Similarities and differences of myocardial metabolic characteristics between HFpEF and HFrEF mice based on LC-MS/MS metabolomics / 中华心血管病杂志

Objective: To reveal the similarities and differences in myocardial metabolic characteristics between heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF) mice using metabolomics. Methods: The experimental mice were divided into 4 groups, including control, HFpEF, sham and HFrEF groups (10 mice in each group). High fat diet and Nω-nitroarginine methyl ester hydrochloride (L-NAME) were applied to construct a"two-hit"HFpEF mouse model. Transverse aortic constriction (TAC) surgery was used to construct the HFrEF mouse model. The differential expression of metabolites in the myocardium of HFpEF and HFrEF mice was detected by untargeted metabolomics (UHPLC-QE-MS). Variable importance in projection>1 and P<0.05 were used as criteria to screen and classify the differentially expressed metabolites between the mice models. KEGG functional enrichment and pathway impact analysis demonstrated significantly altered metabolic pathways in both HFpEF and HFrEF mice. Results: One hundred and nine differentially expressed metabolites were detected in HFpEF mice, and 270 differentially expressed metabolites were detected in HFrEF mice. Compared with the control group, the most significantly changed metabolite in HFpEF mice was glycerophospholipids, while HFrEF mice presented with the largest proportion of carboxylic acids and their derivatives. KEGG enrichment and pathway impact analysis showed that the differentially expressed metabolites in HFpEF mice were mainly enriched in pathways such as biosynthesis of unsaturated fatty acids, ether lipid metabolism, amino sugar and nucleotide sugar metabolism, glycerophospholipid metabolism, arachidonic acid metabolism and arginine and proline metabolism. The differentially expressed metabolites in HFrEF mice were mainly enriched in arginine and proline metabolism, glycine, serine and threonine metabolism, pantothenate and CoA biosynthesis, glycerophospholipid metabolism, nicotinate and nicotinamide metabolism and arachidonic acid metabolism, etc. Conclusions: HFpEF mice have a significantly different myocardial metabolite expression profile compared with HFrEF mice. In addition, biosynthesis of unsaturated fatty acids, arachidonic acid metabolism, glycerophospholipid metabolism and arginine and proline metabolism are significantly altered in both HFpEF and HFrEF mice, suggesting that these metabolic pathways may play an important role in disease progression in both types of heart failure.

Analysis of family mutation type and two-hit hypothesis of a familial VHL Syndrome patient / 现代泌尿外科杂志

【Objective】 To explore the mutation type, clinical characteristics, molecular genetics and the two-hit type of a patient with familial Von Hippel Lindau (VHL) syndrome. 【Methods】 The data of the patient were collected. DNA was extracted from the peripheral blood and renal cell carcinoma sample. The VHL gene germline mutation site was detected with high throughput sequencing next generation sequencing (NGS). The two-hit site was identified with UCSCXena database, methylation-specific PCR (MSP) and microsatellite stability detection. 【Results】 The mutation site of the embryo line was located in c.500G>A R167Q mutation. The patient had single nucleotide polymorphism, but no clear loss of heterozygosity, methylation or system mutation. 【Conclusion】 The germline mutation in exon 3 is the basis for the clinical features of this familial renal cell carcinoma proband. The identification of the two-hit site is key to the occurrence of the disease, which is significant for the diagnosis and treatment. The use of the databases can guide the screening of mutations and methylation sites in familial renal cell carcinoma.

Berberine alleviates myocardial diastolic dysfunction by modulating Drp1-mediated mitochondrial fission and Ca2+ homeostasis in a murine model of HFpEF / 医学前沿

Heart failure with preserved ejection fraction (HFpEF) displays normal or near-normal left ventricular ejection fraction, diastolic dysfunction, cardiac hypertrophy, and poor exercise capacity. Berberine, an isoquinoline alkaloid, possesses cardiovascular benefits. Adult male mice were assigned to chow or high-fat diet with L-NAME ("two-hit" model) for 15 weeks. Diastolic function was assessed using echocardiography and noninvasive Doppler technique. Myocardial morphology, mitochondrial ultrastructure, and cardiomyocyte mechanical properties were evaluated. Proteomics analysis, autophagic flux, and intracellular Ca2+ were also assessed in chow and HFpEF mice. The results show exercise intolerance and cardiac diastolic dysfunction in "two-hit"-induced HFpEF model, in which unfavorable geometric changes such as increased cell size, interstitial fibrosis, and mitochondrial swelling occurred in the myocardium. Diastolic dysfunction was indicated by the elevated E value, mitral E/A ratio, and E/e' ratio, decreased e' value and maximal velocity of re-lengthening (-dL/dt), and prolonged re-lengthening in HFpEF mice. The effects of these processes were alleviated by berberine. Moreover, berberine ameliorated autophagic flux, alleviated Drp1 mitochondrial localization, mitochondrial Ca2+ overload and fragmentation, and promoted intracellular Ca2+ reuptake into sarcoplasmic reticulum by regulating phospholamban and SERCA2a. Finally, berberine alleviated diastolic dysfunction in "two-hit" diet-induced HFpEF model possibly because of the promotion of autophagic flux, inhibition of mitochondrial fragmentation, and cytosolic Ca2+ overload.

