multiplication capacity and invasiveness of cells in group A treated with COE and inhibitor XAV939 significantly declined, while the apoptotic rate increased (p < 0.05).Conclusion: COE reverses drug resistance in chemotherapy by inhibiting the expression of Wnt/β-catenin pathway in LC cells. Therefore, COE has potentials for use along with chemotherapeutic agents in the management of liver cancer.