Five-layer-funnel filtering mode discovers effective components of Chinese medicine formulas: Zhishi-Xiebai-Guizhi decoction as a case study.

BACKGROUND: How to screen and identify the effective components in the complex substance system is one of the core issues in achieving the modernization of traditional Chinese medicine (TCM) formulas. However, it is still challenging to systematically screen out the effective components from the hundreds or thousands of components in a TCM formula. PURPOSE: An innovative five-layer-funnel filtering mode stepwise integrating chemical profile, quantitative analysis, xenobiotic profile, network pharmacology and bioactivity evaluation was successfully presented to discover the effective components and implemented on a case study of Zhishi-Xiebai-Guizhi decoction (ZXG), a well-known TCM formula for coronary heart disease (CHD). METHODS: Initially, the chemical profile of ZXG was systemically characterized. Subsequently, the representative constituents were quantitatively analyzed. In the third step, the multi-component xenobiotics profile of ZXG was systemically delineated, and the prototypes absorbed into the blood were identified and designated as the primary bioavailable components. Next, an integrated network of "bioavailable components-CHD targets-pathways-therapeutic effects" was constructed, and the crucial bioavailable components of ZXG against CHD were screened out. Lastly, the bioactivities of crucial bioavailable components were further evaluated to pinpoint effective components. RESULTS: First of all, the chemical profile of ZXG was systemically characterized with the detection of 201 components. Secondly, 37 representative components were quantified to comprehensively describe its content distribution characteristics. Thirdly, among the quantified components, 24 bioavailable components of ZXG were identified based on the multi-component xenobiotic profile. Fourthly, an integrated network led to the identification of 11 crucial bioavailable components against CHD. Ultimately, 9 components (honokiol, magnolol, naringenin, magnoflorine, hesperidin, hesperetin, naringin, neohesperidin and narirutin) exhibiting myocardial protection in vitro were identified as effective components of ZXG for the first time. CONCLUSION: Overall, this innovative strategy successfully identified the effective components of ZXG for the first time. It could not only significantly contribute to elucidating the therapeutic mechanism of ZXG in the treatment of CHD, but also serve as a helpful reference for the systematic discovery of effective components as well as ideal quality markers in the quality assessment of TCM formulas.

Farnesoid X Receptor Protects Murine Lung against IL-6-promoted Ferroptosis Induced by Polyriboinosinic-Polyribocytidylic Acid.

Various infections trigger a storm of proinflammatory cytokines in which IL-6 acts as a major contributor and leads to diffuse alveolar damage in patients. However, the metabolic regulatory mechanisms of IL-6 in lung injury remain unclear. Polyriboinosinic-polyribocytidylic acid [poly(I:C)] activates pattern recognition receptors involved in viral sensing and is widely used in alternative animal models of RNA virus-infected lung injury. In this study, intratracheal instillation of poly(I:C) with or without an IL-6-neutralizing antibody model was combined with metabonomics, transcriptomics, and so forth to explore the underlying molecular mechanisms of IL-6-exacerbated lung injury. We found that poly(I:C) increased the IL-6 concentration, and the upregulated IL-6 further induced lung ferroptosis, especially in alveolar epithelial type II cells. Meanwhile, lung regeneration was impaired. Mechanistically, metabolomic analysis showed that poly(I:C) significantly decreased glycolytic metabolites and increased bile acid intermediate metabolites that inhibited the bile acid nuclear receptor farnesoid X receptor (FXR), which could be reversed by IL-6-neutralizing antibody. In the ferroptosis microenvironment, IL-6 receptor monoclonal antibody tocilizumab increased FXR expression and subsequently increased the Yes-associated protein (YAP) concentration by enhancing PKM2 in A549 cells. FXR agonist GW4064 and liquiritin, a potential natural herbal ingredient as an FXR regulator, significantly attenuated lung tissue inflammation and ferroptosis while promoting pulmonary regeneration. Together, the findings of the present study provide the evidence that IL-6 promotes ferroptosis and impairs regeneration of alveolar epithelial type II cells during poly(I:C)-induced murine lung injury by regulating the FXR-PKM2-YAP axis. Targeting FXR represents a promising therapeutic strategy for IL-6-associated inflammatory lung injury.

Venous Thrombosis Assay in a Mouse Model of Cancer.