Derivative 6 as an additional chromosomal abnormality along with t(15;17): A case report

Acute promyelocytic leukemia (APL) is a type of acute myeloid leukemia (AML) characterized by the presence of t(15;17)(q22;q21) translocation leading to fusion between PML and RARa gene. Treatment combining all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has dramatically improved the prognosis of APL. We report a rare finding of primary clone of t(15;17) followed by a sequential clonal evolution of additional derivative chromosome 6 formation by a two hit mechanism. Our case showed a good clinical response with a four years and nine months event free survival after ATRA and ATO combination therapy in spite of existence of three chromosomal abnormalities stating that targeted therapy overcomes the adverse effects of additional genetic markers. However, close monitoring with assessment for long term prognostic behavior is required.

Transfusion-related acute lung injury / 中国小儿急救医学

Transfusion-related acute lung injury (TRALI) is an acute and fatal complication of blood product transfusion.TRALI is a syndrome, which diagnosed by the basis of clinical signs.The pathogenesis of TRALI is unclear and the hypothesis of two-hit model is generally accepted.At present, there is no specific treatment for TRALI.Treatment of the patient with TRALI is mainly supportive.The rational transfusions can avoid the occurrence of TRALI.The incidence of TRALI has been decreased by making the mainly measure of avoiding transfusions of plasma from multiparous female donors.

The protective role of raltegravir in experimental acute lung injury in vitro and in vivo

Disruption of pulmonary endothelial permeability and associated barrier integrity increase the severity of acute respiratory distress syndrome (ARDS). This study investigated the potential ability of the human immunodeficiency virus-1 (HIV-1) integrase inhibitor raltegravir to protect against acute lung injury (ALI) and the underlying mechanisms. Accordingly, the impact of raltegravir treatment on an in vitro lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cell (HPMEC) model of ALI and an in vivo LPS-induced two-hit ALI rat model was examined. In the rat model system, raltegravir treatment alleviated ALI-associated histopathological changes, reduced microvascular permeability, decreased Evans blue dye extravasation, suppressed the expression of inflammatory proteins including HMGB1, TLR4, p-NF-κB, NLRP3, and MPO, and promoted the upregulation of protective proteins including claudin 18.1, VE-cadherin, and aquaporin 5 as measured via western blotting. Immunohistochemical staining further confirmed the ability of raltegravir treatment to reverse LPS-induced pulmonary changes in NLRP3, claudin 18.1, and aquaporin 5 expression. Furthermore, in vitro analyses of HPMECs reaffirmed the ability of raltegravir to attenuate LPS-induced declines in VE-cadherin and claudin 18.1 expression while simultaneously inhibiting NLRP3 activation and reducing the expression of HMGB1, TLR4, and NF-kB, thus decreasing overall vascular permeability. Overall, our findings suggested that raltegravir may represent a viable approach to treating experimental ALI that functions by maintaining pulmonary microvascular integrity.

Caracterização de ectasia subclínica com análise integrada da tomografia e biomecânica da córnea / Characterization of subclinical ectasia with integrated corneal tomography and biomechanics assessments

RESUMO O artigo tem como objetivo descrever de forma prospectiva diferentes casos de ectasias altamente assimétricas (very asymmetric ectasia, VAE) para diferenciar formas subclínicas ou "frustas" do ceratocone (forme fruste keratoconus - FFKC) de casos de doença ectásica unilateral. O Caso 1 é um paciente de 39 anos, que admitiu ter coçado intensamente apenas o olho direito (OD) na juventude, se apresentando com ectasia unilateral tratada com sucesso por meio de implante de anel intraestromal em OD. O olho esquerdo (OE) apresentou-se normal ao exame completo por meio de propedêutica multimodal e acuidade visual não corrigida (AVsc) de 20/20, estável por mais de 5 anos, com TBI (tomography and biomechanical index) de 0.02. No Caso 2 é um paciente de 15 anos com ectasia clínica em OD, e OE com topografia normal, mas alterações tomográficas e biomecânicas, incluindo o TBI 0,56, caracterizarando a doença subclínica (FFKC). O Caso 3 é a mãe do paciente do Caso 2, de 46 anos, que se apresentou com presbiopia, sem qualquer histórico oftalmológico relevante. A AVsc foi de 20/20 em cada olho, topografia de Placido com leve encurvamento inferior, mas sem sinais definitivos de ectasia. A avaliação biomecânica e tomográfica revelou sinais de ceratocone em ambos os olhos, com TBI de 1,0 e 0,99. Esses três casos estão de acordo com a definição do consenso global e a hipótese de dois acertos (two-hit hypothesis), que ceratocone é uma doença bilateral, mas ectasia pode ocorrer por causa estritamente mecânica unilateralmente (ou em qualquer olho). A relevância da propedêutica multimodal é destacada, destacando-se a integração do estudo biomecânico e tomográfico com imagens de Scheimpflug.

