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OBJECTIVE To investigate the protective effects and mechanism of ziyuglycoside Ⅰ on acute lung injury in sepsis rats based on network pharmacology， and conduct experimental verification. METHODS The network pharmacology was used to predict the potential target of ziyuglycoside Ⅰ in the treatment of acute lung injury following sepsis. The rat model of sepsis was reproduced by cecum ligation and puncture for experimental verification. Totally 192 SD rats were randomly divided into the sham operation group （Sham group）， sepsis group （Sep group）， conventional therapy group （CT group） and ziyuglycoside Ⅰ group （Zg Ⅰ group）， respectively. Sham group and Sep group were given sterile normal saline， and CT group and ZgⅠ group were given relevant volume of Ringer’s solution and ziyuglycoside Ⅰ. The arterial blood gas， serum inflammatory factors， lung wet/dry mass ratio， pathological changes of lung tissue， pulmonary vascular permeability， the expressions of pulmonary vein tight junction protein 1 （ZO-1） and vascular endothelial cadherin （VE-cadherin） protein and 72-hour survival were observed in each group. RESULTS Results of network pharmacology showed that there were 47 potential targets of ziyuglycoside Ⅰ in the treatment of sepsis. The results of gene ontology function enrichment analysis and Kyoto encyclopedia of genes and genomes pathway enrichment analysis showed that the mechanism could 598486924@qq.com be correlated with biological processes such as positive regulation of reactive oxygen species metabolism， wound healing， regulation of endothelial cell proliferation， cell activation， blood vessel development， response to oxidative stress， etc.， and with signaling pathway such as apoptosis， tight junction， HIF-1 signaling pathway， etc. The results of experimental verification showed that compared with Sham group， pH value and the level of partial arterial oxygen pressure were decreased significantly in Sep group （P＜0.05）， while the level of partial pressure of carbon dioxide， serum levels of tumor necrosis factor α， interleukin 6 were increased significantly （P＜0.05）； the ratio of lung wet/dry mass was increased significantly （P＜0.05）； the protein expressions of ZO-1 and VE-cadherin were decreased significantly （P＜0.05）； 72 h survival rate decreased，the survival time was significantly shortened （P＜0.05）； the results of pathological observation of lung tissue showed that the rats’ alveoli were extensively ruptured， the alveolar wall was thickened and accompanied with edema， and there was obvious inflammatory cell infiltration； the results of pulmonary vascular permeability observation showed that the lung surface of rats was dark， with a large amount of Evans blue exudation， and the left lower lung was obviously dark blue. Compared with Sep group， the levels of above indexes almost were reversed significantly in CT group and ZgⅠ group （P＜0.05）； the lung histopathology and pulmonary vascular permeability were significantly improved， and the recovery degree of ZgⅠ group was greater than that of CT group， which was close to the results of Sham group. CONCLUSIONS Ziyuglycoside Ⅰ can significantly reduce inflammatory reaction and acute lung injury in septic rats， which is related to vascular function and tight junction signal pathway.
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Trauma, a major public hazard in modern society, results in 3.5-5.8 million deaths each year. Governments all over the world pay high attention to the prevention and treatment of trauma, and have established corresponding emergency rescue organizations and rescue teams, with corresponding emergency rescue modes and prevention and treatment measures proposed. China has made great achievement in trauma care. The author reviewes the progress in trauma in domestic and abroad during "13th Five-Year Plan" period in aspects of the injury assessment, on-the-spot emergency care, early treatment, and early warning and intervention of associated complications, so as to provide references for trauma care.
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Objective:To explore and establish the scoring method of injury assessment in rats with trauma combined with seawater immersion, so as to provide a reference for injury assessment in the special environment of trauma combined with seawater immersion.Methods:Sixty-four SD rats were divided into two groups according to the random number table, including hemorrhagic shock group and compound injury group, with 32 rats per group. Each group was divided into trauma combined with seawater immersion group and simple trauma group, with 16 rats per group. In trauma combined with seawater immersion group, the hemorrhagic shock model was placed in 15℃ seawater for 1 hour to start bleeding, and the blood loss was 30% of the total blood volume. The composite injury model caused 10% Ⅱ degree burns and was incised along the vental midline with a length of about 2 cm, and then placed in 15℃ seawater for 