Baicalin alleviates angiotensin II-induced cardiomyocyte apoptosis and autophagy and modulates the AMPK/mTOR pathway.

As a main extraction compound from Scutellaria baicalensis Georgi, Baicalin exhibits various biological activities. However, the underlying mechanism of Baicalin on hypertension-induced heart injury remains unclear. In vivo, mice were infused with angiotensin II (Ang II; 500 ng/kg/min) or saline using osmotic pumps, followed by intragastrically administrated with Baicalin (5 mg/kg/day) for 4 weeks. In vitro, H9C2 cells were stimulated with Ang II (1 µM) and treated with Baicalin (12.5, 25 and 50 µM). Baicalin treatment significantly attenuated the decrease in left ventricular ejection fraction and left ventricular fractional shortening, increase in left ventricular mass, left ventricular systolic volume and left ventricular diastolic volume of Ang II infused mice. Moreover, Baicalin treatment reversed 314 differentially expressed transcripts in the cardiac tissues of Ang II infused mice, and enriched multiple enriched signalling pathways (including apoptosis, autophagy, AMPK/mTOR signalling pathway). Consistently, Baicalin treatment significantly alleviated Ang II-induced cell apoptosis in vivo and in vitro. Baicalin treatment reversed the up-regulation of Bax, cleaved-caspase 3, cleaved-caspase 9, and the down-regulation of Bcl-2. Meanwhile, Baicalin treatment alleviated Ang II-induced increase of autophagosomes, restored autophagic flux, and down-regulated LC3II, Beclin 1, as well as up-regulated SQSTM1/p62 expression. Furthermore, autophagy inhibitor 3-methyladenine treatment alleviated the increase of autophagosomes and the up-regulation of Beclin 1, LC3II, Bax, cleaved-caspase 3, cleaved-caspase 9, down-regulation of SQSTM1/p62 and Bcl-2 expression after Ang II treated, which similar to co-treatment with Baicalin. Baicalin treatment reduced the ratio of p-AMPK/AMPK, while increased the ratio of p-mTOR/mTOR. Baicalin alleviated Ang II-induced cardiomyocyte apoptosis and autophagy, which might be related to the inhibition of the AMPK/mTOR pathway.

Efficacy and safety of Qingda granule versus valsartan capsule in Chinese grade 1 hypertensive patients with low-moderate risk: A randomized, double-blind, double dummy, non-inferiority, multi-center trial.

BACKGROUND: The efficacy and safety of Qingda granule (QDG) in managing blood pressure (BP) among grade 1 hypertensive patients with low-moderate risk remain uncertain. METHODS: In the randomized, double-blind, double dummy, non-inferiority and multicenter trial, 552 patients with grade 1 hypertension at low-moderate risk were assigned at a ratio of 1:1 to receive either QDG or valsartan for 4 weeks, followed up by a subsequent 4 weeks. RESULTS: Post-treatment, clinic systolic/diastolic BPs (SBP/DBP) were reduced by a mean change of 9.18/4.04 mm Hg in the QDG group and 9.85/5.05 mm Hg in the valsartan group (SBP P = 0.47, DBP P = 0.16). Similarly, 24-hour, daytime and nighttime BPs were proportional in both groups (P > 0.05) after 4 weeks treatment. After discontinuing medications for 4 weeks, the mean reduction of clinic SBP/DBP were 0.29/0.57 mm Hg in the QDG group compared to -1.59/-0.48 mm Hg in the valsartan group (SBP P = 0.04, DBP P = 0.04). Simultaneously, the 24-hour SBP/DBP were reduced by 0.9/0.31 mm Hg in the QDG group and -1.66/-1.08 mm Hg in the valsartan group (SBP P = 0.006, DBP P = 0.02). And similar results were observed regarding the outcomes of daytime and nighttime BPs. There was no difference in occurrence of adverse events between two groups (P > 0.05). CONCLUSION: QDG proves to be efficacious for grade 1 hypertension at a low-to-medium risk, even after discontinuation of the medication for 4 weeks. These findings provide a promising option for managing grade 1 hypertension and suggest the potential for maintaining stable BP through intermittent administration of QDG. TRIAL REGISTRATION: ChiCTR2000033890.

Gastrodin attenuates angiotensin II-induced vascular contraction and MLCK/p-MLC2 pathway activation.

