Exploring the correlation analysis of immune microenvironment, mutation burden and prognosis of papillary thyroid carcinoma based on Estimate algorithm.

Background: This study aimed to investigate the correlation between immune infiltration and tumor mutational burden (TMB) in papillary thyroid carcinoma. Methods: Transcriptome sequencing data sets, gene mutation data sets, and clinical data sets were downloaded from The Cancer Genome Atlas (TCGA) database. The immune and papillary carcinoma stromal scores were calculated using the "Estimate" package of R software. The relationship between the immune score, stromal score, TMB, and papillary thyroid carcinoma progression-free survival was analyzed. Pearson's test was used to analyze the correlation between the immune score, stromal score, and TMB. Results: The stromal score and immune score of papillary thyroid carcinoma tissue were lower than those of normal thyroid tissue (P=0.008 and P<0.001, respectively). There was no significant difference in progression-free survival between the high stromal and low stromal score groups (P=0.075). The progression-free survival of the high immune score group was better than that of the low immune score group (P=0.029), and the progression-free survival of the low TMB group was better than that of the high TMB group (P<0.001). The high immune score, low TMB group had the best prognosis (P=0.003). Univariate Cox analysis showed that age, pathological stage, and TMB were risk factors for progression-free survival [hazard ratio (HR) >1, P<0.05], and that the immune score was a protective factor for progression-free survival (HR <1, P<0.05). Multivariate Cox analysis showed that age and TMB were independent risk factors for progression-free survival (HR >1, P<0.05), and that the immune score was an independent protective factor for progression-free survival (HR <1, P<0.05). Correlation analysis showed that the immune and stromal score were both negatively correlated with TMB (r=-0.26, P=0.031 and r=-0.41, P=0.028, respectively). Conclusions: The immune and stromal scores of papillary thyroid carcinoma were negatively correlated with TMB. Thyroid cancer gene mutations inhibit immune cell infiltration and alter the thyroid cancer microenvironment. The immune score was an independent protective factor for progression-free survival, while TMB was an independent risk factor, both of which can be used for clinical prognosis assessment. Combined immunological and genomic analysis of papillary thyroid carcinoma can reveal potential prognostic markers and therapeutic targets and provide clues for the tumor immune escape mechanism.

[High mobility group box 1 contributes to the endoplasmic reticulum stress of liver in rats with trauma].

OBJECTIVE: To investigate the role of high mobility group box 1 (HMGB1) in hepatic endoplasmic reticulum stress (ERS) in rats with trauma. METHODS: Sixty SPF Sprague-Dawley (SD) rats were randomly divided into groups (n = 6). The rat model of liver injury following traumatic stress was established by continuous compressing the bilateral hind-limbs of rats for 3 hours and then intermittent compressing and decompressing for 30 minutes respectively three times with standard weight of 15 kg. The experiment 1 was divided into two groups: control group and 6, 18, 30 hours after crush. The experiment 2 was divided into control group, crush model group (18 hours after crush), HMGB1 inhibitor sodium butyrate (SB) or ethyl pyruvate (EP) groups, and SB or EP treatment groups (500 mg/kg SB solution or 40 mg/kg EP solution was injected intraperitoneally after 3 hours crush). The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were measured with automatic biochemistry analyzer. Histopathological severity of liver injury was assessed by hematoxylin and eosin (HE) staining. The expressions of HMGB1 and ERS-related proteins were detected with Western Blot. The expression and translocation of HMGB1 in liver tissue were evaluated by immuno-histochemical technique. RESULTS: (1) Compared with the control group, the pathological changes of liver injury, the levels of AST and ALT in serum and protein expression of HMGB1 as well as ERS-related proteins such as glucose regulated protein 78 (GRP78), caspase-12, and inositol-requiring enzyme 1α (IRE1α) in liver tissue were significantly increased after traumatic stress, and reached the peak at 18 hours. The expression of C/EBP-homologous protein (CHOP) was increased in a time-dependent manner and peaked at 30 hours after crush. Immunohistochemistry showed that HMGB1 expression increased at 6 hours after crush, some HMGB1 shifted from nucleus to cytoplasm, and the expression was more obvious at 18 hours. (2) Compared with crush model group, the expressions of HMGB1 and ERS-related proteins were significantly decreased following the administration of HMGB1 inhibitors SB or EP (HMGB1/ß-actin: 0.703±0.213, 0.512±0.075 vs. 1.041±0.186; GRP78/ß-actin: 0.614±0.052, 0.450±0.115 vs. 0.847±0.120; caspase-12/ß-actin: 0.636±0.066, 0.812±0.142 vs. 1.086±0.130; CHOP/ß-actin: 0.314±0.046, 0.621±0.123 vs. 0.996±0.764; IRE1α/ß-actin: 0.473±0.033, 0.519±0.094 vs. 0.742±0.054, all P < 0.05), the levels of serum AST and ALT were significantly decreased [AST (U/L): 1 030.50±427.73, 1 414.50±347.86 vs. 2 122.20±322.76; ALT (U/L): 285.75±11.30, 368.50±80.58 vs. 473.80±33.54, all P < 0.01], the degree of acute liver injury was reduced. Only SB or EP could not affect the parameters mentioned above. CONCLUSIONS: HMGB1-ERS pathway was involved in mediating traumatic stress-induced acute liver injury in rats.