ABSTRACT The article aims to prospectively describe different cases of highly asymmetric ectasia (very asymmetric ectasia, VAE) to differentiate subclinical or "frustrated" forms of keratoconus (forme fruste keratoconus - FFKC) from cases of unilateral ectatic disease. Case 1 is a 39-year-old patient with unilateral ectasia treated with an intrastromal ring implant. The contralateral eye was normal due to multimodal propaedeutics, stable for more than 3 years, with a TBI of 0.02. The patient admitted to having intensely scratched only his right eye in his youth. In Case 2, a 15-year-old patient with clinical ectasia in the right eye, had a left eye with normal topography and tomographic and biomechanical changes characterizing FFKC. Case 3 is the mother of the case 2 patient, aged 46, who presented with presbyopia, without any relevant ophthalmological history. Uncorrected visual acuity of 20/20 in each eye, Placido topography with slight lower curving, but without definitive signs of ectasia. The biomechanical and tomographic evaluation revealed signs of keratoconus in both eyes. These three cases are in accordance with the definition of the global consensus: keratoconus is a bilateral disease, but ectasia can occur because of strictly mechanical unilateral (in any eye). The relevance of multimodal refractive imaging is highlighted, with a focus on integrating biomechanical and tomographic assessments with Scheimpflug images.

Research advances in the pathogenesis of nonalcoholic fatty liver disease / 临床肝胆病杂志

The development and progression of nonalcoholic fatty liver disease (NAFLD) have complex potential mechanisms. The traditional “two-hit” pathophysiological theory has been challenged, and in recent years, an increasing number of studies have been performed to investigate the interaction between insulin resistance, adipokines, and other unknown pathogenic factors in various organs. This article summarizes the factors of the liver, intestinal tract, hypothalamus, and extracellular cysts, as well as genetic factors, with an emphasis on the synergistic mechanism of action of the liver and extrahepatic organs in the pathogenesis of NAFLD, in order to provide a reference for obtaining new insights into NAFLD regulatory network and determining new targets for the prevention and treatment of NAFLD.

The role of oxidative stress and nuclear factor erythroid 2-related factor 2 in nonalcoholic fatty liver disease / 临床肝胆病杂志

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease around the world, and its incidence rate is increasing year by year. Oxidative stress is the second hit in the classic “two-hit” pathogenesis of NAFLD, which is currently recognized as one of the pathogeneses of NAFLD. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a group of positive regulators that protect hepatocytes against oxidative stress. It is a key factor for cellular anti-oxidative stress and a key transcription factor that antagonizes liver oxidative stress. It plays an important role in the development and progression of NAFLD and may be a potential treatment target for improving NAFLD. This article reviews the role of oxidative stress and the Nrf2 pathway in the pathogenesis of NAFLD.

Sepsis and cirrhosis in growing animals: description of a new experimental model and its pathological and immunological reliability

OBJECTIVES: In cirrhotic children, infection events and sepsis are more frequent and more severe due to immune dysfunction. The objectives of the current study were therefore to develop an experimental model of infection and sepsis in cirrhotic weaning growing rats, by the use of bile duct ligation (BDL) and cecal ligation and puncture (CLP). Additionally, the correlation of the clinico-histopathological data and serial cytokine levels in septic cirrhotic and non-cirrhotic animals was studied. METHODS: Young Wistar rats of age 21 days and of weight between 70-90 g were divided into 12 groups according to the surgical procedure performed: sham (sacrificed after 2 or 4 weeks), BDL (sacrificed after 2 or 4 weeks), CLP (2- or 4-week old animals sacrificed after 12 or 24 hours), BDL+CLP (2- or 4-week old animals sacrificed after 12 hours). Histopathological studies and determination of serum levels of cytokines IL-1 beta, IL-10, and TNF-alpha, for studies of systemic infection, were performed. Murine sepsis scores (MSS) based on the clinical aspects just before euthanasia were also included. RESULTS: A transitory increase in IL-1, IL-10, and TNF-alpha levels was observed, with different patterns according to the groups. Two-hit groups tended to present with higher values of serum cytokines and histopathological scores than their septic non-cirrhotic counterparts. There was a correlation between mortality rate and MSS (p<0.0001). CONCLUSION: The model is feasible and may be utilized in studies on liver cirrhosis and infection in growing animals.