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis (CAT). Deep venous thrombosis is a common complication in cancer survivors and can be potentially fatal. The current murine venous thrombosis models typically involve a complete or partial mechanical occlusion of the inferior vena cava (IVC) using a suture. This procedure induces a total or partial stasis of blood and endothelial damage, triggering thrombogenesis. The current models have limitations such as higher variability in clot weights, significant mortality rate, and prolonged learning curve. This report introduces surgical refinements using vascular clips to address some of these limitations. Using a syngeneic colon cancer xenograft mouse model, we employed customized vascular clips to ligate the infrarenal vena cava. These clips allow residual lip space similar to a 5-0 polypropylene suture after IVC ligations. Mice with the suture method served as controls. The vascular clip method resulted in a consistent reproducible partial vascular occlusion and greater clot weights with less variability than the suture method. The larger clot weights, greater clot mass, and clot to the IVC luminal surface area were expected due to the higher pressure profile of the vascular clips compared to a 6-0 polypropylene suture. The approach was validated by gray scale ultrasonography, which revealed consistently greater clot mass in the infrarenal vena cava with vascular clips compared to the suture method. These observations were further substantiated with the immunofluorescence staining. This study offers an improved method to generate a venous thrombosis model in mice, which can be employed to deepen the mechanistic understanding of CAT and in translational research such as drug discovery.

Chanling Gao suppresses colorectal cancer via PI3K/Akt/mTOR pathway modulation and enhances quality of survival.

The Chinese medicine formula Chanling Gao (CLG) exhibits significant tumor inhibitory effects in colorectal cancer (CRC) nude mice. However, the detailed mechanisms remain elusive. CRC in situ nude mouse models were treated with CLG. Small animal magnetic resonance imaging (MRI) tracked tumor progression, and overall health metrics such as food and water intake, body weight, and survival were monitored. Posttreatment, tissues and blood were analyzed for indicators of tumor inhibition and systemic effects. Changes in vital organs were observed via stereoscope and hematoxylin-eosin staining. Immunohistochemistry quantified HIF-1α and P70S6K1 protein expression in xenografts. Double labeling was used to statistically analyze vascular endothelial growth factor (VEGF) and CD31 neovascularization. Enzyme-linked immunosorbent assay was used to determine the levels of VEGF, MMP-2, MMP-9, IL-6, and IL-10 in serum, tumors, and liver. Western blotting was used to assess the expression of the PI3K/Akt/mTOR signaling pathway-related factors TGF-ß1 and smad4 in liver tissues. CLG inhibited tumor growth, improved overall health metrics, and ameliorated abnormal blood cell counts in CRC nude mice. CLG significantly reduced tumor neovascularization and VEGF expression in tumors and blood. It also suppressed HIF-1α, EGFR, p-PI3K, Akt, p-Akt, and p-mTOR expression in tumors while enhancing PTEN oncogene expression. Systemic improvements were noted, with CLG limiting liver metastasis, reducing pro-inflammatory cytokines IL-6 and IL-10 in liver tissues, decreasing MMP-2 in blood and MMP-2 and MMP-9 in tumors, and inhibiting TGF-ß1 expression in liver tissues. CLG can enhance survival quality and inhibit tumor growth in CRC nude mice, likely through the regulation of the PI3K/Akt/mTOR signaling pathway.

Sorafenib induces cardiotoxicity through RBM20-mediated alternative splicing of sarcomeric and mitochondrial genes.

Sorafenib, a multi-targeted tyrosine kinase inhibitor, is a first-line treatment for advanced solid tumors, but it induces many adverse cardiovascular events, including myocardial infarction and heart failure. These cardiac defects can be mediated by alternative splicing of genes critical for heart function. Whether alternative splicing plays a role in sorafenib-induced cardiotoxicity remains unclear. Transcriptome of rat hearts or human cardiomyocytes treated with sorafenib was analyzed and validated to define alternatively spliced genes and their impact on cardiotoxicity. In rats, sorafenib caused severe cardiotoxicity with decreased left ventricular systolic pressure, elongated sarcomere, enlarged mitochondria and decreased ATP. This was associated with alternative splicing of hundreds of genes in the hearts, many of which were targets of a cardiac specific splicing factor, RBM20. Sorafenib inhibited RBM20 expression in both rat hearts and human cardiomyocytes. The splicing of RBM20's targets, SLC25A3 and FHOD3, was altered into fetal isoforms with decreased function. Upregulation of RBM20 during sorafenib treatment reversed the pathogenic splicing of SLC25A3 and FHOD3, and enhanced the phosphate transport into mitochondria by SLC25A3, ATP synthesis and cell survival.We envision this regulation may happen in many drug-induced cardiotoxicity, and represent a potential druggable pathway for mitigating sorafenib-induced cardiotoxicity.