[The Role of Hydrogen Sulfide in Acute Liver Injury Induced by Traumatic Stress in Rats].

OBJECTIVE: To explore the role of hydrogen sulfide (H2S) in acute liver injury induced by crushing hind limbs of rats. METHODS: The rats were randomly divided into the following groups: control, crushing, H2S donor sodium hydrosulfide (NaHS) + crushing, H2S inhibitor propargylglycine (PAG) + crushing group. The acute liver injury model was established by 'crushing the hind limbs of rats with standard weight. Rats were sacrificed at 30 min and 120 min after the crush. The activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by colorimetric method, and the content of H2S in plasma and the contents of malondialdehyde (MDA), protein carbonyl, glutathione (GSH) in the liver and the activity of H2S generating enzyme (cystathionine y-lyase, CSE) were determined by chemical method. The expression of CSE mRNA in liver was detected by RT-PCR. RESULTS: For crush injury group, the levels of AST and ALT in serum, MDA and protein carbonyl in liver increased. The levels of GSH, CSE, CSE mRNA in liver and H2S in serum decreased. The administration of NaHS before limbs crush could attenuate the changes of liver injury, but the pre-treatment with PAG could exacerbate the changes. CONCLUSION: The decrease of H2S production could involve in mediating the acute liver injury induced by traumatic stress in rats.

[Effects of HO-1 on Lipopolysaccharide-induced Endoplasmic Reticulum Stress of Rat Hepatocytes].

OBJECTIVE: To investigate effects of antioxidant stress protein heme oxygenase-1 (HO-1) on lipopolysaccharide (LPS)-induced endoplasmic reticulum stress (ERS) of rat hepatocytes. METHODS: The BRL cells (rat hepatocyte cell line) were cultured. The hepatocytes were treated with LPS, LPS+HO-1 siRNA, HO-1 siRNA and PBS solution, respectively. The cell viability was measured by trypan blue exclusion test. The apoptosis cells were detected by the fluorescent dye Hoechst 33258. Expressions of GRP78, CHOP, caspase-12 and HO-1 were detected by Western blotting. RESULTS: LPS caused an increase of HO-1 protein expression of rat hepatocytes in a dose-dependent and time-dependent manner, a up-regulation of GRP78, CHOP and caspase-12, a decrease in cell viability, and an increase in apoptosis rate of hepatocytes. Pretreatment of HO-1 siRNA inhibited the up-regulation of LPS-induced HO-1, however, aggravated ERS and cellular injury. CONCLUSION: HO-1 inhibites ERS-mediated cellular injury of rat hepatocytes induced by LPS.

