Changes in gut microbiota linked to a prevention of cardiac remodeling induced by hypertension in spontaneously hypertensive rats fed a pawpaw fruit diet.

Objective: Dietary intake of fruit is associated with lower incidence of hypertension and cardiovascular risk. Papaya is a kind of delicious fruit and reported has dietary therapeutic effects, such as digestive stimulation and hypotensive efficacy. However, the mechanism of pawpaw involved have not been elucidated. Here, we illustrate that the effect of pawpaw on the gut microbiota and the prevention of cardiac remodeling. Methods: Gut microbiome, cardiac structure/function, and blood pressure were examined in SHR and WKY groups. The intestinal barrier was tested with histopathologic; immunostaining and Western blot were used to measure the tight junction protein level; Gpr41 was tested by RT-PCR, and inflammatory factors were detected with ELISA. Results: We observed a significant decrease in microbial richness, diversity, and evenness is the spontaneously hypertensive rat (SHR), in addition to an increased Firmicutes/Bacteroidetes (F/B) ratio. These changes were accompanied by decreased in acetate and butyrate-producing bacteria. Compared with SHR, treatment with pawpaw at the dosage of 10 g/kg for 12 weeks significantly reduced the blood pressure, cardiac fibrosis and cardiac hypertrophy, while the ratio of F/B decreased. We also found that the concentration of short-chain fatty acids (SCFAs) was increased in SHR fed with pawpaw compared with that in control group, while the gut barrier was restored and level of proinflammatory cytokines in the serum were decreased. Conclusions: Pawpaw, rich of high fiber, led to changes in the gut microbiota that played a protective role in the development of cardiac remodeling. The potential mechanism of pawpaw may explained by the generation of one of the main metabolites of the gut microbiota, the short-chain fatty acid acetate, increasing tight junction protein level occluding to enhance the gut barrier for less releasing the inflammation cytokines, and upregulating G-protein-coupled receptor 41 (GPR41) to reduce blood pressure.

B13, a well-tolerated inhibitor of hedgehog pathway, exhibited potent anti-tumor effects against colorectal carcinoma in vitro and in vivo.

Abnormal activation of Hedgehog (Hh) signaling pathway mediates the genesis and progression of various tumors [1]. Currently, three drugs targeting the Hh signaling component Smoothened (Smo) have been marketed for the clinical treatment of basal cell tumors or acute myeloid leukemia. However, drug resistance is a common problem in those drugs, so the study of Smo inhibitors that can overcome drug resistance has important guiding significance for clinical adjuvant drugs. MTT assay, clone formation assay and EdU assay were used to detect the proliferation inhibitory activity of the drugs on tumor cells. The effect of B13 on cell cycle and apoptosis were detected by flow cytometry. An acute toxicity test was used to detect the toxicity of B13 in vivo, and xenograft tumor model was used to detect the efficacy of B13 in vivo. The binding of B13 to Smo was studied by BODIPY-cyclopamine competitive binding assay and molecular docking. The effect of B13 on the expression and localization of downstream target gene Gli1/2 of Smo was investigated by Western Blot and immunofluorescence assay. SmoD473H mutant cell line was constructed to study the effect of B13 against drug resistance. (1) B13 had the strongest inhibitory activity against colorectal cancer cells. (2) B13 can effectively inhibit the clone formation and EdU positive rate of colon cancer cells. (3) B13 can block the cell cycle in the G2/M phase and cell apoptosis. (4) B13 has low toxicity in vivo, and its efficacy in vivo is better than that of the Vismodegib. (5) Molecular docking and BODIPY-cyclopamine experiments showed that B13 could bind to Smo protein. (6) B13 can inhibit the protein expression of Gli1, the downstream of Smo, and inhibit its entry into the nucleus. (7) B13 could inhibit the expression of Gli1 in the HEK293 cells with SmoD473H, and the molecular docking results showed that B13 could bind SmoD473H protein. B13 with the best anti-tumor activity was screened out by MTT assay. In vitro, pharmacodynamics experiments showed that B13 could effectively inhibit the proliferation and metastasis of colorectal cancer cells, induce cell cycle arrest, and induce cell apoptosis. In vivo pharmacodynamics experiments showed that B13 was superior to Vismodegib in antitumor activity and had low toxicity in vivo. Mechanism studies have shown that B13 can bind Smo protein, inhibit the expression of downstream Gli1 and its entry into the nucleus. Notably, B13 overcomes resistance caused by SmoD473H mutations.