Redox Dysregulation of Vascular Smooth Muscle Sirtuin-1 in Thoracic Aortic Aneurysm in Marfan Syndrome.

BACKGROUND: Thoracic aortic aneurysms (TAAs) are abnormal aortic dilatations and a major cardiovascular complication of Marfan syndrome. We previously demonstrated a critical role for vascular smooth muscle (VSM) SirT1 (sirtuin-1), a lysine deacetylase, against maladaptive aortic remodeling associated with chronic oxidative stress and aberrant activation of MMPs (matrix metalloproteinases). METHODS: In this study, we investigated whether redox dysregulation of SirT1 contributed to the pathogenesis of TAA using fibrillin-1 hypomorphic mice (Fbn1mgR/mgR), an established model of Marfan syndrome prone to aortic dissection/rupture. RESULTS: Oxidative stress markers 3-nitrotyrosine and 4-hydroxynonenal were significantly elevated in aortas of patients with Marfan syndrome. Moreover, reversible oxidative post-translational modifications (rOPTM) of protein cysteines, particularly S-glutathionylation, were dramatically increased in aortas of Fbn1mgR/mgR mice, before induction of severe oxidative stress markers. Fbn1mgR/mgR aortas and VSM cells exhibited an increase in rOPTM of SirT1, coinciding with the upregulation of acetylated proteins, an index of decreased SirT1 activity, and increased MMP2/9 activity. Mechanistically, we demonstrated that TGFß (transforming growth factor beta), which was increased in Fbn1mgR/mgR aortas, stimulated rOPTM of SirT1, decreasing its deacetylase activity in VSM cells. VSM cell-specific deletion of SirT1 in Fbn1mgR/mgR mice (SMKO-Fbn1mgR/mgR) caused a dramatic increase in aortic MMP2 expression and worsened TAA progression, leading to aortic rupture in 50% of SMKO-Fbn1mgR/mgR mice, compared with 25% of Fbn1mgR/mgR mice. rOPTM of SirT1, rOPTM-mediated inhibition of SirT1 activity, and increased MMP2/9 activity were all exacerbated by the deletion of Glrx (glutaredoxin-1), a specific deglutathionylation enzyme, while being corrected by overexpression of Glrx or of an oxidation-resistant SirT1 mutant in VSM cells. CONCLUSIONS: Our novel findings strongly suggest a causal role of S-glutathionylation of SirT1 in the pathogenesis of TAA. Prevention or reversal of SirT1 rOPTM may be a novel therapeutic strategy to prevent TAA and TAA dissection/ruptures in individuals with Marfan syndrome, for which, thus far, no targeted therapy has been developed.

Targeting hepatic serine-arginine protein kinase 2 ameliorates alcohol-associated liver disease by alternative splicing control of lipogenesis.

BACKGROUND AND AIMS: Lipid accumulation induced by alcohol consumption is not only an early pathophysiological response but also a prerequisite for the progression of alcohol-associated liver disease (ALD). Alternative splicing regulates gene expression and protein diversity; dysregulation of this process is implicated in human liver diseases. However, how the alternative splicing regulation of lipid metabolism contributes to the pathogenesis of ALD remains undefined. APPROACH AND RESULTS: Serine-arginine-rich protein kinase 2 (SRPK2), a key kinase controlling alternative splicing, is activated in hepatocytes in response to alcohol, in mice with chronic-plus-binge alcohol feeding, and in patients with ALD. Such induction activates sterol regulatory element-binding protein 1 and promotes lipogenesis in ALD. Overexpression of FGF21 in transgenic mice abolishes alcohol-mediated induction of SRPK2 and its associated steatosis, lipotoxicity, and inflammation; these alcohol-induced pathologies are exacerbated in FGF21 knockout mice. Mechanistically, SRPK2 is required for alcohol-mediated impairment of serine-arginine splicing factor 10, which generates exon 7 inclusion in lipin 1 and triggers concurrent induction of lipogenic regulators-lipin 1ß and sterol regulatory element-binding protein 1. FGF21 suppresses alcohol-induced SRPK2 accumulation through mammalian target of rapamycin complex 1 inhibition-dependent degradation of SRPK2. Silencing SRPK2 rescues alcohol-induced splicing dysregulation and liver injury in FGF21 knockout mice. CONCLUSIONS: These studies reveal that (1) the regulation of alternative splicing by SRPK2 is implicated in lipogenesis in humans with ALD; (2) FGF21 is a key hepatokine that ameliorates ALD pathologies largely by inhibiting SRPK2; and (3) targeting SRPK2 signaling by FGF21 may offer potential therapeutic approaches to combat ALD.