[Nitric oxide mediated TNF-α, IL-1ß gene expression in liver induced by crush injury of rat's soft tissues].

OBJECTIVE: To explore the effect of nitric oxide (NO) on the gene expression of hepatic TNF-α and IL-1ß by crush injury of rat's soft tissues. METHODS: Rats were randomly divided into sham group, crush group, crush+aminoguanidine (AG) group, and crush+L-arginine (L-Arg) group. Activities of ALT and AST as well as NO level in serum were measured. Gene expressions of TNF-α and IL-1ß were detected with RT-PCR. RESULTS: Obvious increase in TNF-α and IL-1ß mRNA expression was detected in the crush group compared with the sham group (P<0.05). After pretreated L-Arg, expressions of TNF-α and IL-1ß mRNA were markedly increased (P<0.05). After pretreated AG, those indices obviously decreased (P<0.05). Activities of ALT and AST enhanced and NO level increased in the crush group compared with the sham group (P<0.05). Pretreatment with L-Arg or AG led to substantial increased or reduced activities of ALT and AST as well as NO levels, respectively. CONCLUSION: Endogenous NO mediated TNF-α, IL-1ß mRNA up expression in liver induced by increased production of NO after crush injury of rat's soft tissues.

Negative modulation of NO for diaphragmatic contractile reduction induced by sepsis and restraint position.

In practice of forensic medicine, potential disease can be associated with fatal asphyxia in restraint position. Research has demonstrated that nitric oxide (NO) and nitric oxide synthase (NOS) are plentifully distributed in skeletal muscle, contributing to the regulation of contractile and relaxation. In the current study, respiratory functions, indices of diaphragmatic biomechanical functions ex vivo, as well as NO levels in serum, the expressions of diaphragmatic inducible NOS (iNOS) mRNA, and the effects of L-NNA on contractility of the diaphragm were observed in sepsis induced by cecal ligation and puncture (CLP) under the condition of restraint position. The results showed that in the CLP12-18h rats, respiratory dysfunctions; indices of diaphragmatic biomechanical functions (Pt, +dT/dt(max), -dT/dt(max), CT, Po, force over the full range of the force-frequency relationship and fatigue resistance) declined progressively; the NO level in serum, and iNOS mRNA expression in the diaphragm increased progressively; force increased significantly at all stimulation frequencies after L-NNA pre-incubation. Restraint position 1 h in CLP12 h rats resulted in severe respiratory dysfunctions after relative stable respiratory functions, almost all the indices of diaphragmatic biomechanical functions declined further, whereas little change took place in NO level in serum and diaphragmatic iNOS mRNA expression; and the effects of L-NNA were lack of statistical significance compared with those of CLP12 h, but differed from CLP18 h group. These results suggest that restraint position and sepsis act together in a synergistic manner to aggravate the great reduction of diaphragmatic contractility via, at least in part, the negative modulation of NO, which may contribute to the pathogenesis of positional asphyxia.

[Expression of CaMK II delta in cerebral cortex following traumatic brain injury].

OBJECTIVE: To observe the time-course expression of calcium-calmodulin dependent protein kinase II delta (CaMK II delta) in cerebral cortex after traumatic brain injury (TBI). METHODS: The TBI rat model was established. The expression of CaMK II delta in cerebral cortex around injured area was tested by Western blotting and immunohistochemical staining. RESULTS: Western blotting revealed expression of CaMK II delta in normal rat brain cortex. It gradually increased after TBI, peaked after 3 days, and then returned to normal level. The result of immunohistochemical staining was consistent with that of Western blotting. CONCLUSION: The expression of CaMK II delta around injured area after TBI increased initially and then decreased. It could be used as a new indicator for wound age determination following TBI.

[Endoplasmic reticulum stress mediates lipopolysaccharide-induced apoptosis in rat hepatocyte].

