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.

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.

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.

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.

[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.

Post-treatment with yiqifumai injection and its main ingredients attenuates lipopolysaccharide-induced microvascular disturbance in mesentery and ileum.

OBJECTIVE: To investigate the effect of Yiqifumai injection (YQFM), a compound Chinese medicine, and its main active ingredients on lipopolysaccharide (LPS)-induced microvascular disturbance in mesentery and ileum. METHODS: Rats were infused with LPS (5 mg/kg/h) for 90 min. Thirty minutes after initiation of LPS administration, YQFM (160 mg/kg/h), Rb1 (5 mg/kg/h), Sch (2.5 mg/kg/h), or Rb1+Sch (5 mg/kg/h + 2.5 mg/kg/h) was infused until 90 min. Human umbilical vein endothelial cells (HUVECs) were incubated with LPS (100 ng/ml) for 90 min. YQFM (1 mg/ml), Rb1 (100 µM), Sch (100 µM), or Rb1+Sch (200 µM) was added 30 min after initiation of LPS stimulation. RESULTS: Yiqifumai injection and Rb1+Sch inhibited mesenteric venule hyperpermeability, suppressed microvillar erosion and submucosal edema, and protected claudin-5 from downregulation and interleukin-1ß from upregulation in ileal tissues after LPS. Study in HUVECs confirmed the effect of YQFM and Rb1+Sch on JAM-1 after LPS and revealed a similar effect on other junction proteins. Moreover, YQFM and Rb1+Sch attenuated the dysfunctional energy metabolism and the activation of TLR-4/Src/NF-κB signaling with Rb1 and Sch being partially effective. CONCLUSION: These results demonstrated the beneficial effect of post-treatment with YQFM, which is attributable to its main ingredient Rb1 and Sch, and likely mediated by targeting TLR-4/Src/NF-κB signaling pathway.

The ameliorating effects of Bushen Tiaoxue Granules and Kunling Wan on impaired angiogenesis and endometrial receptivity in rats following controlled ovarian hyperstimulation.

OBJECTIVE: To investigate the effects of Bushen Tiaoxue Granules and Kunling Wan, the two Chinese medicines, on vascular dysfunction and the impairment of endometrial receptivity caused by controlled ovarian hyperstimulation and its underlying mechanism. METHODS: Female Sprague Dawley rats with regular estrous cycle were enrolled and given Bushen Tiaoxue Granules or Kunling Wan by gavage for 12 days, and then, controlled ovarian hyperstimulation model was induced. We assessed endometrial microvessels, endometrial blood flow, levels of estradiol and progesterone in serum, vascular endothelial growth factor A upstream molecules estrogen and progesterone receptors in the endometrium, and pregnancy outcome. RESULTS: Pre-treatment of Bushen Tiaoxue Granules or Kunling Wan increases endometrial blood flow of controlled ovarian hyperstimulation rats, up-regulates vascular endothelial growth factor A and microvessels, improves the endometrial morphology of controlled ovarian hyperstimulation rats during implantation, decreases the super physiological concentration of estradiol and progesterone in serum, and increases the expression of vascular endothelial growth factor A upstream molecules estrogen and progesterone receptors in the endometrium. In addition, Bushen Tiaoxue Granules or Kunling Wan elevates the lysophosphatidic acid receptor 3 that participates in vascularization and increases the expression of leukemia inhibitory factor through up-regulating the expression of p53 in the endometrium, ultimately affecting pregnancy outcome. CONCLUSION: This study demonstrated Bushen Tiaoxue Granules or Kunling Wan as a potential strategy for prevention of impairment in angiogenesis and endometrial receptivity induced by controlled ovarian hyperstimulation.

Costunolide Plays an Anti-Neuroinflammation Role in Lipopolysaccharide-Induced BV2 Microglial Activation by Targeting Cyclin-Dependent Kinase 2.