Salvianolic acid D: A potent molecule that protects against heart failure induced by hypertension via Ras signalling pathway and PI3K/Akt signalling pathway.

Ethnopharmacological relevance: Salvianolic acid D (Sal D) is a natural substance extracted from Radix Salviae that performs a cardiovascular benefit. However, the protective mechanism of Sal-D for heart failure remains uncertain. Aim of the study: In this study, we aim to evaluate the effect of Sal D on heart failure and elucidate its underlying mechanisms. Materials and methods: Using the spontaneously hypertensive rats (SHR) as a cardiac remodelling model, the cardioprotective effect of Sal D was evaluated. Employing bioinformatics analysis, the related mechanisms of Sal D treatment on heart failure were identified and validated by Western blot and polymerase chain reaction. Results: The results showed that Sal D significantly improved cardiac function and attenuated cardiac hypertrophy. Besides, Sal D impaired mitochondrial structure and restored the energy charge of cardiomyocytes managed by angiotensin II. Bioinformatics analysis suggested that Sal D might improve heart failure by modulating the Ras and PI3K/AKT signalling pathways verified in vitro and in vivo. Conclusion: In summary, Sal D can improve the heart function of SHR by inhibiting the Ras signalling pathway and activating the PI3K/AKT signalling pathway.

Kaempferol inhibits renal fibrosis by suppression of the sonic hedgehog signaling pathway.

BACKGROUND: Most chronic kidney diseases (CKDs) develop to end-stage renal disease (ESRD), which is characterized by fibrosis and permanent tissue and function loss. As a result, better and more effective remedies are essential. Kaempferol (KAE) is a common flavonoid extracted from plants. It can control the progression of kidney fibrosis and the epithelial-to-mesenchymal transition (EMT) of the renal tubular system. PURPOSE: We aim to investigate the effect of KAE therapy on extracellular matrix deposition and stimulation of EMT in vitro and in vivo to elucidate the treatment mechanisms regulating these effects. STUDY DESIGN: Chronic hypertension-induced kidney fibrosis was studied in spontaneously hypertensive rats with chronic kidney disease. Biochemical analysis, histological staining, and the expression level of relative proteins were used to assess the effect of KAE on renal function and fibrosis. The direct impact of KAE on proliferation and migration was evaluated using human renal tubular epithelial cells (HK-2) induced by transforming growth factor-ß1 (TGF-ß1), which can then induce EMT. The molecular mechanism of KAE was verified using co-IP assay and immunofluorescence. RESULTS: KAE could reduce blood pressure and decrease the extracellular matrix (ECM) components (including collagen I and collagen Ш), TGF-ß1, and α-SMA in the kidneys of hypertension-induced rats with chronic kidney disease. Moreover, in HK-2 cell treated with TGF-ß1, KAE administration significantly suppressed proliferation, migration, and EMT via increasing the expression of E-cadherin, while reducing the N-cadherin and α-SMA. Sufu was exceedingly repressed in HK-2 cells treated with TGF-ß1. KAE inhibited the activation of Shh and Gli through increasing the expression of Sufu, thereby blocking the nuclear translocation of Gli1 in vitro. CONCLUSION: KAE ameliorated kidney fibrosis and EMT by inhibiting the sonic hedgehog signaling pathway, thereby to attenuate the pathological progression of hypertensive kidney fibrosis.

Enhanced antitumor efficacy by combining afatinib with MDV3100 in castration-resistant prostate cancer.

