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.

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.

"Comparison and evaluation of three ""two-hit"" animal models of acute respiratory distress syndrome induced by different methods" / 中国实验动物学报

Objective To find an ideal animal model of acute respiratory distress syndrome (ARDS) through investigating the characteristics of three two-hit animal models of ARDS.Methods Forty-eight SD rats were randomly divided into 4 groups: Control group [2.5 mL/kg normal saline (NS) i.v.given at 0 min and 30 min];OA+OA group [0.5 mL/kg oleic acid (OA) i.v.given at 0 min and 30 min];LPS+LPS group [2.5 mg/kg lipopolysaccharide (LPS) i.v.given at 0 min and 30 min];and OA+LPS group [0.5 mL/kg OA i.v.given at 0 min and 2.5 mg/kg LPS,i.v.given at 30 min].The samples were collected at 5 h after the second drug injection.White blood cells count (WBC),polymorphonuclear leukocyte ratio (PMN%),total protein concentration,tumor necrosis factor α (TNF-α) level in bronchoalveolar lavage fluid (BALF),arterial blood gas analysis and lung wet-dry weight ratio (W/D) were measured,respectively.Pathological changes in the lung tissues were observed and histological scores were evaluated.Results Compared with those in the control group,PaCO2,WBC,PMN%,total protein concentration and TNF-α levels in BALF were significantly increased,while PaO2 was dramatically decreased (P<0.01) in the OA+OA,LPS+LPS and OA+LPS groups.The levels of protein concentration in BALF and lung W/D ratio in the OA+LPS group were significantly higher than these in the LPS+LPS group (P<0.05 for all),but had no statistically significant difference compared with these in the OA+OA group.The levels of WBC,PMN% and TNF-α in BALF in the OA+LPS group were significantly higher than those in the OA+OA group (P<0.05),but not significantly different from those in the LPS+LPS group.The most typical pathological changes and the highest pathological scores were found in the OA+LPS group.Conclusions All the three different methods including OA+OA,LPS+LPS,and OA+LPS can be used to establish “two-hit” animal models of acute respiratory distress syndrome.The “two-hit” animal model of acute respiratory distress syndrome induced by OA+LPS is more closer to clinical ARDS and is useful for studies on the pathophysiology of ARDS,and is an ideal “two-hit” animal model of ARDS.

"Reproduction of a model of ""two-hit"" sepsis model with complication of pneumonia in rat" / 中华危重病急救医学

ObjectiveTo reproduce a clinically relevant two-hit model of sepsis complicated by pneumonia and to explore the correlation between two-hit and immune state.Methods Eighty-one male Sprague-Dawley ( SD ) rats were divided into groups according to the random number table. Forty-five male rats were assigned respectively to sepsis-alone group, pneumonia 4 days and 7 days after sepsis groups, respectively. Survival rate of each group was observed. Another group of 36 male rats were divided into normal control group, sepsis-alone for 1, 4 and 7 days groups, and sepsis complicated by pneumonia for 4 days and 7 days after sepsis groups, each group consisted of 6 rats. Cecal ligation and puncture (CLP) was done in rats, andStreptococcus pneumoniae suspension (bacteria count 1×1010 cfu/mL) was injected via the nose on the 4th day or 7th day after CLP. Rats were sacrificed at corresponding time points, and 1 day after challenge ofStreptococcus pneumoniae on the 4 days or 7 days post CLP for the collection of blood and tissue samples to make bacterial count of the blood, splenocyte count, biochemical indices, cytokines concentration, pathological changes in spleen and apoptotic cells.Results① Compared with the rats of sepsis-alone group, the rats in pneumonia 4 days after CLP group had poor survival rate (4 vs. 11,χ2 = 6.533,P = 0.011), while no difference was found between pneumonia 7 days after CLP group and sepsis-alone group (9 vs. 11,χ2 = 0.600,P = 0.439).② The blood bacterial count and all the biochemical indexes were sharply increased on 1 day post-CLP in the rats of sepsis-alone group, and then they gradually lowered. Compared with the rats of 1 day post-CLP, the proportion of splenocytes were decreased on the 4th day post-CLP [dendritic cells (DC): (0.69±0.09)% vs. (0.87±0.31)%, CD4+T cells: (21.05±2.89)% vs. (24.84±4.59)%, CD8+ T cells: (10.62±1.79)% vs. (13.40±1.31)%, allP 0.05). The same trend of changes, with slight reduction in splenocytes and biochemical indices were found between the groups of sepsis followed by pneumonia and sepsis-alone, but no significant difference was found. The level of HMGB1 in the 4-day group of sepsis with complication of pneumonia was further decreased compared with sepsis-alone group (μg/L:1.17±0.74 vs. 1.76±0.71,P 0.05).Conclusions The mortality of this two-hit model with complication of pneumonia 4 days after CLP was significantly higher than that of single sepsis model. The ability of bacteria clearance was decreased, and immunocyte apoptosis was exacerbated. These findings may be with the result of the occurrence of immunoparalysis in the mid stage of sepsis. The two-hit model reproduced on 7 days after CLP might suggest reconstruction of host immune function, and maybe associated with the recovery of immune response.