1 hour. The death and survival time were recorded.The survival time significantly longer than 4 hours out of water was recorded as survival, and significantly shorter than 4 hours out of water was recorded as death. Data were observed within 9 hours after injury, including the changes of physiological indexes (respiration, blood pressure, anal temperature) and arterial blood gas (blood glucose, pH value, blood lactic acid, arterial oxygen partial pressure, arterial carbon dioxide partial pressure, bicarbate, sodium ion, chloride ion, calcium ion, potassium ion). Each index were compared between trauma combined with seawater immersion group and simple trauma group. According to the survival situation of all the trauma combined with seawater immersion group at 4 hours out of water, the rats were divided into survival group and death group. The indicators affecting survival were screened, and then the scatter plot of each index corresponding to the mortality rate was established. According to the trend of each index in different interval in the scatter chart, the score table of injury condition was established.Results:The total mortality was 28% (9/32) in trauma combined with seawater immersion group, and was 6% (2/32) in simple trauma group ( P<0.05). The survival time in trauma combined with seawater immersion group [(8.1±3.7)hours] was shorter than that in simple trauma group [(11.3±4.8)hours] ( P<0.05). In trauma combined with seawater immersion group, the respiratory rate[(58.8±2.9)times/min] was slower than that in simple trauma group [(100.4±7.2)times/min], blood pressure [(80.0±25.1)mmHg] was lower than that in simple trauma group [(89.8±18.1)mmHg], and anal temperature [22.4(20.1, 25.0)℃] was significantly lower than that in sample trauma group [31.7(30.5, 33.2)℃], pH value (7.1±0.1) was lower than that in simple trauma group (7.3±0.1), and arterial oxygen partial pressure [(196.3±34.1)mmHg], arterial carbon dioxide partial pressure [45.5(35.1, 51.1)mmHg], serum sodium [145(142, 148)mmol/L], serum chlorine [120(115, 125)mmol/L], serum calcium [(1.3±0.1)mmol/L]as well as serum potassium [(3.6±0.8)mmol/L] were higher than those in simple trauma group [(149.4±22.6)mmHg, 29.7(25.6, 34.5)mmHg, 142(139, 144)mmol/L, 118(114, 121)mmol/L, (1.2±0.1)mmol/L, (3.3±0.6)mmol/L] (all P<0.05). There were no significances in other indexes between the two groups ( P>0.05). In death group, the breathing[36(30, 36)times/min], blood pressure [(43.1±21.8)mmHg], anal temperature [(20.0±1.9)℃], pH value (7.1±0.1), and bicarbonate [(12.3±2.2)mmol/L] were significantly inhibited or suppressed compared with survival group [60(48, 78)times/min, (86.6±19.3)mmHg, (23.0±3.1)℃, 7.2±0.1, (14.6±2.3)mmol/L (all P<0.05). While the two groups showed no significant differences in other indices ( P>0.05). Therefore, the respiration, blood pressure, rectal temperature, pH value and bicarbonate that significantly affect the survival of rats were screened. According to the death rate corresponding to different intervals, a score value was assigned to the interval as the weight of its impact on survival, namely on the severity of the injury, and an injury score table for trauma combined with seawater immersion in rats was established. The injury scoring scale <6 points indicated no death, 6-9 points indicated the mortality of 50%, ≥9 points indicated the mortality of 71%. The 6 points and 9 points were cutoff value of the scale. It can be considered that the scale of <6 points was classified as minor injury, 6-9 points as moderate injury, and ≥9 points as severe injury. Conclusions:The seawater immersion can result in reduced survival time and increased early mortality, manifested as respiratory depression, more serious blood loss, severe hypothermia, severe metabolic acidosis, water and electrolyte disorders (high sodium, high chlorine, high calcium, and high potassium), etc. According to the respiration, blood pressure, anal temperature, pH value and bicarbate, which affect the survival of rats, the injury rating scale of rats with trauma combined with seawater immersion can be established by using the scatter chart. The predicted mortality rate by using the rating scale was roughly consistent with the actual mortality rate, so the injury rating scale basically had a good prediction and hint for the trauma rats combined with seawater immersion.
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Tuberculous pleurisy (TP) is a common disease, especially in areas with high prevalence of tuberculosis. However, because of the lack of bacterial characteristics in pleural effusion, the detection sensitivity of traditional smear and culture method is low, and the pleural biopsy has certain risks; therefore, the diagnosis of tuberculous pleurisy is challenging. Due to the limitations of traditional detection methods, to find a fast, accurate and effective method for TP diagnosis is important. This article reviews the progress of molecular, biochemical and immunological methods for diagnosis of TP.