CONTEXT: Gastrodin has been used as antihypertension therapy in China; however, the mechanisms underlying the effects of gastrodin have yet to be fully elucidated. OBJECTIVE: To explore the therapeutic efficiency of gastrodin as an antihypertensive and determine the mechanisms underlying this effect. MATERIALS AND METHODS: C57BL/6 mice were continuously administered angiotensin II (Ang II) (500 ng/kg/min) to induce hypertension. Mice were randomly divided into control, Ang II and Ang II + gastrodin groups. Mice received intragastric administration of gastrodin (5 mg/kg) or double distilled water once a day for 4 weeks. Blood pressure, pulse wave velocity (PWV), thickness of the abdominal aorta, pathological morphology and differential expression transcripts (DETs) were assessed. Abdominal aorta rings and primary isolated vascular smooth muscle cells were subjected to Ang II stimulation to induce hypertension as ex vivo and in vitro models, respectively. Vascular ring tension, release of Ca2+ and levels of proteins involved in the myosin light chain kinase (MLCK)/phospho-myosin light chain 2 (p-MLC2) pathway were determined. RESULTS: Gastrodin treatment attenuated increases in blood pressure, PWV and thickness of the abdominal aorta. Treatment with gastrodin resulted in 2785 DETs and the enrichment of vascular contraction and calcium signalling pathways. Gastrodin treatment attenuated Ang II-induced vasoconstriction, produced a norepinephrine-precontracted vasodilation effect (attenuated by verapamil), and reduced intracellular Ca2+ release. Furthermore, gastrodin suppressed activation of the MLCK/p-MLC2 pathway in vivo and in vitro. CONCLUSIONS: Gastrodin treatment lowers blood pressure, suppresses Ang II-induced vascular contraction and MLCK/p-MLC2 pathway activation, thereby demonstrating the mechanisms underlying the therapeutic efficacy of gastrodin as an antihypertensive.

Role of MicroRNAs and their corresponding ACE2/Apelin signaling pathways in hypertension.

Hypertension is controlled via the alteration of microRNAs (miRNAs), their therapeutic targets angiotensin II type I receptor (AT1R) and cross talk of signaling pathways. The stimulation of the Ang II/AT1R pathway by deregulation of miRNAs, has also been linked to cardiac remodeling as well as the pathophysiology of high blood pressure. As miRNAs have been associated to ACE2/Apelin and Mitogen-activated protein kinases (MAPK) signaling, it has revealed an utmost protective impact over hypertension and cardiovascular system. The ACE2-coupled intermodulation between RAAS, Apelin system, MAPK signaling pathways, and miRNAs reveal the practicalities of high blood pressure. The research of miRNAs may ultimately lead to the expansion of an innovative treatment strategy for hypertension, which indicates the need to explore them further at the molecular level. Therefore, here we have focused on the mechanistic importance of miRNAs in hypertension, ACE2/Apelin signaling as well as their biological functions, with a focus on interplay and crosstalk between ACE2/Apelin signaling, miRNAs, and hypertension, and the progress in miRNA-based diagnostic techniques with the goal of facilitating the development of new hypertension-controlling therapeutics.

Upregulation of SNTB1 correlates with poor prognosis and promotes cell growth by negative regulating PKN2 in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most highly malignant tumors and has a complicated pathogenesis. A preliminary study identified syntrophin beta 1 (SNTB1) as a potential oncogene in CRC. However, the clinical significance, biological function, and underlying mechanisms of SNTB1 in CRC remain largely unknown. Thus, the present study aimed to investigate the role of SNTB1 in CRC. METHODS: The expression profile of SNTB1 in CRC samples was evaluated by database analysis, cDNA array, tissue microarray, quantitative real-time PCR (qPCR), and immunohistochemistry. SNTB1 expression in human CRC cells was silenced using short hairpin RNAs (shRNA)/small interfering RNAs (siRNA) and its mRNA and protein levels were assessed by qPCR and/or western blotting. Cell viability, survival, cell cycle, and apoptosis were determined by the CCK-8 assay, colony formation, and flow cytometry assays, respectively. A xenograft nude mouse model of CRC was established to validate the roles of SNTB1 in vivo. Immunohistochemistry and TUNEL staining were used to determine the expression of SNTB1, PCNA, and cell apoptosis in tissue samples. Isobaric tag for relative and absolute quantification (iTRAQ) was used to analyze the differentially expressed proteins after knockdown of SNTB1 in CRC cells. Silence of protein kinase N2 (PKN2) using si-PNK2 was performed for rescue experiments. RESULTS: SNTB1 expression was increased in CRC tissues compared with adjacent noncancerous tissues and the increased SNTB1 expression was associated with shorter overall survival of CRC patients. Silencing of SNTB1 suppressed cell viability and survival, induced cell cycle arrest and apoptosis in vitro, and inhibited the growth of CRC cells in vivo. Further elucidation of the regulation of STNB1 on CRC growth by iTRAQ analysis identified 210 up-regulated and 55 down-regulated proteins in CRC cells after SNTB knockdown. A PPI network analysis identified PKN2 as a hub protein and was up-regulated in CRC cells after SNTB1 knockdown. Western-blot analysis further confirmed that SNTB1 knockdown significantly up-regulated PKN2 protein expression in CRC cells and decreased the phosphorylation of both ERK1/2 and AKT. Moreover, rescue experiments indicated that PKN2 knockdown significantly rescued SNTB1 knockdown-mediated decrease in cell viability, survival, and increase of cell cycle arrest at G0/G1 phase and apoptosis of CRC cells. CONCLUSIONS: These findings indicate that SNTB1 is overexpressed in CRC. Elevated SNTB1 levels are correlated with shorter patient survival. Importantly, SNTB1 promotes tumor growth and progression of CRC, possibly by reducing the expression of PKN2 and activating the ERK and AKT signaling pathway. Our study highlights the potential of SNTB1 as a new prognostic factor and therapeutic target for CRC.