Effect of μ-opioid receptor agonist on acute lung injury induced by trauma-LPS two hits in rat model / 临床麻醉学杂志

Objective To explore the effect of μ-opioid receptor agonist on acute lung injury in-duced by trauma-LPS two hits in rat model.Methods Thirty-two aduld SD rats,were randomized in four groups(n=8):normal control group(group N),blank control group(group B),DAMGO group (group D)and DAMGO+CTOP group(group DC).A rat model was made by intraperitoneal injection of LPS at 6 hours after fracturing bilateral thighbone.Group N didn’t receive trauma and LPS,only anesthesia.The rats in group D received 200 μg/kg of DAMGO,group DC received 600 μg/kg of CTOP and 200 μg/kg of DAMGO,group B obtained the same amount of saline.6 hours after treat-ment,the arterial blood was collected for blood gas analysis,the lungs was harvested to observe lung tissue pathology change and dry-weight/wet-weight ratio,and the levels of MDA,TNF-α,IL-6,SOD activities in lung tissue were determined.Results The results of pathological observation showed that there was obvious inflammatory reaction in lung tissues after two-hits.Compared with group D, PaO2 ,pH and dry-weight/wet-weight ratio were significantly lower in group B and group DC(P <0.05),The score of Smith were significantly increased(P <0.05).The levels of MDA,TNF-α,IL-6 in lung tissue were significantly reduced in group D than those in group B and group DC(P <0.05), SOD activities in group D were significantly higher than those in group B and group DC(P <0.05 ). Conclusion μ-Opioid receptor DAMGO agonist has protective effect on acute lung injury induced by trauma-LPS two hits in rat model.

High mobility group box 1 as a mediator of endotoxin administration after hemorrhagic shock-primed lung injury