OBJECTIVE: To investigate the role of endoplasmic reticulum stress (ERS) in lipopolysaccharide (LPS)-induced hepatocyte apoptosis. METHODS: Cells of the rat hepatocyte line BRL were cultured. The hepatocytes were treated with LPS, ERS inducer thapsigargin (TG), and ERS inhibitor 4-phenylbutyric acid (4-PBA), respectively or in their different combination. The cell viability was measured by MTT assay. The cyto-nuclear morphological changes of apoptosis cells were detected by the fluorescent dye Hoechst 33258. The apoptosis rate was assessed by flow cytometry with Annexin V-FITC/PI double-staining. Expressions of GRP78 as ERS marker protein, CHOP, caspase-12 and cleaved-caspase-3 as ERS related protein were detected by Western blotting. RESULTS: LPS could cause a decrease in cell viability and an increase in apoptosis rate in a dose- and time-dependent manner. The expression of GRP78, CHOP, caspase-12 and cleaved-caspase-3 proteins were significantly increased with LPS treatment. TG led to a marked decrease in cell viability and an increase in apoptosis rate, which aggravated the hepatocyte injury induced by LPS; whereas 4-PBA alleviated LPS-induced apoptosis. CONCLUSION: ERS mediates LPS-induced hepatocyte injuries, indicating that ERS may play a vital role in the pathogenesis of LPS-induced hepatocyte injuries.

[Changes of MDA, SOD, TNF-alpha, and IL-1beta in rat brain tissue after concussion].

OBJECTIVE: To observe the changes of malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) in rat brain tissue and to explore the mechanism of secondary cerebral injury after brain concussion. METHODS: The brain concussion model was established with the pathological changes of rat brain tissue by Weil stain. The expressions of MDA and SOD in brain tissue were examined by photochemical method. The expressions of TNF-alpha and IL-1beta in cerebral cortex and hippocampus were examined by immunochemistry. RESULTS: Nerve myelin sheath showed disorder, disruption, gryposis and swelling by Weil stain. Above changes were more severe at 12h. The quantity of MDA in rat brain tissue after concussion was significantly higher than that in the control group. The activity of SOD was significantly lower than that in the control group. The expressions of TNF-alpha and IL-1beta increased more significantly in cerebral cortex and hippocampus in rat brain tissue after concussion than that in the control group. CONCLUSION: Oxidative stress and inflammatory injury in the rat brain tissue, which may play an important role in secondary cerebral injury after concussion.

[Myocardial expression of Spry1 and MAPK proteins of viral myocarditis].

OBJECTIVE: To discuss the myocardial expression of Spry1 and MAPK proteins of viral myocarditis (VMC), to reveal its mechanism of sudden death, and to provide guides for forensic identification of sudden cardiac death. METHODS: Thirty Balb/c male mice were randomly divided into VMC group and control group, inoculated intraperitoneally with Coxsackievirus B3 and Eagel's solution, respectively. After the mice were sacrificed, the cardiac tissues of the mice were taken to proceed regular pathological examination. The changes of Spry1 protein, Spry1 mRNA and MAPK protein were detected by immunohistochemistry, Western blotting and real-time PCR. RESULTS: Under light microscope, the pathologic changes included myocardial interstitial edema, inflammatory cells infiltration, myocardial necrosis, and focal and patchy necrosis of myocardial fiber in VMC group. The expression of Spry1 protein in VMC group was lower than that in control group (P < 0.05). There was slightly decreased expression of Spry1 of the mRNA level in VMC group (P > 0.05). But the MAPK protein expression in VMC group was higher than that in control group (P < 0.05). CONCLUSION: The pathway of MAPK/ERK involving Spry1 protein accelerates the expression of collagen, which may contribute to arrhythmia, heart failure and even sudden cardiac death.

Cystathionine ß-synthase-derived hydrogen sulfide regulates lipopolysaccharide-induced apoptosis of the BRL rat hepatic cell line in vitro.