Background: Patients with prostate cancer often develop resistance to androgen deprivation therapy, a condition called castration-resistant prostate cancer (CRPC). Enzalutamide (MDV3100) can prolong the survival of patients with CRPC after chemotherapy, but ∼50% of patients eventually relapse and develop resistance to MDV3100. Thus, it is necessary to explore new treatment methods to improve the therapeutic effect of MDV3100. Tyrosine kinases play an essential role in the pathogenesis of CRPC. Methods: MTT assay was used to detect the inhibitory effects of MDV3100 and tyrosine kinase inhibitor on prostate cancer cells. CompuSyn version 1.0 was used to calculate the combination index (CI) values using the Chou-Talalay method. Clone formation and EdU assay were used to detect the effect of afatinib combined with MDV3100 on the proliferation of 22Rv1 cells. RT-qPCR and Western blot were used to explore the mechanism of drug combination. The aim of the present study was to determine the effects of several tyrosine kinase inhibitors (TKIs) when used in combination with MDV3100 in vitro. Results: The results demonstrated that TKIs combined with MDV3100 exerted a synergistic effect on a variety of PCa cells. Afatinib combined with MDV3100 could suppress the proliferation and migration of 22RV1 cells, as well as cause their cell cycle arrest and apoptosis. Mechanistically, afatinib effectively reduced the protein expression levels of HER2 and HER3 and inhibited EGFR phosphorylation, thereby enhancing the effect of MDV3100 and suppressing CRPC. Conclusions: These findings suggested that afatinib treatment improved the effect of MDV3100 on 22RV1 cells, highlighting this drug as a potential therapeutic strategy for patients with CRPC.

5-arylalkynyl-2-benzoyl thiophene: a novel microtubule inhibitor exhibits antitumor activity without neurological toxicity.

The composition of microtubules involving several steps, including the polymerization and depolymerization of α-tubulin and ß-tubulin heterodimers. Microtubule-targeting agents can increase or inhibit microtubule polymerization, thereby disrupting the dynamic process and stalling cells in G2/M phase. Microtubule-targeting agents are generally cytotoxic, which neurological toxicity being one of the significant adverse events associated. We recently reported a novel 5-arylalkynyl-2-benzoyl thiophene (PST-3) that exhibited broad-spectrum cellular cytotoxicity and in vivo potency with high safety. PST-3 was a substrate of p-gp, which could not cross the blood-brain barrier and lead to less neurotoxicity. The antitumor activities in vitro demonstrated that PST-3 combined with the colchicine-binding site on microtubule, induces morphological changes, disrupts microtubule networks, inhibits polymerization of tubulin, arrests breast cancer cells in the G2/M phase of the cell cycle and induces apoptosis. Evaluation of the antitumor effect in vivo demonstrated that PST-3 elicited MDA-MB-468 tumor %T/C of 11.75%, whereas elicited MCF7 tumor %T/C of 44.38% in breast cancer xenograft models. Besides, in vivo experiments of a higher dose (60 mg/kg) of PST-3 treatment for 21 days did not produce any significant neurotoxicity. These results provide evidence that PST-3 might possess the potential to be developed into a new microtubule inhibitor without neurological toxicity.

Modulation of transforming growth factor-beta signaling pathway mediates the effects of Kangxian Formula on cardiac remodeling.

ETHNOPHARMACOLOGICAL RELEVANCE: Kangxian formula (KXF) is a traditional Chinese medicine which shows effective outcomes in treating cardiac remodeling induced by hypertension. However, the exact effects and the mechanisms involved remain obscure. AIM OF THE STUDY: In this study, we aimed to identify the therapeutic role of KXF in vivo and in vitro, and investigate the mechanism of KXF on hypertension induced cardiac remodeling. MATERIALS AND METHODS: After quality control of KXF using fingerprint, blood pressure, cardiac structure/function indexes, and degree of myocardial collagen were measured in vivo. Moreover, the proliferation, migration, and fibroblast-to-myofibroblast transformation (FMT) of cardiac fibroblasts (CFBs) were determined. Using gene chip, the related mechanisms of KXF treatment on cardiac remodeling were identified and further validated by western blot and polymerase chain reaction. RESULTS: A stable quality control standard of KXF was established in this study. KXF administration ameliorated systolic/diastolic blood pressure, cardiac damages, and cardiac fibrosis in vivo. The proliferation, migration, and FMT of CFBs were also inhibited by the treatment of KXF medicated serum. Furthermore, KXF reduced the protein level of transforming growth factor-beta (TGF-ß) receptors Ⅰ, Ⅱ, Tak1, p38, Smad2/3, and Smad4 and the expression of mRNA, which are the hub proteins in the TGF-ß signaling pathway. CONCLUSION: Our findings suggest that KXF attenuates cardiac remodeling by improving cardiac damages, attenuating cardiac fibrosis, and inhibiting the activity of CFBs. In addition, KXF ameliorates cardiac remodeling partially through modulating the TGF-ß signaling pathway. These data provide insights and mechanisms into the wide application of KXF in clinical practice.