Death-associated protein kinase 1 suppresses hepatocellular carcinoma cell migration and invasion by upregulation of DEAD-box helicase 20.

Death-associated protein kinase 1 (DAPK) is a calcium/calmodulin kinase that plays a vital role as a suppressor gene in various cancers. Yet its role and target gene independent of p53 is still unknown in hepatocellular carcinoma (HCC). In this study, we discovered that DAPK suppressed HCC cell migration and invasion instead of proliferation or colony formation. Using a proteomics approach, we identified DEAD-box helicase 20 (DDX20) as an important downstream target of DAPK in HCC cells and critical for DAPK-mediated inhibition of HCC cell migration and invasion. Using integrin inhibitor RGD and GTPase activity assays, we discovered that DDX20 suppressed HCC cell migration and invasion through the CDC42-integrin pathway, which was previously reported as an important downstream pathway of DAPK in cancer. Further research using cycloheximide found that DAPK attenuates the proteasomal degradation of DDX20 protein, which is dependent on the kinase activity of DAPK. Our results shed light on new functions and regulation for both DAPK and DDX20 in carcinogenesis and identifies new potential therapeutic targets for HCC.

Antihypertensive and Vasodilatory Effects of Qingda Granules by Suppression of Calcium Influx and the AKT Pathway.

The Qingda granule (QDG) formulation was simplified from the Qingxuan Jiangya Decoction, which has been used in China to treat hypertension for decades. However, the molecular mechanisms of QDG in antihypertension remain largely unknown. Therefore, we evaluated the therapeutic efficacy of QDG against elevated blood pressure and explored its underlying mechanism. QDG treatment decreased elevated blood pressure and increased the vascular elasticity of thoracic aortic rings to KCl stimulation in spontaneously hypertensive rats. QDG treatment increased the relaxation of isolated thoracic aortic rings precontracted with norepinephrine (NE) or KCl in an endothelium-independent manner, which was attenuated by treatment with verapamil, but not by treatment with TEA, 4-AP, Gli, or BaCl2. Moreover, QDG pretreatment attenuated the CaCl2-induced constriction of isolated thoracic aortic rings in K- or NE-containing Ca-free solutions. In addition, QDG pretreatment significantly inhibited the influx of Ca in A7r5 cells induced by a K- or NE-containing Ca solution and decreased the levels of p-AKT but had no effect on levels of total AKT protein in isolated thoracic aortic rings. Considering these results, QDG treatment attenuated elevated blood pressure and promoted the vasorelaxation of thoracic aortic rings by inhibiting the influx of Ca and activating the AKT pathway.

Qingxuan Jiangya Decoction Reverses Vascular Remodeling by Inducing Vascular Smooth Muscle Cell Apoptosis in Spontaneously Hypertensive Rats.

Qingxuan Jiangya Decoction (QXJYD), a traditional Chinese medicine formula prescribed by academician Ke-ji Chen, has been used in China to clinically treat hypertension for decades of years. However, the molecular mechanisms of its action remain largely unknown. In this study, we examined the therapeutic efficacy of QXJYD against elevated systolic blood pressure in the spontaneously hypertensive rat (SHR) model, and investigated the underlying molecular mechanisms. We found that oral administration of QXJYD significantly reduced the elevation of systolic blood pressure in SHR but had no effect on body weight change. Additionally, QXJYD treatment significantly decreased the media thickness and ratio of media thickness/lumen diameter in the carotid arteries of SHR. Moreover, QXJYD remarkably promoted apoptosis of vascular smooth muscle cells and reduced the expression of anti-apoptotic B-cell leukemia/lymphoma 2. Furthermore, QXJYD significantly decreased the plasma Angiotensin II level in SHR. Collectively, our findings suggest that reversing vascular remodeling via inducing VSMC apoptosis could be one of the mechanisms whereby QXJYD treats hypertension.

