低分子肝素抗炎性凝血效应对脓毒症大鼠急性肺损伤的保护作用
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摘要
【目的】
1.动态观察盲肠结扎穿孔(cecal ligation puncture, CLP)脓毒症模型大鼠肺组织高迁移率族蛋白B1(high mobility group boxl portein,HMGB1)mRNA和蛋白表达水平,研究低分子肝素对肺组织HMGB1基因和蛋白表达的影响,探讨低分子肝素对脓毒症大鼠急性肺损伤的保护作用及其可能机制,为脓毒症患者器官功能保护提供新的理论依据
2.观察低分子肝素对脓毒症大鼠血浆TNF-a及IL-6的含量的影响,探讨低分子肝素对脓毒症早晚期炎症释放的作用。
【方法】
144只SPF级雄性成年SD大鼠(体重200-220g)被随机分为假手术组(48只)、盲肠结扎穿孔术(CLP)后常规治疗组(48只)及常规加低分子肝素治疗组(48只),分别施以假手术(A组)或CLP(B组和C组)。A组开腹后轻拉盲肠后将其回纳腹腔,逐层缝合腹壁,其余各组均采用CLP术制作大鼠脓毒症模型术后立即腹腔注射生理盐水(NS)2ml+头孢曲松(30mg/kg)(A、B组)；生理盐水(NS)2 ml+头孢曲松(30mg/kg)+低分子肝素(150 U/kg)(C组)。以术后3、6、12、18、24、48小时为观察点,观察各组大鼠术后活动、进食、竖毛、腹泻、眼球凹陷、呼吸等情况；于各观察点每组随机处死大鼠5只,每组留18只不杀用以观察5天生存率。采用逆转录-聚合酶链反应(RT-PCR)技术测肺组织]HMGB-1mRNA的表达；采用Western blot法检测肺组织HMGB-1蛋白的表达；采用酶联免疫吸附法测定血浆TNF-α及IL-6的含量：用分光光度比色法检测肺组织髓过氧化物酶(myeloperoxidese,MPO);热干法称取肺组织湿重/干重比值(Wetweight/Dry weight);并在活杀前行脓毒症大鼠评分。观察低分子肝素对脓毒症大鼠严重程度评分、生存时间、血浆TNF-α及IL-6的含量、肺组织HMGB1 mRNA与蛋白表达、MPO活性以及WW/DW比值的影响。
【结果】
1.A组各时间点脓毒症评分差异无显著统计学意义,B、C组脓毒症评分较A组同期水平明显升高(P<0.01),从6h开始增高,24h达到高峰并维持至48h,48h评分仍显著高于组内6h(P<0.05)；C组各时间点评分均显著低于B组同期水平(P<0.05)。A、B、C组半数生存时间依次为120h、20.4h、28h；A、B、C两两组间差异均有统计学意义(P<0.05)。
2.A组各时间点大鼠HMGB1mRNA和HMGBl蛋白均有微量表达,差异无统计学意义(P>0.05)。B、C组HMGB1mRNA和HMGB1蛋白从6h开始表达增多,以12-24h为显著,48h开始下降但仍高于A组。C组同期水平较B组低(P<0.05),但仍比A组高且有显著性差异(P<0.05)。
3.A组大鼠血浆TNF-α水平在术后各时间点差异无显著性,B组大鼠血浆TNF-α水平于术后6h达到峰值,12h开始明显下降,24 h逐渐恢复至3h水平。C组各时间点血浆TNF-α水平低于B组(P<0.05),但仍显著高于A组(P<0.05)。A组大鼠血浆IL-6水平在术后各时间点差异无显著性,B组大鼠血浆IL-6水平于术后3h达到峰值,18h开始明显下降,24 h逐渐恢复至3h水平。C组各时间点血浆IL-6水平低于B组(P<0.05),但仍显著高于A组(P<0.05)。
4.A组各时间点MPO活性无明显差异,B组CLP后3小时MPO活性开始升高,24h达峰值,48h开始有所回落,但与A组同期水平差异仍有显著性(P<0.05),C组较B组同期水平低(P<0.05),但较A组显著升高(P<0.05)。
5.A组肺组织WW／DW比值较B、C低,差异有显著性(P<0.05),C组比值介于A组与B组之间,各组组间差异具显著性(P<0.05)。
6.光镜下B组肺组织可见明显急性肺损伤表现：充血、水肿、炎症细胞浸润；C组较B组病理损伤明显减轻,且肺损伤病理评分明显降低(P<0.05)。
【结论】
1.低分子肝素能够抑制CLP引起的脓毒症过程中的早晚期炎症介质释放。
2.低分子肝素能够显著抑制脓毒症大鼠肺组织HMGB1蛋白及mRNA水平表达；通过抑制肺组织HMGB1,下调脓毒症大鼠肺组织MOP活性,减轻肺组织PMN浸润,减缓CLP诱导的肺损伤程度,提高脓毒症大鼠的生存率。
3.低分子肝素对脓毒症引起的肺损伤的保护作用可能是通过其抗炎性凝血效应对凝血-炎症轴影响而实现的。
[Objective]
1.To find the possible mechanism of low molecular weight heparin protective effect on ALI of septic rats,this study investigated the effect of low molecular weight heparin on mRNA and protein expression of high mobility group box1(HMGB1) in lung tissue of septic rats which induced by cecal ligation puncture (CLP).