Pathogenesis of Minimal Change Nephrotic Syndrome: A Review of the Underlying Molecular Mechanisms

Nephrotic syndrome (NS) is the most common glomerular disorder in childhood, and a vast majority of cases are idiopathic. The precise cause of this common childhood disease is not fully elucidated despite significant advancements in our understanding of podocyte biology. Idiopathic NS has been considered “a disorder of T-cell function” mediated by a circulating factor that alters podocyte function resulting in massive proteinuria since the last four decades. Several circulatory factors released from T-cells are considered to be involved in pathophysiology of NS; however, a single presumptive factor has not been defined yet. Extended evidence obtained by advances in the pathobiology of podocytes has implicated podocytes as critical regulator of glomerular protein filtration and podocytopathy. The candidate molecules as pathological mediators of steroid-dependent NS are CD80 (also known as B7-1), hemopexin, and angiopoietin-like 4. The “two-hit” hypothesis proposes that the expression of CD80 on podocytes and ineffective inhibition of podocyte CD80 due to regulatory T-cell dysfunction or impaired autoregulation by podocytes results in NS. Recent studies suggest that not only T cells but also other immune cells and podocytes are involved in the pathogenesis of MCNS.

Advances in pathogenesis of minimal change nephrotic syndrome / 国际儿科学杂志

Minimal change nephrotic syndrome (MCNS) is the most common nephrotic syndrome among children.Although the details of pathogenesis remain unknown,it is widely considered that upregulated expression of T lymphocyte cell cytokines contributes to the initiation of MCNS.Moreover,studies had revealed that altered number and function disorder of B lymphocyte cells could change the functions of antigen presentation,participating in the onset of MCNS by affecting the function of T lymphocyte cells.Recently,CD80 has emerged as a popular research topic which exerts its effects via the change of podocytes morphology,thereby affecting the glomerular permeability.However,neither immune disorder nor podocyte dysfunction is poorly demonstrated to be associated with the pathogenesis of MCNS,the hypothesis such as "a two hit disorder" and "γδT cells exacerbate podocyte injury via the CD28/B7-1-phosphor-SRC kinase pathway" are raised.In the current review,we summarized the related investigations to help us to understand the mechanisms and pathogenesis of MCNS.

Advances in pathogenesis of minimal change nephrotic syndrome / 国际儿科学杂志

Minimal change nephrotic syndrome (MCNS) is the most common nephrotic syndrome among children.Although the details of pathogenesis remain unknown, it is widely considered that upregulated expression of T lymphocyte cell cytokines contributes to the initiation of MCNS.Moreover, studies had revealed that altered number and function disorder of B lymphocyte cells could change the functions of antigen presentation, participating in the onset of MCNS by affecting the function of T lymphocyte cells.Recently, CD80 has emerged as a popular research topic which exerts its effects via the change of podocytes morphology, thereby affecting the glomerular permeability.However, neither immune disorder nor podocyte dysfunction is poorly demonstrated to be associated with the pathogenesis of MCNS, the hypothesis such as "a 'two hit disorder" and "γδT cells exacerbate podocyte injury via the CD28/B7-1-phosphor-SRC kinase pathway" are raised.In the current review, we summarized the related investigations to help us to understand the mechanisms and pathogenesis of MCNS.

Protective effect of dexmedetomidine against organ dysfunction in a two-hit model of hemorrhage/resuscitation and endotoxemia in rats