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Objective To investigate the early resuscitation effect of hemoglobin-based oxygen carriers (HBOC) in rats with uncontrolled hemorrhagic shock.Methods 170 Sprague-Dawley (SD) rats were randomly divided into five groups:lactate Ringer solution (LR) control group,whole blood control group,and 0.5％,2.0％,5.0％ HBOC groups,with 34 rats in each group.The uncontrolled hemorrhagic shock model in SD rats was reproduced by cutting off the splenic artery branch,and induced mean arterial pressure (MAP) reducing to 40 mmHg (1 mmHg =0.133 kPa).The corresponding solution was infused after model reproduction in each group,maintaining MAP at 50 mmHg for 1 hour,then completely ligating and hemostasis,and maintaining MAP at 70 mmHg for 1 hour and 80 mmHg for 1 hour respectively,after maintaining MAP 80 mmHg,all were supplemented with LR to 2 times blood loss volume.The survival rate and blood loss rate were observed in 16 rats in each group,hemodynamics parameters including MAP,left ventricular systolic pressure (LVSP) and the maximum rate of left ventricular pressure rise (+dp/dtmax) were determined in another 10 rats,and cardiac output (CO) and tissue oxygen supply (DO2) were observed in the rest 8 rats.Results ① When resuscitation by LR alone,the blood loss rate of animals was as high as 60％ to 70％.Compared with the LR control group,whole blood recovery could significantly reduce the blood loss rate before hemostasis in uncontrolled hemorrhagic shock rats [(46.6 ± 4.5)％ vs.(62.3 ± 4.0)％,P ＜ 0.01];0.5％,2.0％,5.0％ HBOC could significantly decrease the blood loss rate,especially in 5.0％ HBOC group with significant difference as compared with that in the LR control group [(45.6±4.1)％ vs.(62.3±4.0)％,P ＜ 0.01].② When LR was used alone for resuscitation,the rats died quickly and survived for a short time.Only one rat survived for 12 hours,and no rat survived for more than 24 hours.Compared with the LR control group,whole blood resuscitation could improve the survival rate of uncontrolled hemorrhagic shock rats,and the survival time was significantly prolonged (hours:20.4± 4.6 vs.3.5 ± 1.1,P ＜ 0.01);0.5％,2.0％ and 5.0％ HBOC also significantly prolonged the survival time of rats.The 5.0％ HBOC group had the best effect,4 rats survived in 24 hours,and the survival time was significantly longer than that of the LR control group (hours:18.4 ± 4.0 vs.3.5 ± 1.1,P ＜ 0.01),and it was the same as the whole blood control group.③ Compared with pre-shock,CO,DO2 and hemodynamic parameters of uncontrolled hemorrhagic shock rats were significantly decreased,and the above parameters were gradually increased with the prolongation of rehydration time.Compared with the LR control group,whole blood resuscitation could significantly increase CO and DO2,and improve hemodynamics in rats with uncontrolled hemorrhagic shock at different time points.Three concentrations of HBOC could also increase CO,DO2 and other hemodynamic parameters of rats at 1 hour of maintaining MAP of 80 mmHg after hemostasis and 1 hour and 2 hours after resuscitation.The effect of 5.0％ HBOC group was more significant than that of the LR control group with statistically significant difference [CO (× 10-3,L/min):72.84±2.84 vs.63.11±2.38 at 1 hour of maintaining MAP of 80 mmHg,70.25±4.55 vs.59.88 ± 9.31 at 1 hour after resuscitation,71.51 ± 2.90 vs.53.24 ± 6.32 at 2 hours after resuscitation;DO2 (L· min-1 · m-2):271.9± 13.5 vs.159.1 ±25.4 at 1 hour of maintaining MAP of 80 mmHg,261.0± 15.0 vs.145.7±20.1 at 1 hour after resuscitation,249.6± 12.0 vs.107.4± 18.2 at 2 hours after resuscitation;MAP (mmHg):82.1±2.1 vs.74.0±2.8 at 1 hour of maintaining MAP of 80 mmHg,107.5±9.3 vs.64.0±5.7 at 1 hour after resuscitation,104.0±9.7 vs.73.0±4.2 at 2 hours after resuscitation;LVSP (mmHg):128.6±7.9 vs.103.8±0.8 at 1 hour of maintaining MAP of 80 mmHg,129.3±± 15.0 vs.99.4±0.0 at 1 hour after resuscitation,127.5± 11.3 vs.97.4±0.0 at 2 hours after resuscitation;+dp/dt max (mmHg/s):6 534.2±± 787.6 vs.5 074.0± 71.7 at 1 hour of maintaining MAP of 80 mmHg,5 961.5 ±± 545.4 vs.4 934.5 ± 510.2 at 1 hour after resuscitation,5 897.4± 350.5 vs.4 534.7 ±489.2 at 2 hours after resuscitation,all P ＜ 0.05].Conclusions HBOC infusion prolonged the survival time,increased survival rate,and improved hemodynamics,cardiac function and tissue oxygen supply in a dose-dependent manner in the early stage of uncontrolled hemorrhagic shock.The recovery effect of 5.0％ HBOC was similar to that of the whole blood.