2. Observe the low molecular weight heparin effect on plasma TNF-a and IL-6 of septic rats,discussing its influence on releasing of early and late mediators of inflammation during the sepsis.
[Methods]
144 sepcific-pathogen free(SPF) healthy male Sprague-Dawley(SD) rats were randomly divided into three groups as follow:Sham operation group(A),nomal treatment group (B),the LMWH treatment group(C),n=48.Group A received a sham operation and the other groups were underwent CLP operation.Groups A and B accepted intraperitoneal injection(i.p.)of normal saline(NS) at a dose of 2.0ml/kg and ceftriaxone(30mg/kg),Group C were intraperitoneal injection additional LMWH(150U/kg). Observe points were made at3、6、12、18、24、48h,the rats were anesthesized and killed, mortality、lungs wet/dry ratio and pathologic change were determined.HMGB-1 mRNA and protein of lung tissues were measured by RT-PCR and Western blot.TNF-a and IL-6 of blood plasma calculated by ELSIA.MPO activity in lung homogenate was detected according to the kit instruction.The right upper lobe were removed when they were killed,then weighted(wet weight)and dried in a 80℃oven until weight was constant(dry weight).The ratio of wet weight/dry weight of the lung was calculated.The effect of LMWH on the septic severity score、survival time,expression of HMGB1 mRNA.HMGB1、MPO activity.WW/DW ratio、TNF-αand IL-6 were observed.
[Result]
1.The septic severity score of Group A at defferent time had no statistics significant difference,the septic severity score of Group were obviousy higher than Group A,the different between each other had ststistics significance(P<0.01);the septic severity score reached to the peak during 12h-24h,and it was still higher than the 6h level.The septic severity score of Group C was remarkably lower than Group B.The half survival time of Group A、B、C were 120h、28h、20.4h respectively,and there were remarkably ststistics significance between Group A、B、C.
2.Group A had very weakly expression of HMGB1 mRNA and HMGB1 on lung tissue at all time points,and no statistics significance were observed.The accrescence of HMGB1 mRNA and HMGB1 of Group B、C were detected since 6h,and reached the peak at time 12-24h,and the decrease were detected since 48h,but still higher than Group A even at time point of 48h.Group C was remarkably lower than Group B (P<0.05).
3.Group A had very weakly expression TNF-a in plasma of at all time points,and no statistics significance were observed.The accrescence of TNF-a content in plasma of Group B、C reached the peak at time 6h, and the decrease were detected since 12h,and get the same level as 3h at time 24h.Group B、C level higher than Group A even at time point of 24h.Group C was remarkably lower than Group B (P<0.05). Group A had very weakly expression IL-6 in plasma of at all time points,and no statistics significance were observed. The accrescence of IL-6 content in plasma of Group B、C reached the peak at time 3h, and the decrease were detected since 18h,and get the same level as 3h at time 24h.Group B、C level higher than Group A even at time point of 24h.Group C was remarkably lower than Group B (P<0.05).
4.The MPO activity of Group A had no statistics significance between each other,the activity of Group B、C begin to increase since 3h,and also reach the peak at point 24h,and the decrease could be detected since 48h.Group C are remarkably lower than Group B(P<0.05),there were dtill higher than the basic of Group A (P<0.05).
5.The WW/DW ratio of Group A was remarkably lower than Group B,. C(P<0.05),and GroupC were remarkably lower than Group B (P<0.05).
6.Through light microscope,there were obviously manifest of ALI on lung tissue of B Group,including:hyperemia、edma、congestion and inflammation.Group C had lightet lung injury and the lung injury score of them lower compared to Group B(P<0.05).
[Conclusion]
1.The LMWH can inhibit the release of early and late mediators of inflammation in the course of sepsis which induced by CLP.
2.The LMWH can significantly inhibit the mRNA、protein expression of HMGB-1 on lung tissue;attenuates the degree of acute lung injury induced by CLP by down regulation of MPO activity and WW/DW ratio. LMWH may through inhibition of HMGB1 of lung tissue to lessen the PMN infiltration and improve the survive rate of septic rats by CLP.
3.The protective effection of LMWH in acute lung injury induced by CLP may through anti-inflammatory influence on the axis of coagulation and inflammation.