Hyperactivation of microglia in the brain is closely related to neuroinflammation and leads to neuronal dysfunction. Costunolide (CTL) is a natural sesquiterpene lactone with wide pharmacological activities including anti-inflammation and antioxidation. In this study, we found that CTL significantly inhibited the production of inflammatory mediators including nitric oxide, IL-6, TNF-α, and PGE2 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Moreover, CTL effectively attenuated IKKß/NF-κB signaling pathway activation. To identify direct cellular target of CTL, we performed high-throughput reverse virtual screening assay using scPDB protein structure library, and found cyclin-dependent kinase 2 (CDK2) was the most specific binding protein for CTL. We further confirmed the binding ability of CTL with CDK2 using cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) assays. Surface plasmon resonance analysis also supported that CTL specifically bound to CDK2 with a dissociation constant at micromole level. Furthermore, knocking down CDK2 obviously reversed the anti-inflammation effect of CTL via AKT/IKKß/NF-κB signaling pathway on BV-2 cells. Collectively, these results indicate that CTL inhibits microglia-mediated neuroinflammation through directly targeting CDK2, and provide insights into the role of CDK2 as a promising anti-neuroinflammation therapeutic target.

[Investigation of modulating effect of Qingfei Paidu Decoction on host metabolism and gut microbiome in rats].

This study is to explore the effect of Qingfei Paidu Decoction(QPD) on the host metabolism and gut microbiome of rats with metabolomics and 16 S rDNA sequencing. Based on 16 S rDNA sequencing of gut microbiome and metabolomics(GC-MS and LC-MS/MS), we systematically studied the serum metabolites profile and gut microbiota composition of rats treated with QPD for continued 5 days by oral gavage. A total of 23 and 43 differential metabolites were identified based on QPD with GC-MS and LC-MS/MS, respectively. The involved metabolic pathways of these differential metabolites included glycerophospholipid metabolism, linoleic acid metabolism, TCA cycle and pyruvate metabolism. Meanwhile, we found that QPD significantly regulated the composition of gut microbiota in rats, such as enriched Romboutsia, Turicibacter, and Clostridium_sensu_stricto_1, and decreased norank_f_Lachnospiraceae. Our current study indicated that short-term intervention of QPD could significantly regulate the host metabolism and gut microbiota composition of rats dose-dependently, suggesting that the clinical efficacy of QPD may be related with the regulation on host metabolism and gut microbiome.

A novel traditional Chinese medicine ameliorates fatigue-induced cardiac hypertrophy and dysfunction via regulation of energy metabolism.

Prolonged exercise and exercise training can adversely affect cardiac function in some individuals. QiShenYiQi Pills (QSYQ), which are a compound Chinese medicine, have been previously shown to improve pressure overload-induced cardiac hypertrophy. We hypothesized that QSYQ can ameliorate as well the fatigue-induced cardiac hypertrophy. This study was to test this hypothesis and underlying mechanism with a focus on its role in energy regulation. Male Sprague-Dawley rats were used to establish exercise adaptation and fatigue model on a motorized rodent treadmill. Echocardiographic analysis and heart function test were performed to assess heart systolic function. Food-intake weight/body weight and heart weight/body weight were assessed, and hematoxylin and eosin staining and immunofluorescence staining of myocardium sections were performed. ATP synthase expression and activity and ATP, ADP, and AMP levels were assessed using Western blot and ELISA. Expression of proteins related to energy metabolism and IGF-1R signaling was determined using Western blot. QSYQ attenuated the food-intake weight/body weight decrease, improved myocardial structure and heart function, and restored the expression and distribution of myocardial connexin 43 after fatigue, concomitant with an increased ATP production and a restoration of metabolism-related protein expression. QSYQ upgraded the expression of IGF-1R, P-AMPK/AMPK, peroxisome proliferator-activated receptor-γ coactivator-1α, nuclear respiratory factor-1, P-phosphatidylinositol 3-kinase (PI3K)/PI3K, and P-Akt/Akt thereby attenuated the dysregulation of IGF-1R signaling after fatigue. QSYQ relieved fatigue-induced cardiac hypertrophy and enhanced heart function, which is correlated with its potential to improve energy metabolism by regulating IGF-1R signaling. NEW & NOTEWORTHY Prolonged exercise may impact some people leading to pathological cardiac hypertrophy. This study using an animal model of fatigue-induced cardiac hypertrophy provides evidence showing the potential of QiShenYiQi Pills, a novel traditional Chinese medicine, to prevent the cardiac adaptive hypertrophy from development to pathological hypertrophy and demonstrates that this effect is correlated with its capacity for regulating energy metabolism through interacting with insulin-like growth factor-1 receptor.