CD31 as a probable responding and gate-keeping protein of the blood-brain barrier and the risk of Alzheimer's disease.

Several studies have shown that an abnormal vascular-immunity link could increase Alzheimer's disease (AD) risk; however, the mechanism is unclear. CD31, also named platelet endothelial cell adhesion molecule (PECAM), is a surface membrane protein of both endothelial and immune cells and plays important roles in the interaction between the vascular and immune systems. In this review, we focus on research regarding CD31 biological actions in the pathological process that may contribute to AD based on the following rationales. First, endothelial, leukocyte and soluble forms of CD31 play multi-roles in regulating transendothelial migration, increasing blood-brain barrier (BBB) permeability and resulting in neuroinflammation. Second, CD31 expressed by endothelial and immune cells dynamically modulates numbers of signaling pathways, including Src family kinases, selected G proteins, and ß-catenin which in turn affect cell-matrix and cell-cell attachment, activation, permeability, survival, and ultimately neuronal cell injury. In endothelia and immune cells, these diverse CD31-mediated pathways act as a critical regulator in the immunity-endothelia-brain axis, thereby mediating AD pathogenesis in ApoE4 carriers, which is the major genetic risk factor for AD. This evidence suggests a novel mechanism and potential drug target for CD31 in the background of genetic vulnerabilities and peripheral inflammation for AD development and progression.

[Application of "major scientific issues and engineering technology difficulties in traditional Chinese medicine(2019-2021)" in national science and t echnology layout].

In order to judge the future development trend of science and technology, plan ahead and lay out the frontier technology fields and directions, China Association of Chinese Medicine(CACM) has launched consultation projects for collecting "major scienti-fic issues and engineering technology difficulties in traditional Chinese medicine(TCM)" for the industry for three consecutive years since 2019. Up to now, 18 projects have been selected as major issues for research, and some experience and achievements have been made. These projects have been applied in important scientific and technological work such as scientific and technological planning and deployment at all levels of national, local, and scientific research institutions, the selection and cultivation of major national scientific and technological projects, and the construction of innovation bases, giving full play to the role of the think tank advisory committee of CACM. This study reviewed the selection of major issues for the first time, systematically combed its application in the national layout of science and technology, and put forward the existing problems and improvement suggestions, aiming to provide new ideas for further improving the selection of major issues and research direction, providing a theoretical basis and decision support for the national scientific and technological layout in the field of TCM, and promoting scientific and technological innovation to facilitate the high quality development of TCM.

CD34+ cell-derived fibroblast-macrophage cross-talk drives limb ischemia recovery through the OSM-ANGPTL signaling axis.

CD34+ cells improve the perfusion and function of ischemic limbs in humans and mice. However, there is no direct evidence of the differentiation potential and functional role of these cells in the ischemic muscle microenvironment. Here, we combined the single-cell RNA sequencing and genetic lineage tracing technology, then provided exact single-cell atlases of normal and ischemic limb tissues in human and mouse, and consequently found that bone marrow (BM)-derived macrophages with antigen-presenting function migrated to the ischemic site, while resident macrophages underwent apoptosis. The macrophage oncostatin M (OSM) regulatory pathway was specifically turned on by ischemia. Simultaneously, BM CD34+-derived proregenerative fibroblasts were recruited to the ischemia niche, where they received macrophage-released OSM and promoted angiopoietin-like protein-associated angiogenesis. These findings provided mechanisms on the cellular events and cell-cell communications during tissue ischemia and regeneration and provided evidence that CD34+ cells serve as fibroblast progenitors promoting tissue regeneration.

Age-dependent loss of hepatic SIRT1 enhances NLRP3 inflammasome signaling and impairs capacity for liver fibrosis resolution.