High mobility group box 1 (HMGB1) was discovered as a novel late-acting cytokine that contributes to acute lung injury (ALI). However, the contribution of HMGB1 to two-hit-induced ALI has not been investigated. To examine the participation of HMGB1 in the pathogenesis of ALI caused by the two-hit hypothesis, endotoxin was injected intratracheally in a hemorrhagic shock-primed ALI mouse model. Concentrations of HMGB1 in the lung of the shock group were markedly increased at 16 h (1.63 ± 0.05, compared to the control group: 1.02 ± 0.03; P < 0.05), with the highest concentration being observed at 24 h. In the sham/lipopolysaccharide group, lung HMGB1 concentrations were found to be markedly increased at 24 h (1.98 ± 0.08, compared to the control group: 1.07 ± 0.03; P < 0.05). Administration of lipopolysaccharide to the hemorrhagic shock group resulted in a notable HMGB1 increase by 4 h, with a further increase by 16 h. Intratracheal lipopolysaccharide injection after hemorrhagic shock resulted in the highest lung leak at 16 h (2.68 ± 0.08, compared to the control group: 1.05 ± 0.04; P < 0.05). Compared to the hemorrhagic shock/lipopolysaccharide mice, blockade of HMGB1 at the same time as lipopolysaccharide injection prevented significantly pulmonary tumor necrosis factor-alpha, interleukin-1beta and myeloperoxidase. Lung leak was also markedly reduced at 16 h; blockade of HMGB1 24 h after lipopolysaccharide injection failed to alter lung leak or myeloperoxidase at 48 h. Our observations suggest that HMGB1 plays a key role as a late mediator when lipopolysaccharide is injected after hemorrhagic shock-primed ALI and the kinetics of its release differs from that of one-hit ALI. The therapeutic window to suppress HMGB1 activity should not be delayed to 24 h after the disease onset.

Mesenteric lymph duct ligation relives hepatic injury subjected to two-hit in rats / 基础医学与临床

Objective To explore the mechanism of mesenteric lymph duct ligation relieving hepatic injury in rats by two-hit of hemorrhage and LPS.Methods Forty-five Wistar rats were divided into three groups: ligation group,non-ligation group and sham group,and the two-hit model was established by hemorrhage and LPS,mesenteric lymph was blocked by ligating mesenteric lymph duct in ligation group.After 24 hours of operation,took out the liver for pathological section,and the hepatocellular apoptosis rate was determined by method of TUNEL,the expression of BCL-2 and BAX protein was determined by immunohistochemical test.At the same time,taking out liver for homogenate of 10 percent,the activity of MPO and ATPase and the contents of TNF-? and IL-6 were determined in hepatic homogenate.Results After two-hit,the hepatocellular apoptosis rate and expression of BAX protein in non-ligation group were significantly increased as compared with sham group and ligation group,and expression of BCL-2 protein was significantly lower.The contents of MPO,TNF-? and IL-6 in hepatic homogenate of non-ligation group were significantly increased than that of sham group,and the activity of ATPase in hepatic homogenate was significantly lower.But the ATPase in hepatic homogenate of ligation group were significantlyincreased and MPO,TNF-? and IL-6 in hepatic homogenate of ligation group were significantly lower as compared with non-ligation group.Conclusion The mechanism of mesenteric lymph duct ligation relieving hepatic injury of rats was related to the mesenteric lymph blockage reduces the TNF-? and IL-6 and improves the expression of BCL-2 protein and the activity of ATPase in liver.

Panaxadiol saponins alleviates acute lung injury in the two-hit rat model with hemorrhagic shock and lipopolysaccharide / 中国病理生理杂志

AIM: To establish the two-hit rat model with hemorrhage and lipopolysaccharide(LPS) and to study the effect of panaxadiol saponins(PDS) against acute lung injury.METHODS: Forty Wistar rats were divided randomly into 4 groups: sham operational group(S);two-hit groups with hemorrhagic shock-LPS(HL);dexamethasone pretreatment group(HLD) and PDS pretreatment group(HLP).The mean arterial blood pressure(MABP) was monitored dynamically by 4-channel physiological meter RM-6000,and pathological alteration of lung tissues was also observed.The levels of various serum enzymes,TNF-? and IL-6 were detected.RESULTS: MABP decreased in two-hit rat model with hemorrhage-LPS.The serum levels of aspartate aminotransferase,alanine aminotransferase,alkaline phosphatase,lactate dehydrogenase,creatine kinase,TNF-? and IL-6 increased significantly.Severe inflammatory change of pulmonary interstitium in HL group was also observed.CONCLUSION: Endotoxin injection following hemorrhage can be used to establish the animal model of acute lung injury.Similar with dexamethasone,PDS prevents lung tissue from seriously damage through increasing MABP and decreasing the level of serum enzymes,TNF-? and IL-6 levels.