miR-1283 Contributes to Endoplasmic Reticulum Stress in the Development of Hypertension Through the Activating Transcription Factor-4 (ATF4)/C/EBP-Homologous Protein (CHOP) Signaling Pathway.

BACKGROUND Hypertension-related microRNA(miR)-1283 and its target gene, activating transcription factor-4 (ATF4), can regulate vascular endothelial dysfunction. This study aimed to explore whether miR-1283 prevents hypertension through targeting ATF4. MATERIAL AND METHODS Transcriptome sequencing was performed after overexpression or inhibition of miR-1283 in human amniotic epithelial cells (HAECs). After miR-1283 was overexpressed or inhibited in HAECs, ATF4+/- and wild-type mice were induced with a high-salt diet. We detected the expression of ATF4, C/EBP-homologous protein (CHOP), BH3-interacting domain death agonist (BID), Bcl-2, Bcl-2-like protein 11 (BIM), Bcl-2-like protein 1 (BCL-X), and caspase-3 by PCR and western blotting. We detected the changes of vasoactive substances including nitric oxide (NO), endothelin 1 (ET-1), endothelial protein C receptor (EPCR), thrombin (TM), and von Willebrand factor (vWF) by ELISA. RESULTS Compared with that of the miR-1283- inhibited group, NO was higher in the miR-1283 overexpression group, while the expression of ET-1, EPCR, TM, and vWF were lower. Similarly, compared with that of the miR-1283 inhibited group, the expression of ATF4, CHOP, BID, BIM, and caspase-3 in the miR-1283 overexpression group was downregulated, while the expression of BCL-2 and BCL-X was upregulated (P<0.05). In vivo experiments showed the lack of ATF4 gene could prevent hypertension in mice induced by high-salt diet and protect endothelial function. CONCLUSIONS The mechanism of regulating blood pressure and endothelial function of the miR-1283/ATF4 axis was related to inhibiting endoplasmic reticulum stress and cell apoptosis through the ATF4/CHOP signaling pathway. Therefore, the miR-1283/ATF4 axis may be a target for the prevention and treatment of hypertension.

Network and 16S rRNA Sequencing-Combined Approach Provides Insightal Evidence of Vitamin K2 for Salt-Sensitive Hypertension.

Vitamin K2 (VK2), found to act to treat hypertension, has been widely used in the food and pharmaceutical industries nowadays. However, the potential targets and molecular mechanisms of VK2 for salt-sensitive hypertension have not been fully investigated. Therefore, the study aimed to investigate the potential molecular mechanisms of VK2 for salt-sensitive hypertension using network pharmacology and 16S rRNA sequencing strategy. The network pharmacology-based findings from KEGG enrichment analysis revealed that VK2-treated salt-sensitive hypertension was mechanically associated with the complement and coagulation cascades, calcium signaling pathway, renin-angiotensin system, etc. A total of 29 different bacteria in an animal experiment after VK2 supplementation were screened and functionally enriched using PICRUSt2. Additionally, 10 signaling pathways were identified in which the renin-angiotensin system was found to be the potential molecular mechanisms with the greatest change in multiple and statistical significance. Moreover, the results of the renin-angiotensin system-related protein expression exhibited VK2-inhibited renin-angiotensin system in salt-induced hypertensive mice, which significantly verified the previous biological and functional prediction analysis. Finally, spearman correlation analysis showed the different bacteria such as Dubosiella, Ileibacterium, etc., had a positive or negative correlation with renin-angiotensin system-related proteins in salt-induced mice. In conclusion, the potential molecular mechanisms of VK2 for salt-sensitive hypertension may be beneficially achieved by the specific inhibition of the renin-angiotensin system, contributing to the development for a new preventive strategy of salt-sensitive hypertension.

ATF4 promotes renal tubulointerstitial fibrosis by suppressing autophagy in diabetic nephropathy.