Our studies indicate that the longevity factor SIRT1 is implicated in metabolic disease; however, whether and how hepatocyte-specific SIRT1 signaling is involved in liver fibrosis remains undefined. We characterized a functional link of age-mediated defects in SIRT1 to the NLRP3 inflammasome during age-related liver fibrosis. In multiple experimental murine models of liver fibrosis, we compared the development of liver fibrosis in young and old mice, as well as in liver-specific SIRT1 knockout (SIRT1 LKO) mice and wild-type (WT) mice. Liver injury, fibrosis, and inflammation were assessed histologically and quantified by real-time PCR analysis. In a model of hepatotoxin-induced liver fibrosis, old mice displayed more severe and persistent liver fibrosis than young mice during liver injury and after injury cessation, as characterized by inhibition of SIRT1, induction of NLRP3, infiltration of macrophages and neutrophils, activation of hepatic stellate cells (HSCs), and excessive deposition and remodeling of the extracellular matrix. Mechanistically, deletion of SIRT1 in hepatocytes resulted in NLRP3 and IL-1ß induction, pro-inflammatory response, and severe liver fibrosis in young mice, mimicking the ability of aging to impair the resolution of established fibrosis. In an aging mouse model, chronic-plus-binge alcohol feeding-induced liver fibrosis was attenuated by treatment with MCC950, a selective NLRP3 inhibitor. NLRP3 inhibition ameliorated alcoholic liver fibrosis in old mice by repressing inflammation and reducing hepatocyte-derived danger signaling-ASK1 and HMGB1. In conclusion, age-dependent SIRT1 defects lead to NLRP3 activation and inflammation, which in turn impairs the capacity to resolve fibrosis during aging.

CX08005, a Protein Tyrosine Phosphatase 1B Inhibitor, Attenuated Hepatic Lipid Accumulation and Microcirculation Dysfunction Associated with Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is one of the common metabolic diseases characterized by hepatic lipid accumulation. Insulin resistance and microcirculation dysfunction are strongly associated with NAFLD. CX08005, an inhibitor of PTP1B with the IC50 of 0.75 ± 0.07 µM, has been proven to directly enhance insulin sensitivity. The present study aimed to investigate the effects of CX08005 on hepatic lipid accumulation and microcirculation dysfunction in both KKAy mice and diet-induced obesity (DIO) mice. Hepatic lipid accumulation was evaluated by hepatic triglyceride determination and B-ultrasound analysis in KKAy mice. Insulin sensitivity and blood lipids were assessed by insulin tolerance test (ITT) and triglyceride (TG)/total cholesterol (TC) contents, respectively. In addition, the hepatic microcirculation was examined in DIO mice by in vivo microscopy. The results showed that CX08005 intervention significantly reduced the TG and echo-intensity attenuation coefficient in the livers of KKAy mice. Furthermore, we found that CX08005 treatment significantly enhanced insulin sensitivity, and decreased plasma TG and/or TC contents in KKAy and DIO mice, respectively. In addition, CX08005 treatment ameliorated hepatic microcirculation dysfunction in DIO mice, as evidenced by increased RBCs velocity and shear rate of the blood flow in central veins and in the interlobular veins, as well as enhanced rate of perfused hepatic sinusoids in central vein area. Additionally, CX08005 administration decreased the adhered leukocytes both in the center veins and in the hepatic sinusoids area. Taken together, CX08005 exhibited beneficial effects on hepatic lipid accumulation and microcirculation dysfunction associated with NAFLD, which was involved with modulating insulin sensitivity and leukocyte recruitment, as well as restoration of normal microcirculatory blood flow.

Circulating Endothelial Progenitor Cells Reduce the Risk of Alzheimer's Disease.

Cerebrovascular damage coexists with Alzheimer's disease (AD) pathology and increases AD risk. However, it is unclear whether endothelial progenitor cells reduce AD risk via cerebrovascular repair. By using the Framingham Heart Study (FHS) offspring cohort, which includes data on different progenitor cells, the incidence of AD dementia, peripheral and cerebrovascular pathologies, and genetic data (n = 1,566), we found that elevated numbers of circulating endothelial progenitor cells with CD34+CD133+ co-expressions had a dose-dependent association with decreased AD risk (HR = 0.67, 95% CI: 0.46-0.96, p = 0.03) after adjusting for age, sex, years of education, and APOE ε4. With stratification, this relationship was only significant among those individuals who had vascular pathologies, especially hypertension (HTN) and cerebral microbleeds (CMB), but not among those individuals who had neither peripheral nor central vascular pathologies. We applied a genome-wide association study (GWAS) and found that the number of CD34+CD133+ cells impacted AD risk depending on the homozygous genotypes of two genes: KIRREL3 rs580382 CC carriers (HR = 0.31, 95% CI: 0.17-0.57, p<0.001), KIRREL3 rs4144611 TT carriers (HR = 0.29, 95% CI: 0.15-0.57, p<0.001), and EXOC6B rs61619102 CC carriers (HR = 0.49, 95% CI: 0.31-0.75, p<0.001) after adjusting for confounders. In contrast, the relationship did not exist in their counterpart genotypes, e.g. KIRREL3 TT/CT or GG/GT carriers and EXOC6B GG/GC carriers. Our findings suggest that circulating CD34+CD133+ endothelial progenitor cells can be therapeutic in reducing AD risk in the presence of cerebrovascular pathology, especially in KIRREL3 and EXOC6B genotype carriers.