Dexmedetomidine (DEX), a selective agonist of α2-adrenergic receptors, has anti-inflammation properties and potential beneficial effects against trauma, shock, or infection. Therefore, this study aimed to investigate whether DEX might protect against multiple-organ dysfunction in a two-hit model of hemorrhage/resuscitation (HS) and subsequent endotoxemia. Eighty Wistar rats were randomized into four groups: NS (normal saline), HS/L (HS plus lipopolysaccharide), HS/L+D (HS/L plus dexmedetomidine), and HS/L+D+Y (HS/L+D plus yohimbine). Six hours after resuscitation, blood gas (PaO2) and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urine nitrogen (BUN), creatinine (Cr), TNF-α, IL-β, IL-6, IL-8, IL-10, and nitric oxide (NO) were measured. The histopathology was assayed by staining. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels and heme oxygenase-1 (HO-1) were assayed. The PaO2 levels in HS/L rats were lower whereas the ALT, AST, BUN, Cr, TNF-α, IL-β, IL-6, IL-8, IL-10, and NO levels were higher compared to the control group. The HS/L+D increased PaO2 and further increased IL-10 and decreased ALT, AST, BUN, Cr, TNF-α, IL-β, IL-6, IL-8, and NO levels of the HS/L groups. In addition, the MDA in the HS/L groups increased whereas SOD activity decreased compared to the control group. Moreover, the HO-1 expression levels were increased by DEX administration in lung, liver, and kidney tissues. Lungs, livers, and kidneys of the HS/L group displayed significant damage, but such damage was attenuated in the HS/L+D group. All of the above-mentioned effects of DEX were partly reversed by yohimbine. DEX reduced multiple organ injury caused by HS/L in rats, which may be mediated, at least in part, by α2-adrenergic receptors.

Mechanistic and therapeutic advances in non-alcoholic fatty liver disease by targeting the gut microbiota / 医学前沿

Non-alcoholic fatty liver disease (NAFLD) is one of the most common metabolic diseases currently in the context of obesity worldwide, which contains a spectrum of chronic liver diseases, including hepatic steatosis, non-alcoholic steatohepatitis and hepatic carcinoma. In addition to the classical "Two-hit" theory, NAFLD has been recognized as a typical gut microbiota-related disease because of the intricate role of gut microbiota in maintaining human health and disease formation. Moreover, gut microbiota is even regarded as a "metabolic organ" that play complementary roles to that of liver in many aspects. The mechanisms underlying gut microbiota-mediated development of NAFLD include modulation of host energy metabolism, insulin sensitivity, and bile acid and choline metabolism. As a result, gut microbiota have been emerging as a novel therapeutic target for NAFLD by manipulating it in various ways, including probiotics, prebiotics, synbiotics, antibiotics, fecal microbiota transplantation, and herbal components. In this review, we summarized the most recent advances in gut microbiota-mediated mechanisms, as well as gut microbiota-targeted therapies on NAFLD.

Effects of terlipressin on blood pressure and survival in septic mice following trauma and its mechanism / 中华危重病急救医学

OBJECTIVE@#To investigate the effects of terlipressin (TP) on blood pressure and survival in septic mice following trauma and its mechanism.@*METHODS@#(1) Survival experiment: 120 male C57BL/6 mice aged 6-8 weeks were enrolled, the posttraumatic sepsis mice model was reproduced by traumatic hemorrhage (bilateral femoral fracture + 45% of total blood loss) followed by cecal ligation and puncture (CLP) after 8 hours. Intraperitoneal injection of TP was used for intervention. Sixty model mice were used to observe the effect of 0.05 μg/g TP at different intervention times (the drug was given immediately after traumatic hemorrhage + the administration was repeated after 6 hours, the drug was given immediately after traumatic hemorrhage + the administration was repeated every 6 hours until the end of the experiment, the drug was given at 4 hours after CLP + the administration was repeated every 6 hours until the end of the experiment) on 48-hour cumulative survival rate of mice with posttraumatic sepsis for finding the best intervention time of TP. The other 60 model mice were used to observe the effect of different TP intervention doses (0.01, 0.05, 0.25 μg/g) at the best intervention time on the 48-hour cumulative survival rate of mice with posttraumatic sepsis for finding the best intervention dose of TP. (2) Intervention experiment: the other 45 mice were enrolled, and they were randomly divided into traumatic hemorrhage + sham group (TH+sham group, only laparotomy without CLP), TH+CLP group, and TH+CLP+TP group (the best intervention time and dose of TP shown by survival experiment were used), with 15 mice in each group. Mean arterial pressure (MAP) of mice was monitored continuously. The orbital whole blood was collected at 2 hours after successful reproduction of the model, and the lung tissues were harvested at 12 hours and 24 hours, respectively. The pathological changes in lung tissue were observed with light microscope. The contents of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in serum and lung tissue were determined by enzyme linked immunosorbent assay (ELISA). The expressions of IL-1β and TNF-α mRNA in lung tissue were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The expressions of nuclear factor-κB p65 (NF-κB p65) in the nucleus and cytoplasm of lung tissue were determined by Western Blot.@*RESULTS@#(1) Survival experiment results showed that the 48-hour cumulative survival rate of mice was highest with TP intervention by 0.05 μg/g administration immediately after traumatic hemorrhage and repeated every 6 hours, which was the best intervention method of TP. (2) Intervention experiment results showed that the pulmonary alveolar wall fracture accompanied by inflammatory cell infiltration was found at 12 hours after the successful reproduction of traumatic sepsis model, and the pathological damage was gradually increased with time prolongation. MAP was decreased sharply after traumatic hemorrhage, and it was continued to decrease after two-hit of CLP. The contents of IL-1β and TNF-α in serum and lung tissue, the expressions of IL-1β and TNF-α mRNA in lung tissue, and expressions of NF-κB p65 protein in cytoplasm and nucleus of TH+CLP group were significantly higher than those in TH+sham group. Compared with TH+CLP group, the pathological changes in lung tissue were improved significantly, and the MAP was decreased gently after TP intervention, the levels of inflammatory mediators in serum were significantly decreased [IL-1β (pg/L): 164.32±25.25 vs. 233.11±23.02, TNF-α (pg/L): 155.56±31.47 vs. 596.38±91.50, both P < 0.05], and their expressions in lung tissue [IL-1β content (ng/mg): 262.68±16.56 vs. 408.15±17.85, IL-1β mRNA (2-Δ ΔCt): 2.63±0.68 vs. 6.22±0.74; TNF-α content (ng/mg): 311.07±17.35 vs. 405.04±24.83, TNF-α mRNA (2-Δ ΔCt): 2.04±0.62 vs. 5.32±0.55, all P < 0.01], and NF-κB p65 protein expressions were significantly down-regulated (gray value: 0.47±0.01 vs. 1.28±0.05 in cytoplasm, 0.45±0.02 vs. 1.95±0.06 in nucleus, both P < 0.01].@*CONCLUSIONS@#The continuous intervention with TP 0.05 μg/g administration immediately after traumatic hemorrhage and repeated every 6 hours could improve the MAP of mice with traumatic sepsis, and improve the prognosis. The mechanism may be related to alleviating the inflammatory response and inhibiting the activation of the NF-κB signaling pathway in the lung tissue.