Nitidine chloride inhibits hepatic cancer growth via modulation of multiple signaling pathways.

BACKGROUND: The development of hepatic cancer is tightly regulated by multiple intracellular signaling pathways. Therefore, most currently-used anti-tumor agents, which typically target single intracellular pathway, might not always be therapeutically effective. Additionally, long-term use of these agents probably generates drug resistance and unacceptable adverse effects. These problems increase the necessity for the development of new chemotherapeutic approaches. Nitidine chloride (NC), a natural benzophenanthridine alkaloid, has been shown to inhibit cancer growth via induction of cell apoptosis and suppression of cancer angiogenesis. But the precise mechanisms of its tumorcidal activity are not well understood. METHODS: To further elucidate the precise mechanisms of its anti-tumor activity, using a hepatic cancer mouse xenograft model, the human hepatic cancer cell lines (HepG2, HCCLM3, Huh7), and umbilical vein endothelial cells (HUVEC), here we evaluate the effect of NC on tumor growth in vivo and in vitro and investigated the underlying molecular mechanisms. RESULTS: We found that NC treatment resulted in significant decrease in tumor volume and tumor weight respectively, but didn't affect body weight changes. Additionally, NC treatment dose- and time-dependently reduced the cell viability of all three hepatic cell lines. Moreover, NC suppressed the activation of STAT3, ERK and SHH pathways; and altered the expression of critical target genes including Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR2. These molecular effects resulted in the promotion of apoptosis, inhibition of cell proliferation and tumor angiogenesis. CONCLUSIONS: Our findings suggest that NC possesses a broad range of anti-cancer activities due to its ability to affect multiple intracellular targets, suggesting that NC could be a novel multi-potent therapeutic agent for the treatment of hepatic cancer and other cancers.

[Effect of bear bile powder on STAT3 pathway in hepatocellular carcinoma xenograft].

OBJECTIVE: To observe the effect of bear bile powder (BBP) on the STAT3 pathway and its downstream target genes of nude mice hepatocellular carcinoma (HCC) xenograft, and to explore its mechanism for treating HCC. METHODS: The subcutaneous xenograft model was established using HepG2 cells. When the subcutaneous transplanted tumor was formed, naked mice were randomly divided into two groups, the BBP group and the control group. Mice in the BBP group were administered with BBP by gastrogavage, once daily for 3 consecutive weeks, while mice in the control group were administered with normal saline by gastrogavage, once daily for 3 consecutive weeks. The body weight and the tumor volume were measured once per week. By the end of medication, the tumor weight was weighed and the tumor inhibition ratio calculated. The apoptosis of the tumor tissue was detected by TdT-mediated dUTP nick end labeling (TUNEL). The expression of Bcl2-associated X protein (Bax), B cell lymphoma/eukemina-2 (Bcl-2), cyclin-dependent protein kinase (CDK4), cyclinD1 were detected by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression levels of signal transducers and transcription activators 3 (p-STAT3), proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, CDK4, and cyclinD1 were determined by immunohistochemistry. RESULTS: BBP could inhibit the tumor volume and tumor weight, showing statistical difference when compared with the control group (P < 0.01). Results of TUNEL showed that BBP could significantly induce the apoptosis of hepatoma carcinoma cells. Results of RT-PCR showed that BBP could up-regulate the expression of Bax and down-regulate mRNA expression of Bcl-2, CDK4, and cyclinD1. Immunohistochemical results showed that BBP could up-regulate the expression of Bax and inhibit the protein expression of p-STAT3, PCNA, Bcl-2, CDK4, and cyclinD1. CONCLUSION: BBP could induce the apoptosis of hepatoma carcinoma cells and inhibit their proliferation by regulating STAT3 pathway.

Erratum: [Corrigendum] Pien Tze Huang inhibits hypoxia­induced epithelial­mesenchymal transition in human colon carcinoma cells through suppression of the HIF­1 pathway.

[This corrects the article DOI: 10.3892/etm.2014.1549.].

Enhancing the therapeutic potential of isoliensinine for hypertension through PEG-PLGA nanoparticle delivery: A comprehensive in vivo and in vitro study.