Hydrogen sulfide (H(2)S), is a member of the novel family of endogenous gaseous transmitters, termed "gasotransmitters exhibiting diverse physiological activities, and is generated in mammalian tissues mainly by cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST) in conjunction with cysteine (aspartate) aminotranferase (CAT). The distributions of these enzymes are species- and tissue-specific. The liver, as the main organ that generates H(2)S in vivo, functions in biotransformation and metabolism. However, the liver is vulnerable to damage from internal and external factors, including inflammatory mediators, drugs and poisons. The present study evaluated the endogenous CBS-H(2)S synthesis regulating lipopolysaccharide (LPS)-induced apoptosis of hepatic cells. The rat hepatic cell line, BRL, was incubated with LPS for various time periods to establish a cell-damage model. Incubation with LPS resulted in a significant increase in CBS expression and H(2)S production. It also stimulated apoptosis and decreased the mitochondrial membrane potential. Pretreatment with the CBS inhibitor aminooxyacetic acid (AOAA) or CBS small interfering RNA (siRNA) decreased LPS-enhanced H(2)S production. Notably, apoptosis increased for a short period and then decreased gradually, while the mitochondrial membrane potential demonstrated the opposite trend. These results showed that endogenous CBS-H(2)S synthesis demonstrated early anti-apoptotic activity and subsequent pro-apoptotic activity in LPS-induced apoptosis. These results suggest a new approach for developing novel drugs for this condition.

An insertion/deletion polymorphism within RERT-lncRNA modulates hepatocellular carcinoma risk.

The Prolyl hydroxylase 1 (EGLN2) is known to affect tumorigenesis by regulating the degradation of hypoxia-inducible factor. Polymorphisms in EGLN2 may facilitate cancer cell survival under hypoxic conditions and directly associate with cancer susceptibility. Here, we examined the contribution of a 4-bp insertion/deletion polymorphism (rs10680577) within the distal promoter of EGLN2 to the risk of hepatocelluar carcinoma (HCC) in Chinese populations. The contribution of rs10680577 to HCC risk was investigated in 623 HCC cases and 1,242 controls and replicated in an independent case-control study consisting of 444 HCC cases and 450 controls. Logistic regression analysis showed that the deletion allele of rs10680577 was significantly associated with increased risk for HCC occurrence in both case-control studies [OR = 1.40; 95% confidence interval (CI) = 1.18-1.66, P < 0.0001; OR = 1.49; 95% CI = 1.18-1.88, P = 0.0007]. Such positive association was more pronounced in current smokers (OR = 3.49, 95% CI = 2.24-5.45) than nonsmokers (OR = 1.24, 95% CI = 1.03-1.50; heterogeneity P = 0.0002). Genotype-phenotype correlation studies showed that the deletion allele was significantly correlated with higher expression of both EGLN2 and RERT-lncRNA [a long noncoding RNA whose sequence overlaps with Ras-related GTP-binding protein 4b (RAB4B) and EGLN2)] in vivo and in vitro. Furthermore, RERT-lncRNA expression was also significantly correlated with EGLN2 expression in vivo, consistent with in vitro gain-of-function study that showed overexpressing RERT-lncRNA upregulated EGLN2. Finally, in silico prediction suggested that the insertion allele could disrupt the structure of RERT-lncRNA. Taken together, our findings provided strong evidence for the hypothesis that rs10680577 contributes to hepatocarcinogenesis, possibly by affecting RERT-lncRNA structure and subsequently EGLN2 expression, making it a promising biomarker for early diagnosis of HCC.

[Effects of restraint position on changes of diaphragmatic mechanical characteristic in rats].