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Objective To observe the protective effects of calcium-sensing receptor (CaSR) inhibitor Calhex231 on traumatic hemorrhagic shock rats. Methods 144 SD rats were divided into six groups by random number table method: normal group, shock group, lactated Ringer's solution (LR) group, LR + Calhex231 0.1 mg/kg group, LR + Calhex231 1 mg/kg group, and LR + Calhex231 5 mg/kg group, with 16 rats in each group for survival observation and 8 rats for hemodynamics test. 64 SD rats were divided into four groups: normal group, shock group, lactated Ringer's solution (LR) group, LR + Calhex231 1 mg/kg group, with 8 rats in each group for detecting organ blood flow and superior mesenteric artery vascular reactivity and the other 8 rats for mesenteric artery vascular reactivity. After the establishment of traumatic hemorrhagic shock model, the shock group did not receive resuscitation, and the LR group was resuscitated with LR equal to two times of the blood loss volume. The three LR + Calhex231 groups with different dosages were firstly given LR of equal volume to that of blood loss, and then the Calhex231 was dissolved into LR (equal to the blood loss volume) to resuscitate. The wound was ligated and sutured immediately after resuscitation. The effect of Calhex231 on animal's 24-hour survival since the beginning of resuscitation was observed. The hemodynamics including the mean arterial blood pressure (MAP), left intraventricular systolic pressure (LVSP), maximal rising, and declining rate of left intraventricular pressure (±dp/dtmax) were observed before shock, at the end of shock, 1 hour after resuscitation, and 2 hours after resuscitation. The effects of Calhex231 on vital organ blood flow and vascular reactivity were observed 2 hours after resuscitation. Results All the shock rats died within 9 hours after the shock model was established. The survival outcomes of LR group rats were slightly improved compared with the shock group rats(P ＜0.05). The survival time and 24 hour survival rate of LR + Calhex231 1 mg/kg group and LR + Calhex231 5 mg/kg group rats were significantly increased compared with the shock group rats (P ＜0.05). The hemodynamic indexes of LR + Calhex231 groups were higher than those of the LR group. The best effect was observed in LR + Calhex231 1 mg/kg group rats (P ＜ 0.01). The MAP, LVSP and ± dp/dtmax were restored to normal level (64.9％, 82.4％, 89.8％, and 77.8％, respectively). Meanwhile, the blood flow in liver and kidney of LR + Calhex231 1 mg/kg group rats were increased from 57.2％ and 41％ to 108.7％ and 95.1％, respectively. The vascular reactivity including superior mesenteric artery and mesenteric artery of LR + Calhex231 1 mg/kg group rats were also increased (P ＜0.01). Conclusions In rats with hemorrhagic shock, the calcium sensitive receptor inhibitor Calhex231 can improve the vascular reactivity, the hemodynamics, and the blood flow of important organs. It plays a role in protecting the cardiovascular function and reducing the mortality after traumatic hemorrhagic shock.
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Objective To investigate the mechanism of interleukin-1β(IL-1β) mediating the vascular calcium desensitization of septic rats by down-regulating Rho kinase activity.Methods Thirty-two SD rats were randomly divided into the sham operation group,cecal ligation and puncture (CLP) 3 h group,CLP 6 h group and CLP 12 h group,8 cases in each group.The septic rat was duplicated by CLP.Then the plasma IL-1β level and calcium sensitivity of superior mesenteric arteries (SMAs) were detected at different time points.Their correlation was analyzed.VSMCc derived from SMAs were cultured and incubated with different concentrations of human recombinant IL-1β for 24 h.Then the influences of IL-1β on the MLC20 phosphorylation level,Rho kinase activity,G protein expression level and RhoGEF activity were observed.Results The calcium sensitivity of SMAs after CLP 3 h began to decrease(P＜0.05),while plasma IL-1β level began to increase after CLP 6 h (P＜0.05),the change trend of SMAs calcium sensitivity was negatively correlated with plasma IL-1β level change(P＜0.05).IL-1β could decrease the phosphorylation level of VSMCs myosin light chain(MLC20) and Rho kinase activity(P＜0.05),up-regulate Gα11 expression and down-regulate Gα12 expression,but had no obvious effect on Gαq and Gα13 expression(P＞0.05).IL-1β could significantly reduce RhoGEF and PDZ-RhoGEF activity(P＜0.05) but significantly increase p63 Rho GEF activity(P＜0.05).Conclusion IL-1β induces the decrease of PDZ-RhoGEF and Rho kinase activity by down-regulating Gα12 expression,causes the decrease of MLC20 phosphorylation level,thus mediates the occurrence of calcium desensitization in septic rat;in addition,IL-1β may cause the increase of p63 RhoGEF activity by upregulating Gα11 expression,thus mediates the increase of vascular calcium sensitivity in septic rats,but the total effect is the decrease of calcium sensitivity.
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Objective To investigate the expression of microRNA (miRNA)-3620 in the plasma of patients with anti-tuberculosis drug-induced hepatotoxicity (ATDH).Methods A total of 35 patients with ATDH and 35 non-ATDH paired individuals were included in this study.Plasma miRNA-3620 levels were detected using real-time Polymerase chain reaction.Comparison between two groups was done with t test.Receiver operation characteristic (ROC) curve analysis was performed to determine the diagnostic value of miRNA-3620 in ATDH.Results The relative expression of plasma miRNA-3620 of patients with ATDH and non-ATDH were 1.65±1.43 and 0.71±0.45, respectively, with significantly statistical difference (t=3.703, P<0.01).The cut off value of miRNA-3620 expression was 1.15 and the area under ROC curve were 0.71(95% CI: 0.43-1.45).Based on this cutoff value, the sensitivity and specificity of miRNA-3620 in diagnosing ATDH were 60.0% and 82.9%, respectively;the positive predictive value was 77.8% and the negative predictive value was 67.4%.Twenty-one ATDH cases and 29 non-ATDH cases was correctly diagnosed, with the accuracy of 71.4%.Conclusion The expression of miRNA-3620 in plasma is significantly increased in ATDH patients.