Discovery of [1,2,4]triazolo[4,3-a]pyridines as potent Smoothened inhibitors targeting the Hedgehog pathway with improved antitumor activity in vivo.

Triple-negative breast cancer (TNBC), a subset of breast cancers, have poorer survival than other breast cancer types. Recent studies have demonstrated that the abnormal Hedgehog (Hh) pathway is activated in TNBC and that these treatment-resistant cancers are sensitive to inhibition of the Hh pathway. Smoothened (Smo) protein is a vital constituent in Hh signaling and an attractive drug target. Vismodegib (VIS) is one of the most widely studied Smo inhibitors. But the clinical application of Smo inhibitors is limited to adult patients with BCC and AML, with many side effects. Therefore, it's necessary to develop novel Smo inhibitor with better profiles. Twenty [1,2,4]triazolo[4,3-a]pyridines were designed, synthesized and screened as Smo inhibitors. Four of these novel compounds showed directly bound to Smo protein with stronger binding affinity than VIS. The new compounds showed broad anti-proliferative activity against cancer cell lines in vitro, especially triple-negative breast cancer cells. Mechanistic studies demonstrated that TPB15 markedly induced cell cycle arrest and apoptosis in MDA-MB-468 cells. TPB15 blocked Smo translocation into the cilia and reduced Smo protein and mRNA expression. Furthermore, the expression of the downstream regulatory factor glioma-associated oncogene 1 (Gli1) was significantly inhibited. Finally, TPB15 demonstrated greater anti-tumor activity in our animal models than VIS with lower toxicity. Hence, these results support further optimization of this novel scaffold to develop improved Smo antagonists.

The Combination of Scutellaria baicalensis Georgi and Sophora japonica L. ameliorate Renal Function by Regulating Gut Microbiota in Spontaneously Hypertensive Rats.

Chronic kidney disease (CKD) is becoming a notable health concern globally. The combination of Scutellaria baicalensis Georgi (SB) and Sophora japonica L. (SJ) has been demonstrated to have anti-hypertensive effects and improve kidney injury clinically. This study aimed to explore the renal protective effect of the combination of SB and SJ against CKD and clarify the potential mechanisms. Male spontaneously hypertensive rats (SHR) were used to induce hypertensive nephropathy and were treated with SB or SJ separately or in combination for 15 weeks, and an antibiotic group was used for a rescue experiment. Blood pressure, serum or urine biochemical markers, serum inflammation factors, short-chain fatty acids (SCFAs), indoxyl sulfate (IS), and oxidative stress indicators were assessed. Western blot analysis was performed to determine the expression of intestinal tight junction proteins, including occludin and ZO-1. The mRNA expression of the SCFAs receptors olfactory 78 (Olfr78) and G protein-coupled receptor 41 (GPR41) was determined by quantitative real-time PCR. Gut microbiota profiles were established via high-throughput sequencing of the V3-V4 region of the bacterial 16S rRNA gene. SB and SJ significantly ameliorated the severity of renal injury induced by hypertension. The combination also decreased the ratio of Firmicutes/Bacteroidetes, increased the relative abundance of Lactobacillus, and reduced that of Clostridiaceae. The intestinal barrier was improved, and the change in dominant bacteria reduced IS accumulation and further inhibited oxidative stress activation in kidneys. SB and SJ increased SCFAs production, inhibited inflammatory factor release, and regulated blood pressure by decreasing the expression of Olfr78 and increasing that of GPR41, then alleviated kidney damage. This research demonstrated the positive effects of SB and SJ in a rat model of hypertensive nephropathy, indicated that the treatment of SB and SJ by improving the intestinal barrier function, increasing SCFAs, reducing inflammation, decreasing IS, and inhibiting oxidative stress reactions.