OBJECTIVE: To observe effects of restraint position on the changes of diaphragmatic mechanical characteristic in rats, and try to explore the role of nitric oxide (NO). METHODS: Rat model of restraint position was established. Rats were divided into control group, restraint position 12h and 24h groups. The markers of respiratory functions in vivo and the biomechanical markers of diaphragmatic characteristic ex vivo were evaluated. Serum NO levels were measured with spectrophotometry. The expressions of nNOS and iNOS mRNA in diaphragm were detected using RT-PCR. RESULTS: Compared with control group, respiratory rate, tidal volume and minute ventilation were significantly decreased in the restraint position 12h and 24h groups. Pt of diaphragm significantly decreased and force-generating capacity reduced at low frequency stimulation in 12h group. Force-generating capacity over the full range reduced at low and high frequency stimulation in 24h group. Pt of diaphragm in control and restraint position groups increased after L-NNA pre-incubation. Force-frequency relationship after L-NNA pre-incubation reduced in 24h group. NO level in serum increased significantly in the restraint position groups. Diaphragmatic nNOS mRNA expression was upregulated significantly in the restraint position groups. CONCLUSION: Restraint position induces the decreasement of diaphragmatic contractility and the decreasement is mediated by NO from diaphragm or circulation blood.

Increased expression of calcium/calmodulin-dependent protein kinase type II subunit δ after rat traumatic brain injury.

Many cellular responses to Ca(2+) signals are mediated by Ca(2+)/calmodulin-dependent enzymes, among which is the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). CaMKII was originally described in rat brain tissue. In rat brain, four different subunits of the kinase have been identified: α, ß, γ, and δ. This study aims to investigate changes of CaMKIIδ after traumatic brain injury and its possible role. Rat traumatic brain injury (TBI) model was established by controlled cortical injury system. In the present study, we mainly investigated the expression and cellular localization of CaMKIIδ after traumatic brain injury. Western blot analysis revealed that CaMKIIδ was present in normal rat brain cortex. It gradually increased, reached a peak at the third day after TBI, and then decreased. Importantly, more CaMKIIδ was colocalized with neuron. In addition, Western blot detection showed that the third day postinjury was also the apoptosis peak indicated by the elevated expression of caspase-3.Importantly, immunohistochemistry analysis revealed that injury-induced expression of CaMKIIδ was colabeled by caspase-3 (apoptosis cells marker). Moreover, pretreatment with the CaMKII inhibitor (KN62) reduced the injury-induced activation of caspase-3. Noticeably, the CaMKII inhibitor KN-62 could reduce TBI-induced cell injury assessed with lesion volume and attenuate behavioral outcome evaluated by motor test. These data suggested that CaMKIIδ may be implicated in the apoptosis of neuron and the recovery of neurological outcomes. However, the inherent mechanisms remained unknown. Further studies are needed to confirm the exact role of CaMKIIδ after brain injury.

[Effect of soft tissue crush injury on tensions of thoracic aortic rings in rats].

OBJECTIVE: To observe the effect of soft tissue crush injury on the tensions of thoracic aortic rings (TARs) in rats and to investigate the potential roles of nitric oxide in the change of the tensions. METHODS: Thirty adult SD rats were randomly divided into control group and crush injury (8 h and 16 h after injury) groups. Two kinds of TARs (one with endothelium and the other without endothelium) in vitro were prepared. In the TARs with endothelium, the tensions induced by phenylephrine (PE), acetylcholine (Ach), calcium ionophore A23187 and angiotensin II (AngI) were measured by the vascular tension detective technique. Then the TARs with endothelium were preincubated with nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA) for 20 minutes, the tensions induced by PE and Ang II were measured again. In the TARs without endothelium, the tensions induced by PE and Ang II were measured by the same method. RESULTS: In the TARs with endothelium, the tension of relaxation induced by cumulative doses of Ach and A23187 decreased significantly in 8 h and 16h groups. The tension of contraction induced by cumulative doses of PE and Ang II also decreased significantly (P<0.05). The tension of contraction increased after the preincubation with L-NNA. In the TARs without endothelium, the tension of contraction induced by PE and Ang II increased comparing to that of TARs with endothelium. CONCLUSION: The soft tissue crush injury can influence the tensions of TARs in rats and the vascular-derived NO can mediate the effects.