Yiqifumai injection and its main ingredients attenuate lipopolysaccharide-induced cerebrovascular hyperpermeability through a multi-pathway mode.

OBJECTIVE: Yiqifumai injection is a compound Chinese medicine used to treat microcirculatory disturbance-related diseases clinically. Our previous study proved that Yiqifumai injection pretreatment inhibited lipopolysaccharide-induced venular albumin leakage in rat mesentery. This study aimed to investigate whether Yiqifumai injection attenuated cerebral microvascular hyperpermeability and corresponding contribution of its main ingredients. METHODS: Rats were challenged by lipopolysaccharide infusion (5 mg/kg/h) for 90 minutes. Yiqifumai injection (160 mg/kg/h), Rb1 (5 mg/kg/h), Sch (2.5 mg/kg/h), and Rb1 (5 mg/kg/h) + Sch (2.5 mg/kg/h) were infused 30 minutes before (pretreatment) or after (post-treatment) lipopolysaccharide administration. RESULTS: Both pretreatment and post-treatment with Yiqifumai injection attenuated cerebral venular albumin leakage during lipopolysaccharide infusion and cerebrovascular hyperpermeability at 72 hours after lipopolysaccharide infusion. Yiqifumai injection restrained the decreased junction protein expression, adenosine triphosphate content, and mitochondria complex I, II, IV, and V activities. Moreover, Yiqifumai injection inhibited toll-like receptor-4 expression, Src phosphorylation, and caveolin-1 expression. Its main ingredients Rb1 and Sch alone worked differently, with Rb1 being more effective for enhancing energy metabolism, while Sch attenuating toll-like receptor-4 expression and Src activation. CONCLUSION: Yiqifumai injection exerts a protective and ameliorated effect on cerebral microvascular hyperpermeability, which is more effective than any of its ingredients, possibly due to the interaction of its main ingredients through a multi-pathway mode.

Effects and mechanisms of QiShenYiQi pills and major ingredients on myocardial microcirculatory disturbance, cardiac injury and fibrosis induced by ischemia-reperfusion.

Coronary heart disease remains a major threaten for public health worldwide, and pharmacological or mechanical coronary reperfusion are currently used for treatment of acute coronary syndrome. However, restoration of blood flow to ischemic myocardium leads to ischemia/reperfusion (I/R) injury. Microcirculatory disturbance and cardiac injury after I/R occur via a complex pathologic process including metabolism impairment in the ischemia phase and oxidative stress in the reperfusion phase. Obviously, any treatment targeting a single link is insufficient to cope with I/R injury. Investigation in the past decade in our laboratory as well as in other's demonstrated the cardioprotection potential of QiShenYiQi Pills (QSYQ) and ingredients in experimental animal models of I/R injury. These results have offered insight into the mechanism thereby QSYQ prevents against cardiac I/R injury in clinic. This review will outline the results with respect to the effect of QSYQ and major bioactive ingredients on I/R-induced microcirculatory disturbance, cardiac injury and fibrosis, with emphasis on the underlying mechanisms.

QiShenYiQi Pills® ameliorates ischemia/reperfusion-induced myocardial fibrosis involving RP S19-mediated TGFß1/Smads signaling pathway.