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Objective To investigate the mechanism of interleukin-1β(IL-1β) mediating the vascular calcium desensitization of septic rats by down-regulating Rho kinase activity.Methods Thirty-two SD rats were randomly divided into the sham operation group,cecal ligation and puncture (CLP) 3 h group,CLP 6 h group and CLP 12 h group,8 cases in each group.The septic rat was duplicated by CLP.Then the plasma IL-1β level and calcium sensitivity of superior mesenteric arteries (SMAs) were detected at different time points.Their correlation was analyzed.VSMCc derived from SMAs were cultured and incubated with different concentrations of human recombinant IL-1β for 24 h.Then the influences of IL-1β on the MLC20 phosphorylation level,Rho kinase activity,G protein expression level and RhoGEF activity were observed.Results The calcium sensitivity of SMAs after CLP 3 h began to decrease(P＜0.05),while plasma IL-1β level began to increase after CLP 6 h (P＜0.05),the change trend of SMAs calcium sensitivity was negatively correlated with plasma IL-1β level change(P＜0.05).IL-1β could decrease the phosphorylation level of VSMCs myosin light chain(MLC20) and Rho kinase activity(P＜0.05),up-regulate Gα11 expression and down-regulate Gα12 expression,but had no obvious effect on Gαq and Gα13 expression(P＞0.05).IL-1β could significantly reduce RhoGEF and PDZ-RhoGEF activity(P＜0.05) but significantly increase p63 Rho GEF activity(P＜0.05).Conclusion IL-1β induces the decrease of PDZ-RhoGEF and Rho kinase activity by down-regulating Gα12 expression,causes the decrease of MLC20 phosphorylation level,thus mediates the occurrence of calcium desensitization in septic rat;in addition,IL-1β may cause the increase of p63 RhoGEF activity by upregulating Gα11 expression,thus mediates the increase of vascular calcium sensitivity in septic rats,but the total effect is the decrease of calcium sensitivity.
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Objective To investigate the pathophysiological features of rat hemorrhagic shock under high temperature conditions.Methods A total of 128 SD rats were assigned to high temperature group and normal temperature group according to the random number table,with 64 rats per group.Rats in high temperature group were pretreated at 342 for 12 h,and in normal temperature group were kept at 25℃.Hemorrhagic shock models in rats were produced by withdrawing 40％ of the total blood volume via the femoral artery.Parameters of the two groups were measured including blood electrolytes (Na +,K +),plasma osmotic pressure,liver function [aspartate aminotransferase (AST),alanine aminotransferase (ALT)],kidney function [creatinine (CREA)],arterial blood gases (pH,PO2 and PCO2) and cardiac function [heart rate (HR),cardiac output (CO),cardiac index (CI) and DO2].Animal survival time and survival rate were detected.Results Before shock,high temperature group versus normal temperature group showed higher detections of Na+ concentration [(142.3 ± 2.2) mmol/L:(139.1 ±1.5) mmol/L],plasma osmotic pressure [(304.8 ± 4.7) mmol/L:(300.0 ± 1.9) mmol/L],HR [(462 ±30) times/min:(402 ± 44) times/min],CO [(0.892 ± 0.190) L/min:(0.713 ± 0.090) L/min] and CI [(0.0030±0.0006)L·min-1 · cm-2:(0.0023 ±0.0002)L· min-1 · cm-2] (P＜0.05).After shock,normal temperature group showed further increased concentrations of Na + and K + and significantly enhanced AST,ALT and CREA compared to normal temperature group (P ＜ 0.05).After shock,CO,CI and DO2 in high temperature group were decreased by 63％,63％ and 69％ respectively compared to those before shock,but less decrease was observed in normal temperature group.Rat survival rate and survival time were significantly reduced in high temperature group compared to normal temperature group (P ＜ 0.05).Conclusion Pathophysiological changes of hemorrhagic shock in rats under high temperature are mainly manifested as impaired cardiac function,significantly increased concentrations of Na + and K + and significantly shortened survival time.
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Objective:To investigate the changes of gene expression profile in lipopolysaccharide (LPS)-stimulated primary Kupffer cells ( KCs ) . Methods: Rat KCs were isolated and purified by means of in situ perfusion and density gradient centrifugation. After being identified by ink phagocytosis and ED2 staining test,KCs were stimulated with LPS. Gene expression profile were studied using gene microarrays,and the most significant upregulated gene was verified using real-time PCR. Results:27 genes were upregulated including Ces1f, Slc17a3, Slc21a4, Hsd17b2, Sorbs2, Ccdc116, Mgam, Myo5b, Etl4, Fabp1, Kif4b, Fosl1, Cyp4a1, Penk, Tmem221,Rpl5,Nr2f1,Hoxb1,Gpr165,Fam90a13p,Kpna6,Irak1bp1,Kcnh1 and 4 unnamed genes and 4 downregulated including Oc90,Tagln,Arxes2 and Olr830 in LPS-stimulated KCs. Among the upregulated genes, Ces1f was the most significant upregulatory gene. Real-time PCR confirmed that the levels of Ces1f were 23. 88 times higher in LPS-stimulated than control cells. Conclusion:There is a significant difference between LPS-stimulated and normal control cells in gene expression profile by microarray analysis,and Ces1f is the most significantly upregulated gene.