Redox-responsive polyurethane-polyurea nanoparticles targeting to aortic endothelium and atherosclerosis.

Aortic endothelial cell dysfunction is an early trigger of atherosclerosis, the major cause of the cardiovascular disease (CVD). Nanomedicines targeting vascular endothelium and lesions hold great promise as therapeutic solutions to vascular disorders. This study investigates the vascular delivery efficacy of polyurethane-polyurea nanocapsules (Puua-NCs) with pH-synchronized shell cationization and redox-triggered release. Fluorescent lipophilic dye DiI was encapsulated into Puua-NCs of variable sizes and concentrations. In vitro cellular uptake studies with human aortic endothelial cells showed that these Puua-NCs were taken up by cells in a dose-dependent manner. In apolipoprotein E-deficient mice fed a Western diet, a model of atherosclerosis, circulating Puua-NCs were stable and accumulated in aortic endothelium and lesions within 24 hours after intravenous administration. Treatment with thiol-reducing and oxidizing reagents disrupted the disulfide bonds on the surface of internalized NCs, triggering disassembly and intracellular cargo release. Ultimately, Puua-NCs are a potential redox-controllable cardiovascular drug delivery system.

Multidisciplinary Progress in Obesity Research.

Obesity is a chronic disease that endangers human health. In recent years, the phenomenon of obesity has become more and more common, and it has become a global epidemic. Obesity is closely associated with many adverse metabolic changes and diseases, such as insulin resistance, type 2 diabetes mellitus, coronary heart disease, nervous system diseases and some malignant tumors, which have caused a huge burden on the country's medical finance. In most countries of the world, the incidence of cancer caused by obesity is increasing year on year. Diabetes associated with obesity can lead to secondary neuropathy. How to treat obesity and its secondary diseases has become an urgent problem for patients, doctors and society. This article will summarize the multidisciplinary research on obesity and its complications.

Cardiotonic Pills® protects from myocardial fibrosis caused by in stent restenosis in miniature pigs.

BACKGROUND: Stent implantation has been increasingly applied for the treatment of obstructive coronary artery disease, which, albeit effective, often harasses patients by in-stent restenosis (ISR). PURPOSE: The present study was to explore the role of compound Chinese medicine Cardiotonic Pills® (CP) in attenuating ISR-evoked myocardial injury and fibrosis. STUDY DESIGN: Chinese miniature pigs were used to establish ISR model by implanting obsolete degradable stents into coronary arteries. Quantitative coronary angiography (QCA) was performed to confirm the success of the model. METHODS: CP was given at 0.2 g/kg daily for 30 days after ISR. On day 30 and 60 after stent implantation, the myocardial infarct and myocardial blood flow (MBF) were assessed. Myocardial histology was evaluated by hematoxylin-eosin and Masson's trichrome staining. The content of ATP, MPO, and the activity of mitochondrial respiratory chain complex Ⅳ were determined by ELISA. Western blot was performed to assess the expression of ATP5D and related signaling proteins, and the mediators of myocardial fibrosis. RESULTS: Treatment with CP diminished myocardial infarct size, retained myocardium structure, attenuated myocardial fibrosis, and restored MBF. CP ameliorated energy metabolism disorder, attenuated TGFß1 up-regulation and reversed its downstream gene expression, such as Smad6 and Smad7, and inhibited the increased expression of MCP-1, PR S19, MMP-2 and MMP-9. CONCLUSION: CP effectively protects myocardial structure and function from ISR challenge, possibly by regulating energy metabolism via inactivation of RhoA/ROCK signaling pathway and inhibition of monocyte chemotaxis and TGF ß1/Smads signaling pathway.

QiShenYiQi Pill Ameliorates Cardiac Fibrosis After Pressure Overload-Induced Cardiac Hypertrophy by Regulating FHL2 and the Macrophage RP S19/TGF-ß1 Signaling Pathway.