QiShenYiQi Pills (QSYQ) is a compound Chinese medicine widely used in China for treatment of cardiovascular disease. However, limited data are available regarding the anti-fibrotic role of QSYQ after ischemia/reperfusion (I/R) injury. This study aimed to investigate the effect of post-treatment with QSYQ on myocardial fibrosis after I/R-induced myocardium injury, and the role of different compounds of QSYQ, focusing especially on the involvement of chemokine ribosomal protein S19 (RP S19) dimer and monocyte migration. Male Sprague-Dawley rats were subjected to left anterior descending coronary artery occlusion for 30 min followed by reperfusion with or without administration of QSYQ (0.6, 1.2, or 1.8 g/kg) once daily by gavage for 6 days. Post-treatment with QSYQ diminished I/R-induced infarct size, alleviated myocardium injury, attenuated myocardial fibrosis after 6 days of reperfusion, and restored heart function and myocardial blood flow after I/R. In addition, the drug significantly inhibited monocyte infiltration and macrophage polarization towards M2, which was attributable to chemokine RP S19 dimer. Moreover, Western blots revealed that QSYQ blocked I/R-induced increase in TGFß1 and TGFßRⅡ and reversed its relevant gene expression, such as Smad3,4,6,7, and inhibited the increase of MMP 2,9 expression. As the major components of QSYQ, astragaloside IV (AsIV), 3,4-dihydroxy-phenyl lactic acid (DLA), and notoginsenoside R1 (R1) were assessed as to the contribution of each of them to the expression of the proteins concerned. The results showed that the effect of AsIV was similar to QSYQ, while DLA and R1 only partly simulated the effect of QSYQ. The results provide evidence for the potential role of QSYQ in treating myocardial fibrosis following I/R injury. This effect may be associated with QSYQ's inhibition effect on monocyte chemotaxis and TGFß1/Smads signaling pathway with different component targeting distinct link (s) of the signaling.

Deficiency of FAM3D (Family With Sequence Similarity 3, Member D), A Novel Chemokine, Attenuates Neutrophil Recruitment and Ameliorates Abdominal Aortic Aneurysm Development.

OBJECTIVE: Chemokine-mediated neutrophil recruitment contributes to the pathogenesis of abdominal aortic aneurysm (AAA) and may serve as a promising therapeutic target. FAM3D (family with sequence similarity 3, member D) is a recently identified novel chemokine. Here, we aimed to explore the role of FAM3D in neutrophil recruitment and AAA development. APPROACH AND RESULTS: FAM3D was markedly upregulated in human AAA tissues, as well as both elastase- and CaPO4-induced mouse aneurysmal aortas. FAM3D deficiency significantly attenuated the development of AAA in both mouse models. Flow cytometry analysis indicated that FAM3D-/- mice exhibited decreased neutrophil infiltration in the aorta during the early stage of AAA formation compared with their wild-type littermates. Moreover, application of FAM3D-neutralizing antibody 6D7 through intraperitoneal injection markedly ameliorated elastase-induced AAA formation and neutrophil infiltration. Further, in vitro coculture experiments with FAM3D-neutralizing antibody 6D7 and in vivo intravital microscopic analysis indicated that endothelial cell-derived FAM3D induced neutrophil recruitment. Mechanistically, FAM3D upregulated and activated Mac-1 (macrophage-1 antigen) in neutrophils, whereas inhibition of FPR1 (formyl peptide receptor 1) or FPR2 significantly blocked FAM3D-induced Mac-1 activation, indicating that the effect of FAM3D was dependent on both FPRs. Moreover, specific inhibitors of FPR signaling related to Gi protein or ß-arrestin inhibited FAM3D-activated Mac-1 in vitro, whereas FAM3D deficiency decreased the activation of both FPR-Gi protein and ß-arrestin signaling in neutrophils in vivo. CONCLUSIONS: FAM3D, as a dual agonist of FPR1 and FPR2, induced Mac-1-mediated neutrophil recruitment and aggravated AAA development through FPR-related Gi protein and ß-arrestin signaling.

Angioedema and Hemorrhage After 4.5-Hour tPA (Tissue-Type Plasminogen Activator) Thrombolysis Ameliorated by T541 via Restoring Brain Microvascular Integrity.