Effects of terlipressin on blood pressure and survival in septic mice following trauma and its mechanism / 中华危重病急救医学

Objective@#To investigate the effects of terlipressin (TP) on blood pressure and survival in septic mice following trauma and its mechanism.@*Methods@#① Survival experiment: 120 male C57BL/6 mice aged 6-8 weeks were enrolled, the posttraumatic sepsis mice model was reproduced by traumatic hemorrhage (bilateral femoral fracture + 45% of total blood loss) followed by cecal ligation and puncture (CLP) after 8 hours. Intraperitoneal injection of TP was used for intervention. Sixty model mice were used to observe the effect of 0.05 μg/g TP at different intervention times (the drug was given immediately after traumatic hemorrhage + the administration was repeated after 6 hours, the drug was given immediately after traumatic hemorrhage + the administration was repeated every 6 hours until the end of the experiment, the drug was given at 4 hours after CLP + the administration was repeated every 6 hours until the end of the experiment) on 48-hour cumulative survival rate of mice with posttraumatic sepsis for finding the best intervention time of TP. The other 60 model mice were used to observe the effect of different TP intervention doses (0.01, 0.05, 0.25 μg/g) at the best intervention time on the 48-hour cumulative survival rate of mice with posttraumatic sepsis for finding the best intervention dose of TP. ② Intervention experiment: the other 45 mice were enrolled, and they were randomly divided into traumatic hemorrhage + sham group (TH+sham group, only laparotomy without CLP), TH+CLP group, and TH+CLP+TP group (the best intervention time and dose of TP shown by survival experiment were used), with 15 mice in each group. Mean arterial pressure (MAP) of mice was monitored continuously. The orbital whole blood was collected at 2 hours after successful reproduction of the model, and the lung tissues were harvested at 12 hours and 24 hours, respectively. The pathological changes in lung tissue were observed with light microscope. The contents of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in serum and lung tissue were determined by enzyme linked immunosorbent assay (ELISA). The expressions of IL-1β and TNF-α mRNA in lung tissue were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The expressions of nuclear factor-κB p65 (NF-κB p65) in the nucleus and cytoplasm of lung tissue were determined by Western Blot.@*Results@#① Survival experiment results showed that the 48-hour cumulative survival rate of mice was highest with TP intervention by 0.05 μg/g administration immediately after traumatic hemorrhage and repeated every 6 hours, which was the best intervention method of TP. ② Intervention experiment results showed that the pulmonary alveolar wall fracture accompanied by inflammatory cell infiltration was found at 12 hours after the successful reproduction of traumatic sepsis model, and the pathological damage was gradually increased with time prolongation. MAP was decreased sharply after traumatic hemorrhage, and it was continued to decrease after two-hit of CLP. The contents of IL-1β and TNF-α in serum and lung tissue, the expressions of IL-1β and TNF-α mRNA in lung tissue, and expressions of NF-κB p65 protein in cytoplasm and nucleus of TH+CLP group were significantly higher than those in TH+sham group. Compared with TH+CLP group, the pathological changes in lung tissue were improved significantly, and the MAP was decreased gently after TP intervention, the levels of inflammatory mediators in serum were significantly decreased [IL-1β (pg/L): 164.32±25.25 vs. 233.11±23.02, TNF-α (pg/L): 155.56±31.47 vs. 596.38±91.50, both P < 0.05], and their expressions in lung tissue [IL-1β content (ng/mg): 262.68±16.56 vs. 408.15±17.85, IL-1β mRNA (2-ΔΔCt): 2.63±0.68 vs. 6.22±0.74; TNF-α content (ng/mg): 311.07±17.35 vs. 405.04±24.83, TNF-α mRNA (2-ΔΔCt): 2.04±0.62 vs. 5.32±0.55, all P < 0.01], and NF-κB p65 protein expressions were significantly down-regulated (gray value: 0.47±0.01 vs. 1.28±0.05 in cytoplasm, 0.45±0.02 vs. 1.95±0.06 in nucleus, both P < 0.01].@*Conclusions@#The continuous intervention with TP 0.05 μg/g administration immediately after traumatic hemorrhage and repeated every 6 hours could improve the MAP of mice with traumatic sepsis, and improve the prognosis. The mechanism may be related to alleviating the inflammatory response and inhibiting the activation of the NF-κB signaling pathway in the lung tissue.