Purpose: Heart failure (HF) is a leading cause of morbidity and mortality worldwide, and it is characterized by cardiac hypertrophy and fibrosis. However, effective treatments are not available to block cardiac fibrosis after cardiac hypertrophy. The QiShenYiQi pill (QSYQ) is an effective treatment for chronic HF. However, the underlying mechanism remains unclear. Methods: In the present study, a pressure overload-induced cardiac hypertrophy model was established in rats by inducing ascending aortic stenosis for 4 weeks. QSYQ was administered for 6 weeks, and its effects on cardiac fibrosis, myocardial apoptosis, RP S19 release, macrophage polarization, TGF-ß1 production, and TGF-ß1/Smad signaling were analyzed. In vitro studies using H9C2, Raw264.7, and RDF cell models were performed to confirm the in vivo study findings and evaluate the contribution to the observed effects of the main ingredients of QSYQ, namely, astragaloside IV, notoginsenoside R1, 3,4-dihydroxyl-phenyl lactic acid, and Dalbergia odorifera T. C. Chen oil. The role of four-and-a-half LIM domains protein 2 (FHL2) in cardiac fibrosis and QSYQ's effects were assessed by small interfering RNAs (siRNAs). Results: QSYQ ameliorated cardiac fibrosis after pressure overload-induced cardiac hypertrophy and attenuated cardiomyocyte apoptosis, low FHL2 expression, and TGF-ß1 release by the injured myocardium. QSYQ also inhibited the following: release of RP S19 from the injured myocardium, activation of C5a receptors in monocytes, polarization of macrophages, and release of TGF-ß1. Moreover, QSYQ downregulated TGF-ßR-II expression induced by TGF-ß1 in fibroblasts and inhibited Smad protein activation and collagen release and deposition. Conclusion: The results showed that QSYQ inhibited myocardial fibrosis after pressure overload, which was mediated by RP S19-TGF-ß1 signaling and decreased FHL2, thus providing support for QSYQ as a promising therapy for blocking myocardial fibrosis.

Ameliorative effect and mechanism of Si-Ni-San on chronic stress-induced diarrhea-irritable bowel syndrome in rats.

Background: Chronic stress-induced diarrhea is a common clinical condition, characterized by an abnormal bowel movement and loose stools, which lacks effective treatment in the clinic. Si-Ni-San (SNS) is a compound traditional Chinese medicine extensively used in China for stress-related diarrhea. However, the mechanism is unclear. Methods: Male Wistar rats (200 ± 20 g) were placed in a restraint cylinder and fixed horizontally for 3 h once daily for 21 consecutive days to establish a chronic restraint stress (CRS) rat model. SNS (0.6944 g/kg or 1.3888 g/kg) was given by gavage 1 h before the restraint once daily for 21 consecutive days. We examined the fecal score, dopamine ß hydroxylase (DßH), and c-fos expression in locus coeruleus, norepinephrine (NE) content in ileum and plasma, expression of α1 adrenergic receptors, MLCK, MLC, and p-MLC in the colon and mesenteric arteries, contraction of isolated mesenteric arteries, The expression of subunit δ of ATP synthase (ATP5D) in intestinal tissues, ATP, ADP, and AMP content in the ileum and colon, occludin expression between ileum epithelial cells, the number of enterochromaffin cells (ECs) and mast cells (MCs) in the ileum, and 5-hydroxytryptamine (5-HT) content in the ileum and plasma. Results: After SNS treatment, the fecal score was improved. The increased expression of DßH and c-fos in locus coeruleus was inhibited. SNS suppressed the increased NE content in the ileum and plasma, down-regulated α1 adrenergic receptors in mesenteric arteries and MLCK, MLC, p-MLC in the colon and mesenteric arteries, and inhibited the contraction of mesenteric arteries. SNS also increased the ATP content in the ileum and colon, inhibited low expression of ATP5D in intestinal tissues, inhibited the decrease of ATP/ADP in the ileum and ATP/AMP in the colon, and up-regulated the occludin expression between ileum epithelial cells. In addition, SNS inhibited the increase of ECs and MCs in the ileum and the increase of 5-HT content in the ileum and plasma. Conclusion: This study demonstrated that SNS could improve CRS-induced abnormal feces in rats. This effect was related to the inhibition of CRS-induced increased expression of DßH and c-fos in the locus coeruleus, NE content in the ileum and plasma, and the contraction of isolated mesenteric arteries; inhibition of energy metabolism abnormality and decreased occludin expression; inhibition of increased ECs and MCs in the ileum, and 5-HT content in the ileum and plasma.