1.动态观察盲肠结扎穿孔(cecal ligation puncture, CLP)脓毒症模型大鼠肺组织高迁移率族蛋白B1(high mobility group boxl portein,HMGB1)mRNA和蛋白表达水平,研究低分子肝素对肺组织HMGB1基因和蛋白表达的影响,探讨低分子肝素对脓毒症大鼠急性肺损伤的保护作用及其可能机制,为脓毒症患者器官功能保护提供新的理论依据
2.观察低分子肝素对脓毒症大鼠血浆TNF-a及IL-6的含量的影响,探讨低分子肝素对脓毒症早晚期炎症释放的作用。
【方法】
144只SPF级雄性成年SD大鼠(体重200-220g)被随机分为假手术组(48只)、盲肠结扎穿孔术(CLP)后常规治疗组(48只)及常规加低分子肝素治疗组(48只),分别施以假手术(A组)或CLP(B组和C组)。A组开腹后轻拉盲肠后将其回纳腹腔,逐层缝合腹壁,其余各组均采用CLP术制作大鼠脓毒症模型术后立即腹腔注射生理盐水(NS)2ml+头孢曲松(30mg/kg)(A、B组)；生理盐水(NS)2 ml+头孢曲松(30mg/kg)+低分子肝素(150 U/kg)(C组)。以术后3、6、12、18、24、48小时为观察点,观察各组大鼠术后活动、进食、竖毛、腹泻、眼球凹陷、呼吸等情况；于各观察点每组随机处死大鼠5只,每组留18只不杀用以观察5天生存率。采用逆转录-聚合酶链反应(RT-PCR)技术测肺组织]HMGB-1mRNA的表达；采用Western blot法检测肺组织HMGB-1蛋白的表达；采用酶联免疫吸附法测定血浆TNF-α及IL-6的含量：用分光光度比色法检测肺组织髓过氧化物酶(myeloperoxidese,MPO);热干法称取肺组织湿重/干重比值(Wetweight/Dry weight);并在活杀前行脓毒症大鼠评分。观察低分子肝素对脓毒症大鼠严重程度评分、生存时间、血浆TNF-α及IL-6的含量、肺组织HMGB1 mRNA与蛋白表达、MPO活性以及WW/DW比值的影响。
【结果】
1.A组各时间点脓毒症评分差异无显著统计学意义,B、C组脓毒症评分较A组同期水平明显升高(P<0.01),从6h开始增高,24h达到高峰并维持至48h,48h评分仍显著高于组内6h(P<0.05)；C组各时间点评分均显著低于B组同期水平(P<0.05)。A、B、C组半数生存时间依次为120h、20.4h、28h；A、B、C两两组间差异均有统计学意义(P<0.05)。
2.A组各时间点大鼠HMGB1mRNA和HMGBl蛋白均有微量表达,差异无统计学意义(P>0.05)。B、C组HMGB1mRNA和HMGB1蛋白从6h开始表达增多,以12-24h为显著,48h开始下降但仍高于A组。C组同期水平较B组低(P<0.05),但仍比A组高且有显著性差异(P<0.05)。
3.A组大鼠血浆TNF-α水平在术后各时间点差异无显著性,B组大鼠血浆TNF-α水平于术后6h达到峰值,12h开始明显下降,24 h逐渐恢复至3h水平。C组各时间点血浆TNF-α水平低于B组(P<0.05),但仍显著高于A组(P<0.05)。A组大鼠血浆IL-6水平在术后各时间点差异无显著性,B组大鼠血浆IL-6水平于术后3h达到峰值,18h开始明显下降,24 h逐渐恢复至3h水平。C组各时间点血浆IL-6水平低于B组(P<0.05),但仍显著高于A组(P<0.05)。
4.A组各时间点MPO活性无明显差异,B组CLP后3小时MPO活性开始升高,24h达峰值,48h开始有所回落,但与A组同期水平差异仍有显著性(P<0.05),C组较B组同期水平低(P<0.05),但较A组显著升高(P<0.05)。
5.A组肺组织WW／DW比值较B、C低,差异有显著性(P<0.05),C组比值介于A组与B组之间,各组组间差异具显著性(P<0.05)。
6.光镜下B组肺组织可见明显急性肺损伤表现：充血、水肿、炎症细胞浸润；C组较B组病理损伤明显减轻,且肺损伤病理评分明显降低(P<0.05)。
【结论】
1.低分子肝素能够抑制CLP引起的脓毒症过程中的早晚期炎症介质释放。
2.低分子肝素能够显著抑制脓毒症大鼠肺组织HMGB1蛋白及mRNA水平表达；通过抑制肺组织HMGB1,下调脓毒症大鼠肺组织MOP活性,减轻肺组织PMN浸润,减缓CLP诱导的肺损伤程度,提高脓毒症大鼠的生存率。
3.低分子肝素对脓毒症引起的肺损伤的保护作用可能是通过其抗炎性凝血效应对凝血-炎症轴影响而实现的。
[Objective]
1.To find the possible mechanism of low molecular weight heparin protective effect on ALI of septic rats,this study investigated the effect of low molecular weight heparin on mRNA and protein expression of high mobility group box1(HMGB1) in lung tissue of septic rats which induced by cecal ligation puncture (CLP).