An insertion/deletion polymorphism in the 3' untranslated region of beta-transducin repeat-containing protein (betaTrCP) is associated with susceptibility for hepatocellular carcinoma in Chinese.

Hepatocellular carcinoma (HCC) is an epithelial cancer which originates from hepatocytes or their progenitors. As a positive regulator of NFkappaB signaling pathway, beta-transducin repeat-containing protein (betaTrCP) is overexpressed and oncogenic in epithelial cancers, suggesting a potential role of betaTrCP in HCC susceptibility. We carried out a case-control study in a Chinese population (256 cases and 367 controls) to estimate the susceptibility to HCC associated with a 9bp insertion/deletion polymorphism (rs16405) in 3' untranslated region of betaTrCP. Using unconditional logistic regression, we found that 9N del/del and 9N ins/del genotypes were significantly associated with decreased HCC risk: OR=0.44 (0.24-0.83) (p=0.004) and OR=0.56 (0.31-1.00) (p=0.034), respectively. Furthermore, in vivo experiments showed that mRNA levels of betaTrCP from HCC tumor tissues were correlated with rs16405 genotypes. HCC tumor tissues with homozygous for 9N ins/ins has the highest level of betaTrCP, which are 3.99 and 7.04-fold higher than heterozygous 9N ins/del and homozygous 9N del/del, respectively. Based on bioinformatics prediction, we found that the risk allele for rs16405 disrupted a binding site for human microRNA-920 which would negatively regulate betaTrCP. We propose a microRNA-920 mediated betaTrCP regulation model depending on rs16405 genotype, which warrants further replication association studies and follow-up functional experiments.

[The expression of cholecystokinin-octapeptide receptor in vascular endothelial cells induced by lipopolysaccharide].

OBJECTIVE: To investigate the expression of cholecystokinin-octapeptide receptor (CCK-R) mRNA, and observe the effect of lipopolysaccharide (LPS) on CCK-AR mRNA and CCK-BR mRNA expression in ECV-304. METHODS: The human umbilical vein endothelial cell line ECV-304 was cultured and treated with LPS in dosage of 0.01, 0.1, 1, 10 mg/L for 2 hours, or treated with LPS in dosage of 1 mg/L for 0.5, 2, 6, 12 hours. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the expression of CCK-AR mRNA and CCK-BR mRNA in ECV-304, and to analyse the sequences of the amplification products. RESULTS: Compared with control group, the expression of CCK-AR mRNA and CCK-BR mRNA was significantly upregulated in a dose-dependence manner after incubated with 0.01, 0.1 and 1 mg/L LPS for 2 hours (all P<0.05). However, the expression of CCK-AR mRNA showed no significant increase,while that of CCK-BR mRNA was increased, after being incubated with 0.01 mg/L LPS. The expressions of CCK-AR mRNA and CCK-BR mRNA in the 10 mg/L LPS group showed no significant difference compared with 1 mg/L LPS group (both P>0.05). The expression of CCK-AR mRNA and CCK-BR mRNA was significantly upregulated in a time-dependence manner after incubated with 1 mg/L LPS from 0.5 hour to 2 hours compared with control group (all P<0.05). After incubated with 1 mg/L LPS for 6 hours, the expression of CCK-AR mRNA and CCK-BR mRNA was significantly decreased compared with 2-hour group, but was still higher than that of control group (both P<0.05). Its expression was decreased further after being incubated with 1 mg/L LPS for 12 hours compared with the 6 hours group (both P<0.05), but showed no significant difference compared with the control group (both P>0.05). CONCLUSION: Both CCK-AR mRNA and CCK-BR mRNA are expressed in ECV-304. LPS can up-regulate the expression of CCK-AR mRNA and CCK-BR mRNA.

[Mechanism of acute liver injury induced by crushing hindlimbs in rabbits].