Background and Purpose- tPA (tissue-type plasminogen activator) is the only recommended intravenous thrombolytic agent for ischemic stroke. However, its application is limited because of increased risk of hemorrhagic transformation beyond the time window. T541 is a Chinese compound medicine with potential to attenuate ischemia and reperfusion injury. This study was to explore whether T541-benefited subjects underwent tPA thrombolysis extending the time window. Methods- Male C57BL/6 N mice were subjected to carotid artery thrombosis by stimulation with 10% FeCl3 followed by 10 mg/kg tPA with/without 20 mg/kg T541 intervention at 4.5 hours. Thrombolysis and cerebral blood flow were observed dynamically until 24 hours after drug treatment. Neurological deficit scores, brain edema and hemorrhage, cerebral microvascular junctions and basement membrane proteins, and energy metabolism in cortex were assessed then. An in vitro hypoxia/reoxygenation model using human cerebral microvascular endothelial cells was used to evaluate effect of T541 on tight junctions and F-actin in the presence of tPA. Results- tPA administered at 4.5 hours after carotid thrombosis resulted in a decrease in thrombus area and survival rate, whereas no benefit on cerebral blood flow. Study at 24 hours after tPA administration revealed a significant angioedema and hemorrhage in the ischemia hemisphere, a decreased expression of junction proteins claudin-5, zonula occludens-1, occludin, junctional adhesion molecule-1 and vascular endothelial cadherin, and collagen IV and laminin. Meanwhile, ADP/ATP, AMP/ATP, and ATP5D (ATP synthase subunit) expression and activities of mitochondria complex I, II, and IV declined, whereas malondialdehyde and 8-Oxo-2'-deoxyguanosine increased and F-actin arrangement disordered. All the insults after tPA treatment were attenuated by addition of T541 dose dependently. Conclusions- The results suggest T541 as a potential remedy to attenuate delayed tPA-related angioedema and hemorrhage and extend time window for tPA treatment. The potential of T541 to upregulate energy metabolism and protect blood-brain barrier is likely attributable to its effects observed.

Polymer-free dual drug-eluting stents evaluated in a porcine model.

BACKGROUND: Although drug-eluting stents have dramatically reduced the rates of restenosis and target lesion revascularization, they are associated with stent thrombosis (ST), a catastrophic event likely due to delayed endothelialization and chronic inflammation caused by the polymer and the metal scaffolds. To increase the safety and efficacy of stents, polymer-free dual drug-eluting stents (DDES) have been developed. METHODS: A total 160 stents (Bare-metal stents (BMS), polymer-free probucol stents (PrES), sirolimus-eluting stents (SES) and DDES) were randomly implanted in the coronary arteries of 80 pigs. 14, 28, 90 and 191 days after implantation, QCA and OCT evaluations were performed in 20 pigs respectively, and the arteries were harvested for scanning electron microscope (SEM), histomorphology, histopathology analyses and for the relative expression of CD31, CD34 and CD133 on mRNA and protein levels. RESULTS: At the 14-day time point, there were significant differences in the strut rate coverage (p = 0.011), with greater coverage in the PrES than in the SES group (53.2%vs. 20.3%, p = 0.002). As well, there were no significant differences in the expression of CD31, CD34 and CD133 between groups in mRNA and protein level. CONCLUSIONS: DDES were as safe as BMS and SES, but they did not further improve the endothelialization of the stented coronary arteries in the porcine model.

Catalpol restores LPS-elicited rat microcirculation disorder by regulation of a network of signaling involving inhibition of TLR-4 and SRC.