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Objective To investigate the effects of calcium sensing receptor (CaSR)on vasorelaxation/vasoconstriction of superior mes-enteric artery (SMA)in rats and its relationship to endothelium.Methods With endothelium-intact and endothelium-denuded SMA rings of rats,the effects of CaSR-specific allosteric modulator cinacalcet on the SMA rings pre-contracted with norepinephrine (NE),and vascular contractile response /relaxation reactivity were observed.Results Cinacalcet had no effects on resting tension of SMA rings with or without endothelium.Cinacalcet caused a significant relaxation in the endothelium-intact SMA rings pre-contraction with NE in a dose-dependent manner.Endothelium denudation abolished cinacalcet-induced vasorelaxation.Pretreatment with cinacalcet for 30 minutes decreased the con-tractile response of endothelium-intact SMA rings to NE,but had no significant influence on relaxation reactivity.In the endothelium-denuded SMA rings,cinacalcet did not affect both vasoconstriction and vasorelaxation.Conclusion CaSR plays an important role in the regulation of the vascular reactivity,and this effect is endothelium-dependent.
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Objective:To investigate the effects of IRF 3 shRNA adenovirus on dynamic changes of early cytokines in LPS-stimulated primary Kupffer cells ( KCs ).Methods: Rat KCs were isolated and purified by means of in situ perfusion.After being infected with adenovirus carrying IRF 3 shRNA for 48 h, KCs were stimulated with LPS.Cell culture supernatants were collected respectively at 0,2,4 and 6 h after LPS stimulation as well as cells at 6 h.Supernatant cytokine secretion levels were detected by enzyme-linked immunosorbent assay ( ELISA).Intracellular gene expressions were tested by RT-PCR and Westeron blot.Results:IRF3 mRNA and protein were induced by LPS ,but suppressed by IRF 3 shRNA adenovirus in LPS-stimulated or non-stimulated KCs.IFN-βsecretions rose in the very early stage ( at 2 h) ,reached the peak at 4 h,and began to reduce but still remained high levels at 6 h after LPS stimulation in KCs.Interference adenovirus pretreatment suppressed IFN-βsecretions ( especially the secretion peak ) at each time point after LPS stimulation.IFN-βsecretions reached normal levels at 6 h after the stimulation in adenovirus-pretreated cells;TNF-αse-cretions rapidly increased in the very early stage and reached the peak at 2 h,then began to decrease gradually ,but remained high levels at 6 h after LPS stimulation in KCs.Interference adenovirus pretreatment inhibited LPS-induced TNF-αsecretions, especially the secretion peak;IL-1βsecretions did not increase untill 4 h, but reached a higher level at 6 h after LPS stimulation.Interference adenovirus suppressed IL-1βsecretions in the early stage of LPS stimulation;IL-10 secretions began to rise in the very early stage ,and gradually increased over time after LPS stimulation in KCs.Pretreatment of adenovirus with IRF 3 shRNA promoted upregulations of IL-10 secretions at each time point of the early of LPS stimulation.Conclusion:IRF3 gene expression can be silenced by IRF 3 shRNA ad-enovirus.IRF3 can promote its downstream signaling molecule IFN-βand pro-inflammatory cytokines including TNF-αand IL-1β,and block anti-inflammation cytokine IL-10 secretions in LPS-stimulated primary KCs.Therefore,IRF3 may play a central role in immune inflammatory injury of liver tissues.
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Objective To study the liver protection of crocetin against paraquat (PQ) poisoning induced acute liver injury in rats. Methods Fifty-four male Wistar rats were randomly divided into control group, exposure group and treatment group, and the rats in each group were subdivided into the 0.5th, 2nd, and 6th day after exposure subgroups (n = 6). The model of acute liver failure induced by PQ poisoning was reproduced by intraperitoneal injection of 20 mg/kg of 20% PQ, and the rats in control group was injected with the same amount of normal saline. The rats in treatment group were given with intraperitoneal injection of 50 mg/kg crocetin after 0.5 day, once a day until they were sacrificed; the other two groups were injected with the same amount of normal saline. The rats in all groups were sacrificed at the corresponding time points, and blood was collected from inferior vena cava and hepatic tissue was harvested. Hematoxylin and eosin (HE) staining was used to observe the pathological changes in liver tissue on the 6th day under light microscope. Enzyme linked immunosorbent assay (ELISA) was used to detect the serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expressions of inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB). The activities of apoptosis related factors, including caspase-8, -9, -12, in hepatic tissue were determined on the 6th day with chromogenic substrate method. Results In the liver tissue of exposed group, extensive infiltration of the inflammatory cells and the diffuse fragments necrosis were visible, and the regeneration of the liver cells was not obvious, and severity of the injury in a time dependent way. In the treatment group, the structure of hepatic artery was visible, and the infiltration of necrosis, congestion and inflammatory cells were not obvious. On the 0.5th, 2nd, and 6th day, serum levels of IL-6 and TNF-α, the mRNA expressions of iNOS and NF-κB in liver tissue, and the caspase-8, -9, -12 activities on the 6th day in the exposure group and treatment group were significantly higher than those in the control group. And the parameters in treatment group were significantly lower than those of the exposure group [IL-6 (ng/L): 188.37±64.21 vs. 376.61±82.42 on the 0.5th day, 287.18±58.69 vs. 432.77±96.28 on the 2nd day, 234.24±10.17 vs. 375.41±37.59 on the 6th day; TNF-α (ng/L): 472.36±76.43 vs. 688.33±102.19 on the 0.5th day, 189.32±87.54 vs. 296.21±89.77 on the 2nd day, 99.28±16.13 vs. 168.41±66.78 on the 6th day; iNOS mRNA (gray value): 2.998±0.801 vs. 3.453±0.026 on the 0.5th day, 3.126±0.306 vs. 5.259±0.153 on the 2nd day, 0.841±0.135 vs. 1.225±0.057 on the 6th day; NF-κB mRNA (gray value): 1.569±0.818 vs. 2.361±0.063 on the 0.5th day, 2.345±0.489 vs. 4.668±0.368 on the 2nd day, 2.348±0.316 vs. 3.972±0.449 on the 6th day; caspase-8 (pmol/mg): 126.77±9.97 vs. 199.18±66.48 on the 6th day; caspase-9 (pmol/mg): 213.12±69.06 vs. 321.62±89.39 on the 6th day; caspase-12 (pmol/mg): 183.46±70.52 vs. 219.68±53.93 on the 6th day, all P < 0.05]. Conclusion Crocetin has protective effect on liver in rats with PQ poisoning, which role is related with reducing the blood level of inflammatory factors, inhibiting the hepatic caspase-8, -9, -12 activities and gene expressions of iNOS and NF-κB.