Regulatory effect of hemichannels protein Pannexin-1 on P2X7 receptor activity in the lungs of mice with lung injury / 中华危重病急救医学

Objective To investigate the regulative effect of hemichannels protein Pannexin-1 on P2X7 receptor activation and caspase-1 mediated inflammatory response in the lungs of mice with lung injury. Methods Sixty male C57BL/6 mice were randomly divided into five groups with 12 mice in each group: sham operation group (Sham group), mechanical ventilation (MV) group, MV + low dose lipopolysaccharide (LPS) group (MLL group), MV + medium and high dose LPS group (MML group) and MV + high dose LPS group (MHL group). A "two-hit" lung injury model was reproduced by MV with high tidal volume combined with LPS injection in airway. All the mice underwent tracheotomy and intubation. After operation, the mice in Sham group were maintained spontaneous breathing, and those in other four groups were put on small animal ventilators to give MV with a large tidal volume of 28 mL/kg. After stable respiration in mice, those in the Sham group and MV group were injected 8 mL/kg of normal saline (NS) into the airway, and those in MLL, MML and MHL groups were given 2, 5 and 8 mg/kg of LPS respectively (diluted with NS into 8 mL/kg). After 4 hours on MV, the mice were sacrificed, and bronchoalveolar lavage fluid (BALF) was extracted to determine intracellular and extracellular ATP concentration. Lung tissue was harvested and water containing ratio of lungs was measured. The degree of lung pathological damage was observed after hematoxylin-eosin (HE) staining, and lung injury score was calculated. The expression of Pannexin-1 protein in lung tissue was calculated with immunohistochemistry. Western Blot and fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) were used to detect the protein and mRNA expressions of Pannexin-1, P2X7 receptor, caspase-1 and interleukin-1β (IL-1β). Results There was no obvious pathological change in lung tissue in Sham group, intracellular ATP concentration was higher than extracellular ATP concentration, water content in lung tissue was lower, Pannexin-1 expression was low in lung tissue by immunohistochemical staining, and Pannexin-1, P2X7 receptor, caspase-1 and IL-1β were only expressed in micro-protein and mRNA in lung tissue. Compared with the Sham group, the alveolar lesions and hemorrhages in the MV group were not obvious, and lung injury score was slightly increased. There was no significant fluctuation between intracellular ATP concentration and extracellular ATP concentration. The water content in lung tissue was increased significantly, while the expressions of Pannexin-1, P2X7 receptor, caspase-1 and IL-1β in lung tissue were increased slightly. After LPS intervention, progressively increased lung exudation, ruptured alveoli, dilated capillaries, and inflammatory cells were found, and lung injury score was increased without significant difference among the three LPS doses groups. With the increase in LPS dosage, the concentration of extracellular ATP in BALF was increased, the concentration of intracellular ATP was decreased, the water containing ratio of lung tissue was increased gradually, and the protein and mRNA expressions of Pannexin-1, P2X7 receptor, caspase-1 and IL-1β in lung tissue were increased gradually in a dose-dependent manner. The parameters in MHL group showed significant differences as compared with those in MV group [lung injury score: 8.25±0.45 vs. 3.50±0.52; intracellular ATP concentration (μmol/L): 198.76±150.77 vs. 896.69±281.11, extracellular ATP concentration (μmol/L): 336.57±90.28 vs. 141.52±42.22; lung water containing rate: (6.37±0.11)% vs. (5.05±0.14)%; Pannexin-1 protein (gray value): 3.20±0.70 vs. 1.54±0.76, Pannexin-1 mRNA (2-ΔΔCT): 7.86±0.86 vs. 2.47±0.92; P2X7 receptor protein (gray value): 3.18±0.88 vs. 1.80±0.72, P2X7 receptor mRNA (2-ΔΔCT): 7.17±0.96 vs. 2.31±0.45; caspase-1 protein (gray value): 3.00±0.45 vs. 0.93±0.51, caspase-1 mRNA (2-ΔΔCT): 4.39±0.91 vs. 2.74±0.41; IL-1β protein (gray value): 2.54±1.08 vs. 1.16±0.53, IL-1β mRNA (2-ΔΔCT): 132.34±41.48 vs. 19.67±8.67; all P < 0.05]. Conclusion Pannexin-1 may be involved in LPS and MV induced lung injury, which may be regulated by intracellular release of ATP to the extracellular site and binding to P2X7 receptor on the cell surface, thereby regulating active caspase-1 production and release, involving in the production of IL-1β and other inflammatory factors eventually which leads to the occurrence and development of lung injury.