AIM: Diabetic nephropathy (DN) is the dominant cause of end-stage renal disease which is characterized by extracellular matrix accumulation. The purpose of this study was to investigate the role of activating transcription factor 4 (ATF4) in regulating renal fibrosis and autophagy in DN. MAIN METHOD: Streptozotocin (STZ) was administered to heterozygous ATF4 knockout (KO) and wild-type (WT) mice via an intraperitoneal injection to induce DN. NRK-52E cells were cultured in high glucose to mimic diabetic pathological. qRT-PCR, western blot, immunofluorescence, histology and electron microscopic analysis were performed. The autophagy flux was observed by tandem mRFP-GFP-LC3 fluorescence microscopy. KEY FINDINGS: DN mice experienced severe renal injury and fibrosis and showed increased expression of ATF4 and inhibition of autophagy in kidney tissues. We found that STZ-induced ATF4 KO mice showed significant improvement in urinary albumin, serum creatinine and blood urea nitrogen and the pathological changes of renal tubulointerstitial fibrosis compared with STZ-induced WT mice. Furthermore, inhibition of ATF4 could restore autophagy in DN mice. Similar results were shown in vitro. Overexpression of ATF4 in NRK-52E cells cultured in high glucose condition suppressed autophagy and upregulated Collagen type 4 (Col-IV) expression, while inhibition of ATF4 could increase the number of the autophagosomes, improve autophagic flux and decrease Col-IV level. SIGNIFICANCE: Our study provided the evidence of a crucial role for ATF4 in inhibiting autophagy against diabetic kidney damage. Suppression of ATF4 may be an effective therapy in restraining renal tubulointerstitial fibrosis in DN.

Gut Microbiota-Related Evidence Provides New Insights Into the Association Between Activating Transcription Factor 4 and Development of Salt-Induced Hypertension in Mice.

Activating transcription factor 4 (ATF4), which regulates genes associated with endoplasmic reticulum stress, apoptosis, autophagy, the gut microbiome, and metabolism, has been implicated in many diseases. However, its mechanistic role in hypertension remains unclear. In the present study, we investigated its role in salt-sensitive hypertensive mice. Wild-type (WT) C57BL/6J mice were used to establish Atf4 knockout (KO) and overexpression mice using CRISPR-Cas9 and lentiviral overexpression vectors. Then, fecal microbiota transplantation (FMT) from Atf4 ± mice and vitamin K2 (VK2) supplementation were separately carried out in high-salt-diet (8% NaCl)-induced mice for 4 weeks. We found that Atf4 KO inhibited and Atf4 overexpression enhanced the increase in blood pressure and endothelial dysfunction induced by high salt intake in mice, while regulating the gut microbiota composition and VK2 expression. It was further verified that ATF4 is involved in the regulation of salt-sensitive hypertension and vascular endothelial function, which is achieved through association with gut microbiota and may be related to VK2 and different bacteria such as Dubosiella. In addition, we found that VK2 supplementation prevents the development of salt-sensitive hypertension and maintains vascular endothelial function; moreover, VK2 supplementation increases the abundance of intestinal Dubosiella and downregulates the relative expression of Atf4 in the thoracic aorta of mice. We conclude that ATF4 plays an important role in regulating gut microbiota and VK2 production, providing new insights into the association between ATF4 and development of salt-induced hypertension in mice, meanwhile contributing to the development for a new preventive strategy of hypertension.

Verification of Resveratrol Inhibits Intestinal Aging by Downregulating ATF4/Chop/Bcl-2/Bax Signaling Pathway: Based on Network Pharmacology and Animal Experiment.

Resveratrol is one of the most well-known drugs used in the treatment of aging. However, the potential mechanisms of resveratrol on intestinal aging have not yet been fully investigated. Herein, we aimed to further explore the pharmacological mechanisms of resveratrol as a therapy for intestinal aging. We performed network construction and enrichment analysis via network pharmacology. Then a further animal experimental validation containing 20 female C57BL/6J (wild type, WT) and 16 female ATF4+/- (knock down, KD) naturally aging mice and oral supplementary resveratrol (44 mg/kg/day) for 30 days were conducted. The expression of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), linear alkylethoxylate (AE), and malondialdehyde (MDA) were measured by ELISA, the observation of pathological changes and apoptosis in intestinal tissue were performed by HE, PAS, and TUNEL staining, the ATF4/Chop/Bcl-2/Bax signaling pathway-related proteins and mRNAs expression were measured by western blotting and real-time PCR. The network pharmacology showed 132 targets of resveratrol on aging. The enrichment analysis showed resveratrol antiaging involved mainly included protein heterodimerization activity, apoptosis, etc. Then ATF4/Chop/Bcl-2/Bax signaling pathway in biological process of apoptosis was selected to verify the potential mechanisms. Animal studies showed resveratrol upregulated the relative expression of SOD, GSH-Px, CAT, AE, whereas it downregulated the relative expression of MDA in intestine compared with the control group. There was also higher relative expression of SOD, GSH-Px, CAT, AE, and lower relative expression of MDA in KD mice than that in WT mice. Moreover, there was higher relative expression of SOD, GSH-Px, CAT, AE, and lower relative expression of MDA in KD mice than that in WT mice after resveratrol treatment. Decreased ATF4, Chop, Bax but increased Bcl-2 proteins and mRNAs expression were determined after resveratrol treatment compared with the control group; lower ATF4, Chop, Bax but higher Bcl-2 proteins and mRNAs expression were found in KD mice than that in WT mice. Additionally, lower relative proteins and mRNAs expression of ATF4, Chop, Bax and higher relative expression of Bcl-2 in KD mice than that in WT mice after resveratrol treatment. These findings demonstrated that resveratrol substantially inhibited intestinal aging via downregulating ATF4/Chop/Bcl-2/Bax signaling pathway.