LPS-induced microvascular hyperpermeability and hemorrhage play a key role in the development of sepsis, the attenuation of which might be an important strategy to prevent sepsis. However, the current clinical therapies have proven to be inefficient in improving the prognosis for patients with sepsis. Catalpol, an iridoid glycoside extracted from the roots of Rehmannia, has been reported to protect against LPS-induced acute lung injury through a Toll-like receptor-4 (TLR-4)-mediated NF-κB signaling pathway. However, it is still unknown whether catalpol can be an effective treatment to ameliorate the LPS-induced microvascular disorder. The present study aimed to investigate the impact of catalpol on LPS-induced mesenteric microvascular disorder and its underlying mechanism. Male Wistar rats were challenged by infusion of LPS (10 mg·kg-1·h-1) through the left femoral vein for 120 min. Post-treatment with catalpol (10 mg/kg) alleviated the LPS-induced microvascular hyperpermeability and hemorrhage; reduced mortality; ameliorated the alteration in the distribution of claudin-5 and the junctional adhesion molecule-1, as well as the degradation of collagen IV and laminin; and attenuated the increase of TLR-4 level, phosphorylations of Src tyrosine kinase, phosphatidyl inositol 3-kinase, focal adhesion kinase, and cathepsin B activation. In vitro study in human umbilical vein endothelial cells verified these results and further revealed that inhibition of TLR-4 and Src each simulated some, but not all, of the effects that catalpol exerted. Besides, surface plasmon resonance showed that catalpol could directly bind to TLR-4 and Src. These results demonstrated that catalpol was able to ameliorate the LPS-induced microvascular barrier damage and hemorrhage by targeting both TLR-4 and Src, thus attenuating the phosphorylation of Src kinase, phosphatidyl inositol 3-kinase, and focal adhesion kinase, as well as cathepsin B activation.

Kudiezi Injection(®) Alleviates Blood-Brain Barrier Disruption After Ischemia-Reperfusion in Rats.

OBJECTIVE: This study was designed to examine the effect of KDZ, on the BBB disruption in rat underwent MCAO and reperfusion. METHODS: Male Sprague-Dawley rats (260-280 g) were subjected to 60 minutes MCAO followed by reperfusion. KDZ (4 mL/kg) was administrated before ischemia. The Evans blue extravasation, albumin leakage, brain water content, TJ proteins, caveolin-1, p-caveolin-1, Src, and p-Src were evaluated. Neurological scores, cerebral infarction, and CBF were assessed. The binding affinity of KDZ to Src was examined. RESULTS: I/R evoked a range of insults including Evans blue extravasation, albumin leakage, brain water content increase, CBF decrease, cerebral infarction, and neurological deficits, all of which were attenuated by KDZ. Meanwhile, KDZ inhibited TJ proteins down-expression, expression of caveolin-1, phosphorylation of caveolin-1 and Src after I/R. In addition, SPR revealed binding of KDZ to Src with high affinity. CONCLUSIONS: KDZ protects BBB from disruption and improves cerebral outcomes following I/R via preventing the degradation of TJ proteins, caveolin-1 expression, and inhibiting p-caveolin-1 and p-Src, which were most likely attributable to the ability of its main ingredients to bind to Src and inhibit its phosphorylation.

Endothelial Cell Redox Regulation of Ischemic Angiogenesis.

The endothelium produces and responds to reactive oxygen and nitrogen species (RONS), providing important redox regulation to the cardiovascular system in physiology and disease. In no other situation are RONS more critical than in the response to tissue ischemia. Here, tissue healing requires growth factor-mediated angiogenesis that is in part dependent on low levels of RONS, which paradoxically must overcome the damaging effects of high levels of RONS generated as a result of ischemia. Although the generation of endothelial cell RONS in hypoxia/reoxygenation is acknowledged, the mechanism for their role in angiogenesis is still poorly understood. During ischemia, the major low molecular weight thiol glutathione (GSH) reacts with RONS and protein cysteines, producing GSH-protein adducts. Recent data indicate that GSH adducts on certain proteins are essential to growth factor responses in endothelial cells. Genetic deletion of the enzyme glutaredoxin-1, which selectively removes GSH protein adducts, improves, whereas its overexpression impairs revascularization of the ischemic hindlimb of mice. Ischemia-induced GSH adducts on specific cysteine residues of several proteins, including p65 NF-kB and the sarcoplasmic reticulum calcium ATPase 2, evidently promote ischemic angiogenesis. Identifying the specific proteins in the redox response to ischemia has provided therapeutic opportunities to improve clinical outcomes of ischemia.