BACKGROUND: Hypertension, a highly prevalent chronic disease, is known to inflict severe damage upon blood vessels. In our previous study, isoliensinine, a kind of bibenzyl isoquinoline alkaloid which isolated from a TCM named Lotus Plumule (Nelumbo nucifera Gaertn), exhibits antihypertensive and vascular smooth muscle proliferation-inhibiting effects, but its application is limited due to poor water solubility and low bioavailability. In this study, we proposed to prepare isoliensinine loaded by PEG-PLGA polymer nanoparticles to increase its efficacy METHOD: We synthesized and thoroughly characterized PEG-PLGA nanoparticles loaded with isoliensinine using a nanoprecipitation method, denoted as, PEG-PLGA@Isoliensinine. Additionally, we conducted comprehensive investigations into the stability of PEG-PLGA@Isoliensinine, in vitro drug release profiles, and in vivo pharmacokinetics. Furthermore, we assessed the antihypertensive efficacy of this nano-system through in vitro experiments on A7R5 cells and in vivo studies using AngII-induced mice. RESULT: The findings reveal that PEG-PLGA@Isoliensinine significantly improves isoliensinine absorption by A7R5 cells and enhances targeted in vivo distribution. This translates to a more effective reduction of AngII-induced hypertension and vascular smooth muscle proliferation. CONCLUSION: In this study, we successfully prepared PEG-PLGA@Isoliensinine by nano-precipitation, and we confirmed that PEG-PLGA@Isoliensinine surpasses free isoliensinine in its effectiveness for the treatment of hypertension, as demonstrated through both in vivo and in vitro experiments. SIGNIFICANCE: This study lays the foundation for isoliensinine's clinical use in hypertension treatment and vascular lesion protection, offering new insights for enhancing the bioavailability of traditional Chinese medicine components. Importantly, no toxicity was observed, affirming the successful implementation of this innovative drug delivery system in vivo and offers a promising strategy for enhancing the effectiveness of Isoliensinine and propose an innovative avenue for developing novel formulations of traditional Chinese medicine monomers.

Effects of Liqi Tongbian decoction on gut microbiota, SCFAs production, and 5-HT pathway in STC rats with Qi Stagnation Pattern.

Slow transit constipation (STC) is a common and debilitating condition characterized by delayed colonic transit and difficulty in fecal expulsion, significantly impacting patients' physical and mental wellbeing as well as their overall quality of life. This study investigates the therapeutic potential of Liqi Tongbian Decoction (LTD) in the treatment of STC, especially in cases involving the context of Qi stagnation, through a multifaceted approach involving the modulation of intestinal flora and short-chain fatty acids (SCFAs). We employed a rat model of STC with Qi Stagnation Pattern, established using the "loperamide + tail-clamping provocation method," to explore the effects of LTD on fecal characteristics, intestinal motility, and colonic pathology. Importantly, LTD exhibited the ability to increase the richness, diversity, and homogeneity of intestinal flora while also modulating the composition of microorganisms. It significantly increased the production of SCFAs, especially butyric acid. Moreover, LTD exerted a substantial influence on the synthesis of serotonin (5-HT) by modulating the expression of tryptophan hydroxylase (TPH) and interacting with the 5-HT4 receptor (5-HT4R), resulting in enhanced colonic motility. Correlation analyses revealed a positive correlation between certain bacterial genera, such as Lachnospiraceae_NK4A136 spp. and Clostridiales spp. and the concentrations of butyric acid and 5-HT. These results suggest a mechanistic link between microbiome composition, SCFAs production, and 5-HT synthesis. These findings highlight the potential of LTD to alleviate STC by facilitating a beneficial interplay among intestinal flora, SCFAs production, and 5-HT-mediated colonic motility, providing novel insights into the management of STC with Qi Stagnation Pattern.

Pien Tze Huang Inhibits Proliferation of Colorectal Cancer Cells through Suppressing PNO1 Expression and Activating p53/p21 Signaling Pathway.

OBJECTIVE: To explore the regulatory effect of Pien Tze Huang (PZH) on targeting partner of NOB1 (PNO1) and it's down-stream mediators in colorectal cancer (CRC) cells. METHODS: Quantitative polymerase chain reaction was performed to determine mRNA levels of PNO1, TP53, and CDKN1A. Western blotting was performed to determine protein levels of PNO1, p53, and p21. HCT-8 cells were transduced with a lentivirus over-expressing PNO1. Colony formation assay was used to detect cell survival in PNO1 overexpression of HCT-8 cells after PZH treatment. Cell-cycle distribution, cell viability and cell apoptosis were performed to identify the effect of PNO1 overexpression on cell proliferation and apoptosis of HCT-8 cells after PZH treatment. Xenograft BALB/c nude mice bearing HCT116 cells transduced with sh-PNO1 or sh-Ctrl lentivirus were evaluated. Western blot assay was performed to detect PNO1, p53, p21 and PCNA expression in tumor sections. Terminal deoxynucleotidyl transferase dUTP nick end labling (TUNEL) assay was used to determine the apoptotic cells in tissues. RESULTS: PZH treatment decreased cell viability, down-regulated PNO1 expression, and up-regulated p53 and p21 expressions in HCT-8 cells (P<0.05). PNO1 overexpression attenuated the effects of PZH treatment, including the expression of p53 and p21, cell growth, cell viability, cell cycle arrest and cell apoptosis in vitro (P<0.05). PNO1 knockdown eliminated the effects of PZH treatment on tumor growth, inhibiting cell proliferation inhibition and apoptosis induction in vivo (P<0.05). Similarly, PNO1 knockdown attenuated the effects of PZH treatment on the down-regulation of PNO1 and up-regulation of p53 and p21 in vivo (P<0.05). CONCLUSION: The mechanism by which PZH induces its CRC anti-proliferative effect is at least in part by regulating the expression of PNO1 and its downstream targets p53 and p21.