Yu-Ping-Feng Formula Ameliorates Alveolar-Capillary Barrier Injury Induced by Exhausted-Exercise via Regulation of Cytoskeleton.

Background: Yu-ping-feng powder (YPF) is a compound traditional Chinese medicine extensively used in China for respiratory diseases. However, the role of YPF in alveolar-capillary barrier dysfunction remains unknown. This study aimed to explore the effect and potential mechanism of YPF on alveolar-capillary barrier injury induced by exhausted exercise. Methods: Male Sprague-Dawley rats were used to establish an exhausted-exercise model by using a motorized rodent treadmill. YPF at doses of 2.18 g/kg was administrated by gavage before exercise training for 10 consecutive days. Food intake-weight/body weight, blood gas analysis, lung water percent content, BALF protein concentration, morphological observation, quantitative proteomics, real-time PCR, and Western blot were performed. A rat pulmonary microvascular endothelial cell line (PMVEC) subjected to hypoxia was applied for assessing the related mechanism. Results: YPF attenuated the decrease of food intake weight/body weight, improved lung swelling and hemorrhage, alleviated the increase of lung water percent content and BALF protein concentration, and inhibited the impairment of lung morphology. In addition, YPF increased the expression of claudin 3, claudin 18, occludin, VE-cadherin, and ß-catenin, attenuated the epithelial and endothelial hyperpermeability in vivo and/or in vitro, and the stress fiber formation in PMVECs after hypoxia. Quantitative proteomics discovered that the effect of YPF implicated the Siah2-ubiquitin-proteasomal pathway, Gng12-PAK1-MLCK, and RhoA/ROCK, which was further confirmed by Western blot. Data are available via ProteomeXchange with identifier PXD032737. Conclusion: YPF ameliorated alveolar-capillary barrier injury induced by exhausted exercise, which is accounted for at least partly by the regulation of cytoskeleton.

Implication of IGF1R signaling in the protective effect of Astragaloside IV on ischemia and reperfusion-induced cardiac microvascular endothelial hyperpermeability.

BACKGROUND: Myocardial ischemia-reperfusion (I/R) causes damage to coronary capillary endothelial barrier and microvascular leakage (MVL), aggravating tissue injury and heart dysfunction. However, the effective strategy for protecting endothelium barrier of cardiac vasculature remains limited. PURPOSE: This study aimed to explore the effect of Astragaloside IV (ASIV) on coronary MVL after cardiac I/R and the underlying mechanism. STUDY DESIGN: Sprague-Dawley (SD) rats were used for assessment of the efficacy of Astragaloside IV in protection of myocardial I/R injury, while human cardiac microvascular endothelial cells were applied to gain more insight into the underlying mechanism. METHODS: Sprague-Dawley rats with or without pretreatment by ASIV at 10 mg/kg were subjected to occlusion of left coronary anterior descending artery followed by reperfusion. Endothelial cells were exposed to hypoxia and re-oxygenation (H/R). The distribution of junction proteins was detected by immunofluorescence staining and confocal microscope, the content of junction proteins was detected by Western blot, the level of adenosine triphosphate (ATP) was detected by ELISA, and the signal pathway related to permeability was detected by siRNA infection. The fluorescence intensity of FITC-albumin and FITC-Dextran was measured to evaluate the permeability of endothelial cells. RESULTS: ASIV exhibited protective effects on capillary damage, myocardium edema, albumin leakage, leucocyte infiltration, and the downregulated expression of endothelial junction proteins after I/R. Moreover, ASIV displayed ability to protect ATP from depletion after I/R or H/R, and the effect of ASIV on regulating vascular permeability and junction proteins was abolished once ATP synthase was inhibited. Notably, ASIV activated the insulin-like growth factor 1 receptor (IGF1R) and downstream signaling after reoxygenation. Knocking IGF1R down abolished the effect of ASIV on restoration of ATP, junction proteins and endothelial barrier after H/R. CONCLUSION: ASIV was potential to prevent MVL after I/R in heart. Moreover, the study for the first time demonstrated that the beneficial role of ASIV depended on promoting production of ATP through activating IGF1R signaling pathway. This result provided novel insight for better understanding the mechanism underlying the potential of ASIV to cope with cardiac I/R injury.