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Objective To investigate the impacts of adenovirus on the transfection efficiency and proliferative activity of primary Kupffer cells (KCs). Methods Rat liver KCs were separated and purified by density gradient centrifugation , and was then transfected with adenovirus carrying green fluorescence protein (GFP) gene at different multiplicity of infection (MOI). After 24 h, the transfection efficiency was evaluated by fluorescence microscope and flow cytometry. The proliferative activity of KCs was assessed by colorimetric method. Results The positive percentages of GFP staining cells were statistically different among different doses of adenovirus (MOI 0, 100, 300, 500, 700 and 900) under fluorescence microscopy or by flow cytometry (P 0.05 for all comparisons) by CCK8 assay. Conclusions KCs can effectively be transfected by GFP adenovirus; and with an increase in virus MOI, the transfection efficiency rises gradually. A higher dose of adenovirus may have a negative effect on cell proliferative.
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Objective To investigate platelet-derived growth factor ( PDGF ) protection on blood flow and mitochondrial function of hemorrhagic shock rats. Methods Ninety-six SD rats were randomly divided into six groups including shock group, lactated ringer's solution (LR) resuscitation group,PDGF treatment groups(1,3. 5,7,15μg/kg). Laster-Doppler and oxygen concentration determination method were applied to observe the protective effect of PDGF treatment on animal survival,blood flow and mitochondrial function in liver and kidney. Re-sults As compared with LR resuscitation group,PDGF treatment increased animal survival rate and also improved blood fiow of liver and kindy,mitochondrial respiration control ration(RCR),of which the group with 3. 5μg/kg had the best result. Conclusion This finding sug-gests that PDGF may be a potential agent to treat acute critical such as hemorrhagic shock.
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Objective To investigate the effects of interferon regulator factor 3 (IRF3) shRNA on the expression of TLR4 downstream signal molecules including IRF3-IFN-β, NF-κB/p38 MAPK-TNF-α/IL-1βand IL-10 in lipopolysaccharide (LPS)-stimulated Kupffer cells (KCs). Methods KCs were isolated from rats by in situ perfusion. The adenovirus strains carrying IRF3 shRNA were used for the transfection of purified KCs. The isolated KCs were randomly divided into four groups including adenovirus(-) LPS(-) treatment group, adenovirus(-) LPS(+) treatment group, adenovirus(+) LPS(-) treatment group and ad-enovirus(+) LPS(+) treatment group. The levels of cytokines in the supernatants of KC culture were detec-ted by enzyme-linked immunosorbent assay ( ELISA ) . Real-time PCR and Western blot assay were per-formed to analyze the expression of related cytokines at mRNA and protein levels, respectively. Results The expression of IRF3 at mRNA and protein levels in primary cultured KCs were induced by LPS. The cel-lular constitutive expression of IRF3 at mRNA level and the LPS-induced expression of IRF3 were signifi-cantly inhibited after transfection of KCs with adenovirus strains carrying IRF3 shRNA. However, the nucle-ar constitutive expression of IRF3 protein was not affected by IRF3 shRNA. The expression of IFN-βat mR-NA and protein levels in KCs were induced by LPS, but were suppressed by the interference with IRF3 shR-NA. No significant changes of the cellular constitutive expression of IFN-βat mRNA and protein levels were observed in IRF3 shRNA-treated KCs. Enhanced expression of proinflammatory cytokines including TNF-αand IL-1β at mRNA and protein levels were detected in LPS-stimulated KCs. Transfection of KCs with ade-novirus strains carrying IRF3 shRNA inhibited the LPS-induced secretion of TNF-α and IL-1β, but neither LPS-induced expression of TNF-α and IL-1β at mRNA level nor cellular constitutive expression of TNF-αand IL-1βat mRNA and protein levels were affected by IRF3 shRNA. The LPS-induced expression of IL-10 at mRNA and protein levels were enhanced in IRF3 shRNA-treated KCs. However, the cellular constitutive expression of IL-10 at mRNA and protein levels were not affected by the adenovirus. The levels of phosphor-NF-κB p65 subunit and phosphor-p38 MAPK protein in the nuclei of KCs were increased upon the stimula-tion with LPS. Treatment of KCs with IRF3 shRNA showed no significant effects on nuclear phosphor-NF-κB p65 subunit and phosphor-p38 MAPK. Conclusion Transfection of LPS-stimulated primary KCs with ade-novirus strains carrying IRF3 shRNA could effectively inhibit the expression of IRF3 and the transduction of downstream signals. IRF3 enhanced the secretion of TNF-αand IL-1β, but inhibited the expression of IL-10 in LPS-treated KCs. The LPS-induced activation of NF-κB and p38 MAPK in KCs were not affected by IRF3 signal.