Neferine ameliorates hypertensive vascular remodeling modulating multiple signaling pathways in spontaneously hypertensive rats.

BACKGROUND: Neferine exhibits therapeutic effects on anti-hypertension. However, the effect of neferine on hypertensive vascular remodeling remains unexplored. Therefore, the current study was to investigate the effect of neferine on hypertensive vascular remodeling and its underlying mechanisms. METHODS: Total 30 male spontaneously hypertensive rats (SHRs) were divided randomly into five groups, including SHR, Neferine-L (2.5 mg/kg/day), Neferine-M (5 mg/kg/day), Neferine-H (10 mg/kg/day), and Valsartan (10 mg/kg/day) groups (n = 6 for each group). Wistar Kyoto (WKY) rats were set as control group (n = 6). Noninvasive blood pressure system, ultrasound, hematoxylin and eosin staining, masson trichrome staining were used to detect the blood pressure, pulse wave velocity (PWV), pathological changes and collagen content in abdominal aortas of SHRs. RNA-sequencing and immunohistochemistry(IHC) analyses were used to identify and verify the differentially expressed transcripts and activation of associated signaling pathways in SHRs. RESULTS: Various concentrations of neferine or valsartan treatment substantially reduced the elevation of blood pressure, PWV, and abdominal aortic thickening of SHRs. RNA-sequencing and KEGG analyses recognized 441 differentially expressed transcripts and several enriched pathways (including PI3K/AKT and TGF-ß/Smad2/3 signaling pathways) after neferine treatment. Masson trichromatic staining and IHC analysis demonstrated that neferine treatment decreased the collagen content and down-regulated the protein expression of PCNA, collagen I & III, and fibronectin, as well as p-PI3K, p-AKT, TGF-ß1 and p-Smad2/3 in abdominal aortic tissues of SHRs. CONCLUSION: Neferine treatment exhibits therapeutic effects on anti-hypertension and reduces vascular remodeling, as well as suppresses the abnormal activation of multiple signaling pathways, including PI3K/AKT and TGF-ß1/Smad2/3 pathways.

Based on network pharmacology, gastrodin attenuates hypertension-induced vascular smooth muscle cell proliferation and PI3K/AKT pathway activation.

The effects and underlying mechanisms of gastrodin treatment on hypertensive vascular dysfunction and proliferation of vascular smooth muscle cells (VSMCs) were determined in vitro and in vivo. Using a pharmacological target network interaction analysis, 151 common targets and a PPI network were identified containing the top 10 hub genes. Kyoto encyclopedia of genes and genomes (KEGG) analysis identified the PI3K/AKT pathway as a significantly enriched pathway. Both spontaneous hypertensive rats (SHRs) and Wistar Kyoto rats were used to assess the therapeutic effects of gastrodin on hypertension. Gastrodin treatment of the SHRs resulted in a marked attenuation of elevated blood pressure, pulse wave velocity, and pathological changes in the abdominal aorta. Moreover, gastrodin treatment significantly inhibited cell growth and downregulated the expression of PCNA as well as the p-PI3K/PI3K and p-AKT/AKT levels in angiotensin II-stimulated VSMCs. Taken together, gastrodin treatment attenuates blood pressure elevation, vascular dysfunction, and proliferation of VSMCs and inhibits the activation of the PI3K/AKT pathway.

Qing Hua Chang Yin alleviates chronic colitis of mice by protecting intestinal barrier function and improving colonic microflora.