2. Observe the low molecular weight heparin effect on plasma TNF-a and IL-6 of septic rats,discussing its influence on releasing of early and late mediators of inflammation during the sepsis.
[Methods]
144 sepcific-pathogen free(SPF) healthy male Sprague-Dawley(SD) rats were randomly divided into three groups as follow:Sham operation group(A),nomal treatment group (B),the LMWH treatment group(C),n=48.Group A received a sham operation and the other groups were underwent CLP operation.Groups A and B accepted intraperitoneal injection(i.p.)of normal saline(NS) at a dose of 2.0ml/kg and ceftriaxone(30mg/kg),Group C were intraperitoneal injection additional LMWH(150U/kg). Observe points were made at3、6、12、18、24、48h,the rats were anesthesized and killed, mortality、lungs wet/dry ratio and pathologic change were determined.HMGB-1 mRNA and protein of lung tissues were measured by RT-PCR and Western blot.TNF-a and IL-6 of blood plasma calculated by ELSIA.MPO activity in lung homogenate was detected according to the kit instruction.The right upper lobe were removed when they were killed,then weighted(wet weight)and dried in a 80℃oven until weight was constant(dry weight).The ratio of wet weight/dry weight of the lung was calculated.The effect of LMWH on the septic severity score、survival time,expression of HMGB1 mRNA.HMGB1、MPO activity.WW/DW ratio、TNF-αand IL-6 were observed.
[Result]
1.The septic severity score of Group A at defferent time had no statistics significant difference,the septic severity score of Group were obviousy higher than Group A,the different between each other had ststistics significance(P<0.01);the septic severity score reached to the peak during 12h-24h,and it was still higher than the 6h level.The septic severity score of Group C was remarkably lower than Group B.The half survival time of Group A、B、C were 120h、28h、20.4h respectively,and there were remarkably ststistics significance between Group A、B、C.
2.Group A had very weakly expression of HMGB1 mRNA and HMGB1 on lung tissue at all time points,and no statistics significance were observed.The accrescence of HMGB1 mRNA and HMGB1 of Group B、C were detected since 6h,and reached the peak at time 12-24h,and the decrease were detected since 48h,but still higher than Group A even at time point of 48h.Group C was remarkably lower than Group B (P<0.05).
3.Group A had very weakly expression TNF-a in plasma of at all time points,and no statistics significance were observed.The accrescence of TNF-a content in plasma of Group B、C reached the peak at time 6h, and the decrease were detected since 12h,and get the same level as 3h at time 24h.Group B、C level higher than Group A even at time point of 24h.Group C was remarkably lower than Group B (P<0.05). Group A had very weakly expression IL-6 in plasma of at all time points,and no statistics significance were observed. The accrescence of IL-6 content in plasma of Group B、C reached the peak at time 3h, and the decrease were detected since 18h,and get the same level as 3h at time 24h.Group B、C level higher than Group A even at time point of 24h.Group C was remarkably lower than Group B (P<0.05).
4.The MPO activity of Group A had no statistics significance between each other,the activity of Group B、C begin to increase since 3h,and also reach the peak at point 24h,and the decrease could be detected since 48h.Group C are remarkably lower than Group B(P<0.05),there were dtill higher than the basic of Group A (P<0.05).
5.The WW/DW ratio of Group A was remarkably lower than Group B,. C(P<0.05),and GroupC were remarkably lower than Group B (P<0.05).
6.Through light microscope,there were obviously manifest of ALI on lung tissue of B Group,including:hyperemia、edma、congestion and inflammation.Group C had lightet lung injury and the lung injury score of them lower compared to Group B(P<0.05).
[Conclusion]
1.The LMWH can inhibit the release of early and late mediators of inflammation in the course of sepsis which induced by CLP.
2.The LMWH can significantly inhibit the mRNA、protein expression of HMGB-1 on lung tissue;attenuates the degree of acute lung injury induced by CLP by down regulation of MPO activity and WW/DW ratio. LMWH may through inhibition of HMGB1 of lung tissue to lessen the PMN infiltration and improve the survive rate of septic rats by CLP.
3.The protective effection of LMWH in acute lung injury induced by CLP may through anti-inflammatory influence on the axis of coagulation and inflammation.
引文
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