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Objective To investigate the beneficial effects of cyclosporin A ( CsA) on traumatic hemorrhagic shock in rats. Methods The traumatic hemorrhagic shock model was adopted in 144 SD rats which were divided into 6 groups: sham-operated group,shock control group,lactated Ringer's solution ( LR) group,CsA 1 mg/kg,5 mg/kg and 10 mg/kg group. The effects of three doses of CsA on the animal survival time and 24 h survival rate were observed,and the effects of CsA on hemodynamic parameters,including mean arterial blood pressure ( MAP) ,left intraventricular systolic pressure ( LVSP) ,left ventricular end-diastolic pressure ( LVEDP) ,maximal change rate of left intraven-tricular pressure ( ± dp/dtmax ) and heart rate ( HR) were also observed. Results CsA at the concentration of 5 mg/kg and 10 mg/kg can significantly increase the survival time and 24 h survival rate of shock rats,the survival rate was increased to 56. 3% from 25% of LR group. After shock,the hemodynamic parameters were significantly decreased including MAP,LVSP and ± dp/dtmax ,LR infusion only improved the hemodynamics to some extent,which were significantly lower than those in sham-operated group. CsA (5 mg/kg and 10 mg/kg) can signifi-cantly improve the hemodynamics of shock rats including LVSP and ± dp/dtmax ,which were increased at 2 h after resuscitation as compared to LR group,and return to about normal levels. 1 mg/kg of CsA also restored the hemodynamic parameters, but there were no significant differences between CsA 1 mg/kg group and LR group. Conclusion CsA has good beneficial effect on traumatic hemorrhagic shock,and 5 mg/kg and 10 mg/kg of CsA showed a better effect.
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Objecitve:To investigate effects of urotensin Ⅱ( UⅡ)/UT system on innate immune inflammatory signal pathway TLR4-IRF3 in the lipopolysaccharide (LPS)-stimulated Kupffer cells (KCs).Methods: Rat KCs were isolated and cultured.Pro-in-flammatory cytokines including IL-6,IFN-βand IFN-γwere assayed by ELISA in culture supernatant of KCs.Cell surface TLR4 were tested with flow cytometry technique.Expression of IRF3 were tested with real-time PCR and Western blot.Results: Significant increases were showed in IL-6, IFN-βand IFN-γsecretion, TLR4-expressed positive rates and IRF3 mRNA levels in KCs after stimulated by LPS,but were inhibited via urantide pretreatment.In addition,LPS induced upregulation of nuclear IRF3 protein and downregulation of cytoplasm IRF3 protein in KCs,which were blocked by urantide pretreatment.Conclusion:UⅡ/UT system mediates immune inflammatory response in part through activating TLR 4-IRF3 pathway in LPS-stimulated KCs.
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Objective To investigate the effects of urotensin Ⅱ/urotensin Ⅱreceptor ( UⅡ/UT) system on the expression of inflammatory signal molecules p 38 mitogen-activated protein kinase ( p38 MAPK) and nuclear factor-κB ( NF-κB ) in lipopolysaccharide ( LPS )-stimulated Kupffer cells ( KCs ) . Methods Rat KCs were isolated and purified by means of in situ perfusion and density gradient centrifuga-tion.The isolated cells were randomly divided into six treatment groups including group 1:UⅡ(-) urantide (-)LPS(-), group 2:UⅡ(+)urantide(-)LPS(-), group 3: UⅡ(-)urantide(+)LPS(-), group 4:UⅡ(-)urantide(-)LPS(+), group 5:UⅡ(+) urantide(-) LPS(+) and group 6:UⅡ(-)urantide(+) LPS(+) .Western blot assay was performed to detect p 38 MAPK/p-p38 MAPK protein and NF-κB p65 sub-unit.The DNA-binding activity of NF-κB was tested by electrophoretic mobility shift assay (EMSA).Re-sults There was no significant difference with the expression of p 38 MAPK protein in KCs among the six groups (P>0.05).The expression of p65 protein and p-p38 MAPK and the DNA-binding activity of NF-κB were significantly enhanced in LPS-stimulated KCs from groups 4, 5 and 6 in comparison with those in group 1 (P0.05), but that were decreased in group 6 than those in group 4 (all P<0.01).Conclusion UⅡ/UT system participated in the activation of p38 MAPK and NF-κB signaling pathways in LPS-stimulated primary Kupffer cells .