Background: Qing Hua Chang Yin (QHCY) is a famous formula of traditional Chinese medicine (TCM) and has been proven to have protective effect on ulcerative colitis. However, its protective effect and potential therapeutic mechanisms in chronic colitis remain unclear. The purpose of this study is to explore the effects and underlying mechanisms of QHCY on dextran sulfate sodium (DSS)-induced chronic colitis mice model. Methods: The chronic colitis model was established by administration of 2% DSS for three consecutive cycles of 7 days with two intervals of 14 days for recovery by drinking water. The experiment lasted 49 days. The DSS + QHCY group received QHCY administration by oral gavage at doses of 1.6 g/kg/d, DSS + Mesalazine group was administrated Mesalazine by oral gavage at doses of 0.2 g/kg/d. The control and DSS group were given equal volume of distilled water. The body weight, stool consistency and blood in stool were monitored every 2 days. The disease activity index (DAI) was calculated. The colon length was measured after the mice were sacrificed. The histomorphology of colonic tissues was checked by the HE and PAS staining. Immunohistochemistry was performed to detect the expressions of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6), tight junction proteins (ZO-1, occludin) and Mucin2 (MUC2). 16S rRNA sequencing analysis was conducted to study the diversity and abundance of gut microbiota changes. Results: QHCY treatment not only significantly attenuated DSS-induced the weight loss, DAI score increase, colon shortening and histological damage in mice, but also decreased the expression of pro-inflammatory cytokines in colonic tissues and increased the expression of ZO-1, occludin, and MUC2. Furthermore, QHCY enhanced the diversity of gut microbes and regulated the structure and composition of intestinal microflora in mice with chronic colitis. Conclusion: QHCY has a therapeutic effect on a murine model of chronic colitis. It can effectively reduce the clinical and pathological manifestations of colitis and prevent alterations in the gut microbiota.

Qingda granule ameliorates vascular remodeling and phenotypic transformation of adventitial fibroblasts via suppressing the TGF-ß1/Smad2/3 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingda granule (QDG) exhibits significant therapeutic effects on high blood pressure, vascular dysfunction, and elevated proliferation of vascular smooth muscle cells by inhibiting multiple pathways. However, the effects and underlying mechanisms of QDG treatment on hypertensive vascular remodeling are unclear. AIM OF THE STUDY: The aim of this study was to determine the role of QDG treatment in hypertensive vascular remodeling in vivo and in vitro. MATERIALS AND METHODS: An ACQUITY UPLC I-Class system coupled with a Xevo XS quadrupole time of flight mass spectrometer was used to characterize the chemical components of QDG. Twenty-five spontaneously hypertensive rats (SHR) were randomly divided into five groups, including SHR (equal volume of double distilled water, ddH2O), SHR + QDG-L (0.45 g/kg/day), SHR + QDG-M (0.9 g/kg/day), SHR + QDG-H (1.8 g/kg/day), and SHR + Valsartan (7.2 mg/kg/day) groups. QDG, Valsartan, and ddH2O were administered intragastrically once a day for 10 weeks. For the control group, ddH2O was intragastrically administered to five Wistar Kyoto rats (WKY group). Vascular function, pathological changes, and collagen deposition in the abdominal aorta were evaluated using animal ultrasound, hematoxylin and eosin and Masson staining, and immunohistochemistry. Isobaric tags for relative and absolute quantification (iTRAQ) was performed to identify differentially expressed proteins (DEPs) in the abdominal aorta, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Cell Counting Kit-8 assays, phalloidin staining, transwell assays, and western-blotting were performed to explore the underlying mechanisms in primary isolated adventitial fibroblasts (AFs) stimulated with transforming growth factor-ß 1 (TGF-ß1) with or without QDG treatment. RESULTS: Twelve compounds were identified from the total ion chromatogram fingerprint of QDG. In the SHR group, QDG treatment significantly attenuated the increased pulse wave velocity, aortic wall thickening, and abdominal aorta pathological changes and decreased Collagen I, Collagen III, and Fibronectin expression. The iTRAQ analysis identified 306 DEPs between SHR and WKY and 147 DEPs between QDG and SHR. GO and KEGG pathway analyses of the DEPs identified multiple pathways and functional processes involving vascular remodeling, including the TGF-ß receptor signaling pathway. QDG treatment significantly attenuated the increased cell migration, actin cytoskeleton remodeling, and Collagen I, Collagen III, and Fibronectin expression in AFs stimulated with TGF-ß1. QDG treatment significantly decreased TGF-ß1 protein expression in abdominal aortic tissues in the SHR group and p-Smad2 and p-Smad3 protein expression in TGF-ß1-stimulated AFs. CONCLUSIONS: QDG treatment attenuated hypertension-induced vascular remodeling of the abdominal aorta and phenotypic transformation of adventitial fibroblasts, at least partly by suppressing TGF-ß1/Smad2/3 signaling.

Gastrodin attenuates renal injury and collagen deposition via suppression of the TGF-ß1/Smad2/3 signaling pathway based on network pharmacology analysis.