Network Pharmacology Identifies the Mechanisms of Action of TaohongSiwu Decoction Against Essential Hypertension.

BACKGROUND TaohongSiwu decoction (THSWT), a traditional herbal formula, has been used to treat cardiovascular and cerebrovascular diseases such as essential hypertension (EH) in China. However, the pharmacological mechanism is not clear. To investigate the mechanisms of THSWT in the treatment of EH, we performed compounds, targets prediction and network analysis using a network pharmacology method. MATERIAL AND METHODS We selected chemical constituents and targets of THSWT according to TCMSP and UniProtKB databases and collected therapeutic targets on EH from Online Mendelian Inheritance in Man (OMIM), Drugbank and DisGeNET databases. The protein-protein interaction (PPI) was analyzed by using String database. Then network was constructed by using Cytoscape_v3.7.1, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed by using Database for Annotation, Visualization and Integrated Discovery (DAVID) software. RESULTS The results of our network pharmacology research showed that the THSWT, composed of 6 Chinese herbs, contained 15 compounds, and 23 genes regulated the main signaling pathways related to EH. Moreover, the PPI network based on targets of THSWT on EH revealed the interaction relationship between targets. These core compounds were 6 of the 15 disease-related compounds in the network, kaempferol, quercetin, luteolin, Myricanone, beta-sitosterol, baicalein, and the core genes contained ADRB2, CALM1, HMOX1, JUN, PPARG, and VEGFA, which were regulated by more than 3 compounds and significantly associated with Calcium signaling pathway, cGMP-PKG signaling pathway, cAMP signaling pathway, PI3K-Akt signaling pathway, Rap1 signaling pathway, and Ras signaling pathway. CONCLUSIONS This network pharmacological study can reveal potential mechanisms of multi-target and multi-component THSWT in the treatment of EH, provide a scientific basis for studying the mechanism.

Bacterial Diversity in Roots, Stems, and Leaves of Chinese Medicinal Plant Paris polyphylla var. yunnanensis.

The root of Paris polyphylla var. yunnanensis, a famous and endangered traditional Chinese herb, has a significant medicinal value. The aim of this study was to analyze the composition and functional characteristics of bacterial endophytes in roots, stems, and leaves of P. polyphylla var. yunnanensis. The 16S rRNA gene sequencing and functional prediction of bacterial endophytes in roots, stems, and leaves of P. polyphylla var. yunnanensis were conducted. The Chao and Shannon indices of the bacteria in roots were significantly higher than those in stems and leaves. The dominant endophyte phyla were Cyanobacteria, Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. The main genera detected in roots were unclassified Cyanobacteria, Rhizobium, Flavobacterium, and Sphingobium; the main genera in stems were norank_c__Cyanobacteria, Bacillus, and Pseudomonas; the main genera in leaves were norank_c__Cyanobacteria and Rhizobium. The microbiota in roots was particularly enriched in functional categories "extracellular structures" and "cytoskeleton" compared with stems and leaves (p < 0.05). Our study reveals the structural and functional characteristics of the endophytic bacteria in roots, stems, and leaves of P. polyphylla var. yunnanensis, which aids in the scientific understanding of this plant.The root of Paris polyphylla var. yunnanensis, a famous and endangered traditional Chinese herb, has a significant medicinal value. The aim of this study was to analyze the composition and functional characteristics of bacterial endophytes in roots, stems, and leaves of P. polyphylla var. yunnanensis. The 16S rRNA gene sequencing and functional prediction of bacterial endophytes in roots, stems, and leaves of P. polyphylla var. yunnanensis were conducted. The Chao and Shannon indices of the bacteria in roots were significantly higher than those in stems and leaves. The dominant endophyte phyla were Cyanobacteria, Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. The main genera detected in roots were unclassified Cyanobacteria, Rhizobium, Flavobacterium, and Sphingobium; the main genera in stems were norank_c__Cyanobacteria, Bacillus, and Pseudomonas; the main genera in leaves were norank_c__Cyanobacteria and Rhizobium. The microbiota in roots was particularly enriched in functional categories "extracellular structures" and "cytoskeleton" compared with stems and leaves (p < 0.05). Our study reveals the structural and functional characteristics of the endophytic bacteria in roots, stems, and leaves of P. polyphylla var. yunnanensis, which aids in the scientific understanding of this plant.