OBJECTIVE: To investigate the role of oxidative stress in acute liver injury during crushing hindlimbs in rabbit. METHODS: The crushing injury model in rabbit was established by intermittent crushing the hind limbs of rabbit with standard weight. The ALT and AST activities were spectrophotometrically measured. The weight ratio (wet/dry,W/D) of livers was measured with scale, and the pathologic changes were observed. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and total anti-oxidant capacity (T-AOC) as well as malondialdehyde (MDA) level were spectrophotometrically measured. RESULTS: As compared with control rabbits, crushing hindlimbs of rabbits induced acute liver injury with the increase in ALT and AST activities in serum,which were 4.31 (P < 0.01) and 10.54 times (P < 0.01) of control group respectively, there were cellular swellings and slight congestion of hepatic sinuses. In addition,crushing hind-limbs elicited significant decrease in SOD,CAT,GSH-Px activity and T-AOC to 17%, 29%, 24% and 21% (P < 0.01) compared with control group respectively, whereas MDA level markedly enhanced. CONCLUSION: Crushing hindlimbs of rabbits induced acute liver injury and significant decrease in anti-oxidant capacity, the latter maybe play an important role in crushing hind-limbs of rabbits-elicited the acute liver injury.

[Change of nitric oxide in local muscle of crush injury hind-limbs in rats].

OBJECTIVE: To investigate the change of nitric oxide (NO) level in local muscles induced by crushing hind-limbs in rats. METHODS: The rat experimental model of hind-limb crushing injury was established by crushing the hind limbs of rats with standard weight for 5 hours, thereafter releving the standard weight for another 5 hours. The rats were randomly divided into sham group, crushing group, crushing and injecting aminoguanidine (AG) group, crushing and injecting L-arginine (L-Arg) group. The NOS activity and NO level in local muscles and serum were spectrophotometrically measured, and iNOS and eNOS protein expressions in local muscles were examined by immunohistochemistry. The weight ratio of wet to dry (W/D) of local muscles was measured and the pathologic changes were observed. RESULTS: The crushing hind-limbs induced serious primary and secondary injuries of local muscles such as rupture and rhadomyolysis of skeletal muscular fibers, interstitial vascular congestion and edema, and marked increase in W/D. The expressions of eNOS and iNOS were upregulated in local muscle in crush group compared with sham group. The NOS activity and NO level in local muscles and serum significantly increased. There was positive relationship between NO level and W/D in local muscles. With the usage of AG and L-arg, the hind-limb injuries seemed alleviated and aggravated, respectively. CONCLUSION: The crushing hind-limbs of rats elicited the upregulation of eNOS and iNOS protein expression, the enhancement of NOS activity and the excess production of NO, the latter of which was involved in the mediation of secondary pathological changes in local muscles.

[Inhibitory effect of cholecystokinin octapeptide on lipopolysaccharide-elicited inducible nitric oxide synthase expression in vascular endothelial cells].

OBJECTIVE: To investigate the effect of cholecystokinin octapeptide (CCK-8) on lipopolysaccharide (LPS)-elicited inducible nitric oxide synthase (iNOS) expression in vascular endothelial cells. METHODS: Human umbilical vein endothelial cell line (ECV-304 cells) was stimulated with vehicle (normal saline) or LPS in the presence (0.01, 0.1, 1 mg/L) or absence (0.1 mg/L) of CCK-8 (10(-6)-10(-8)mol/L). Nitric oxide (NO) level and cellular nitric oxide synthase (NOS) activity were determined with spectrophotometrically. The iNOS expression was detected with immunocytochemical technique and Western blot. RESULTS: Compared with normal saline, LPS significantly induced the upregulation of iNOS protein expression in the cultured ECV-304 cells, and NOS activity in ECV-304 cells and NO level in cultured media were increased. CCK-8 obviously inhibited above-mentioned effect of LPS in a dose-dependent manner. Whereas CCK-8 alone did not showed effect on iNOS protein expression, NO level and cellular NOS activity as compared with those values when vehicle was used. CONCLUSION: CCK-8 inhibited LPS-elicited iNOS expression and NO production in ECV-304 cells.