Background: Gastrodin has been widely used clinically in China as an antihypertensive drug. However, its effect on hypertensive renal injury is yet to be elucidated. The current study aimed to investigate the effects of gastrodin on hypertensive renal injury and its underlying mechanisms by network pharmacology analysis and validation in vivo and in vitro. Methods: A total of 10 spontaneously hypertensive rats (SHRs) were randomly categorized into the following two groups: SHR and SHR + Gastrodin groups. Wistar Kyoto (WKY) rats were used as the control group (n = 5). The SHR + Gastrodin group was intragastrically administered gastrodin (3.5 mg/kg/day), and the rats in both WKY and SHR groups were intragastrically administered an equal amount of double-distilled water for 10 weeks. Hematoxylin-eosin, Masson's trichrome, and Sirius red staining were used to detect the pathological changes and collagen content in the renal tissues. Network pharmacology analysis was performed to explore its potential targets and related pathways. In vitro, the CCK-8 assay was used to determine the cell viability. Immunohistochemistry and western-blotting analyses were employed to assess the protein expression associated with renal fibrosis and transforming growth factor-ß1 (TGF-ß1) pathway-related proteins in the renal tissues or in TGF-ß1-stimulated rat kidney fibroblast cell lines (NRK-49F). Results: Gastrodin treatment attenuates renal injury and pathological alterations in SHRs, including glomerular sclerosis and atrophy, epithelial cell atrophy, and tubular dilation. Gastrodin also reduced the accumulation of collagen in the renal tissues of SHRs, which were confirmed by downregulation of α-SMA, collagen I, collagen III protein expression. Network pharmacology analysis identified TGFB1 and SMAD2 as two of lead candidate targets of gastrodin on against hypertensive renal injury. Consistently, gastrodin treatment downregulated the increase of the protein expression of TGF-ß1, and ratios of both p-Smad2/Smad2 and p-Samd3/Smad3 in renal tissues of SHRs. In vitro, gastrodin (25-100 µM) treatment significantly reversed the upregulation of α-SMA, fibronectin, collagen I, as well as p-Smad2 and p-Smad3 protein expressions without affecting the cell viability of TGF-ß1 stimulated NRK-49F cells. Conclusion: Gastrodin treatment significantly attenuates hypertensive renal injury and renal fibrosis and suppresses TGF-ß1/Smad2/3 signaling in vivo and in vitro.

Isoliensinine Attenuates Renal Fibrosis and Inhibits TGF-ß1/Smad2/3 Signaling Pathway in Spontaneously Hypertensive Rats.

Purpose: This study aimed to investigate the molecular mechanisms of isoliensinine, a kind of bibenzyl isoquinoline alkaloid which isolated from a TCM named Lotus Plumule (Nelumbo nucifera Gaertn), in treating renal interstitial fibrosis (RIF) by using RNA sequencing, KEGG analysis and in vivo experimental approaches. Methods: Spontaneous hypertension rats (SHRs) were randomly assigned into five groups, consisting of SHR, SHR+Isoliensinine-L (2.5 mg/kg/day), SHR+Isoliensinine-M (5 mg/kg/day), SHR+Isoliensinine-H (10 mg/kg/day), and SHR+Valsartan (10 mg/kg/day) groups (n = 6 for each group). A control group of Wistar Kyoto rats (n = 6) was also included. Rats were treated intragastrically with isoliensinine, valsartan, or double-distilled water of equal volume for 10 weeks. To examine the therapeutic impact on hypertensive renal injury, fibrosis, and its underlying mechanisms, multiple techniques were employed, including hematoxylin and eosin staining, Masson trichrome staining, RNA sequencing, gene ontology (GO) function and pathway enrichment analysis and immunohistochemistry. Results: Resultantly, the use of isoliensinine at different concentrations or valsartan showed significant improvement in renal pathological injury in SHRs. RNA sequencing and KEGG analysis uncovered 583 differentially expressed transcripts and pathways enriched in collagen formation and ECM-receptor interaction after treatment with isoliensinine. There was also a reduction in the increase of collagen and upregulation of collagen I & III, TGF-ß1, p-Smad2, and p-Smad3 in the renal tissue of SHRs. Thus, isoliensinine ameliorated renal injury and collagen deposition in hypertensive rats, and inhibiting the activation of the TGF-ß1/Smad2/3 pathway might be one of the underlying mechanisms. Conclusion: This study showed that treatment with isoliensinine effectively reduced the renal injury and fibrosis in SHRs. In addition, isoliensinine inhibited the TGF-ß1/Smad2/3 signaling in-vivo. These findings provided strong evidence for the therapeutic benefits of isoliensinine in combating renal injury and fibrosis.