Beneficial effects of polysaccharides on "two-hit" acute lung injury and endotoxic fever in rats associated with anti-complementary activities

Thunb. is a traditional herb used for clearing heat and eliminating toxins, and has also been used for the treatment of severe acute respiratory syndrome (SARS). the crude polysaccharides (CHCP) exhibited potent anti-complementary activity through both the classical and alternative pathways by acting on components C3 and C4 of the complement system without interfering with the coagulation system. This study was to investigate the preventive effects of CHCP on acute lung injury (ALI) induced by hemorrhagic shock plus lipopolysaccharide (LPS) instillation (two-hit) and LPS-induced fever in rats. CHCP significantly attenuated pulmonary injury in the "two-hit" ALI model by reducing pulmonary edema and protein exudation in bronchoalveolar lavage fluid (BALF). In addition, it reduced the deposit of complement activation products in the lung and improved oxidant-antioxidant imbalance. Moreover, CHCP administration inhibited fever in rats, reduced the number of leukocytes and restored serum complement levels. The inhibition on the inappropriate activation of complement system by CHCP may play an important role in its beneficial effects on inflammatory diseases. The anti-complementary polysaccharides are likely to be among the key substances for the heat-clearing function of .

Saquinavir plus methylprednisolone ameliorates experimental acute lung injury

Glucocorticoid insensitivity is an important barrier to the treatment of several inflammatory diseases, including acute lung injury (ALI). Saquinavir (SQV) is an inhibitor of the human immunodeficiency virus protease, and the therapeutic effects of SQV in ALI accompanied with glucocorticoid insensitivity have not been previously investigated. In this study, the effects of SQV on lipopolysaccharide (LPS)-mediated injury in human pulmonary microvascular endothelial cells (HPMECs), human type I alveolar epithelial cells (AT I), and alveolar macrophages were determined. In addition, the effects of SQV on an LPS-induced ALI model with or without methylprednisolone (MPS) were studied. In LPS-stimulated HPMECs, SQV treatment resulted in a decrease of high mobility group box 1 (HMGB1), phospho-NF-κB (p-NF-κB), and toll-like receptor 4 (TLR4), and an increase of VE-cadherin. Compared to MPS alone, MPS plus SQV attenuated the decrease of glucocorticoid receptor alpha (GRα) and IκBα in LPS-stimulated HPMECs. HMGB1, TLR4, and p-NF-κB expression were also lessened in LPS-stimulated alveolar macrophages with SQV treatment. In addition, SQV reduced the injury in human AT I with a decrease of HMGB1 and p-NF-κB, and with an increase of aquaporin 5 (AQP 5). SQV ameliorated the lung injury caused by LPS in rats with reductions in vascular permeability, myeloperoxidase activity (MPO) and histopathological scores, and with lowered HMGB1, TLR4, and p-NF-κB expression, but with enhanced VE-cadherin expression. By comparison, SQV plus MPS increased GRα and IκBα in lung tissues of rats with ALI. This study demonstrated that SQV prevented experimental ALI and improved glucocorticoid insensitivity by modulating the HMGB1/TLR4 pathway.