Identification of lncRNA NEAT1/miR-21/RRM2 axis as a novel biomarker in breast cancer.

As a major disease that threatens the health of women worldwide, breast cancer (BC) lacks effective molecular markers in the clinic at the same time. We aim at finding a new biomarker of BC. In our study, through the Gene Expression Omnibus database chip, a total of 1393 pairs of microRNA-messenger RNA (miRNA-mRNA) networks and 35754 pairs of long noncoding RNA-miRNA networks were obtained. We found out that NEAT1/miR-21/RRM2 axis may play a role in BC diagnosis and prognosis. The real-time quantitative reverse transcription-polymerase chain reaction test was used to analyze the mRNA level of NEAT1, miR-21, and RRM2. Western blot was used to detect the protein level of RRM2. Through the 5-ethynyl-2'-deoxyuridine assay, the proliferation of MDA-MB-231 cells was detected. Through wound healing and transwell assay, the migration of MDA-MB-231 cells was detected. Altogether, our data indicated that NEAT1, miR-21, and RRM2 were upregulated in several BC cell lines. Overexpressed of miR-21 in MDA-MB-231 cells promote proliferation and migration. Besides, our results demonstrated that overexpressed of miR-21 upregulated the level of RRM2. Accordingly, miR-21/RRM2 might be a new diagnosis and treatment target of BC.

Danshenol A Alleviates Hypertension-Induced Cardiac Remodeling by Ameliorating Mitochondrial Dysfunction and Suppressing Reactive Oxygen Species Production.

Current therapeutic approaches have a limited effect on cardiac remodeling, which is characteristic of cardiac fibrosis and myocardial hypertrophy. In this study, we examined whether Danshenol A (DA), an active ingredient extracted from the traditional Chinese medicine Radix Salviae, can attenuate cardiac remodeling and clarified the underlying mechanisms. Using the spontaneously hypertensive rat (SHR) as a cardiac remodeling model, DA ameliorated blood pressure, cardiac injury, and myocardial collagen volume and improved cardiac function. Bioinformatics analysis revealed that DA might attenuate cardiac remodeling through modulating mitochondrial dysfunction and reactive oxygen species. DA repaired the structure/function of the mitochondria, alleviated oxidative stress in the myocardium, and restored apoptosis of cardiomyocytes induced by angiotensin II. Besides, DA inhibited mitochondrial redox signaling pathways in both the myocardium and cardiomyocytes. Thus, our study suggested that DA attenuates cardiac remodeling induced by hypertension through modulating mitochondrial dysfunction and reactive oxygen species.

Design, synthesis, and biological evaluation of 1-substituted -2-aryl imidazoles targeting tubulin polymerization as potential anticancer agents.

A series of novel 1-substituted-2-aryl imidazoles (SAI) were designed and synthesized based on our previously reported ABI (2-Aryl-4-Benzoyl Imidazole) analogs and on the structure of combretastatin A-4 (CA-4). These compounds showed potent antiproliferative activities against six human cancer cell lines with IC50 values in nano molar range. Among them, compound 3X exhibited the best anticancer activity with an average IC50 value of ∼100 nM. The compound maintains the mechanism of action by inhibiting tubulin polymerization, thus causing cell arrest at G2/M phase and apoptosis. In vivo efficacy studies indicated that 3X was highly effective in suppressing tumor growth in a MDA-MB-468 xenograft model of nude mouse with a TGI (Tumor Growth Suppression) of 77% at 60 mg/kg without causing significant toxicity. In addition, 3X displayed significantly better water solubility (36.70 µg/mL) than CA-4 (2.83 µg/mL). Molecular modeling study indicated that 3X binds well to the colchicine binding site in tubulin. Our results suggest that the novel SAI analogs deserve further investigation as potential anticancer agents.