【摘要】 目的 观察百草枯中毒大鼠肾组织中血红素氧合酶1(HO1)的表达,探讨其病理生理机制。 方法 SD大鼠126只随机分为空白对照组、中毒组和褪黑素组,各42只。中毒组、褪黑素组予以百草枯(25 mg/kg)腹腔注射染毒,对照组予以等量生理盐水腹腔注射,15 min后褪黑素组予以褪黑素(10 mg/kg)腹腔注射,对照组、中毒组予以等量生理盐水腹腔注射。于1、3、6、12 h,1、2、3、5 d时各组随机取6只处死。苏木精〖CD3/5〗伊红（HE）染色观察各组肾组织病理学变化,采用免疫组识化学和RTPCR观察肾组织HO1蛋白和mRNA表达。 结果 ①与对照组相比,中毒组染毒后3 h即可见充血、水肿及空泡变性等病理变化,1 d达顶峰,病理损伤评分3.30±0.31(Plt;0.05),其后缓解趋势不明显；而褪黑素组病理变化明显减轻且缓解趋势明显,1 d时病理损伤评分2.70±0.26,与中毒组相比差异有统计学意义(Plt;0.05)。②与对照组相比,中毒组染毒3 h在皮质部肾小管上皮细胞的细胞膜及细胞浆HO1呈阳性表达,免疫组识化学评分(IHS)3.33±1.75,HO1 mRNA表达增强,与对照组相比差异有统计学意义(Plt;0.05),1 d达顶峰,HIS为7.00±2.00,之后减弱,5 d仍有阳性表达,但与对照组相比差异无统计学意义(Pgt;0.05)；褪黑素组HO1表达较中毒组明显增强,IHS评分6 h~3 d差异具有统计学意义(Plt;0.05),5 d不再有统计学意义(Pgt;005)。 结论 HO1在百草枯中毒大鼠肾组织中呈高表达,褪黑素能明显改善百草枯中毒肾脏病理损伤,增强HO1表达可能是其作用途径之一,而氧化损伤可能是百草枯中毒肾损伤病理生理机制之一。【Abstract】 Objective To investigate the expression of heme oxygenase1 (HO1) in paraquartinduced renal injury in rats and the protective effects of melatonin, and explore possible mechanism of paraquartinduced renal injury. Methods One hundred and twentysix adult healthy SpragneDawley rats were randomly divided into three groups and 42 in each group: control group (A), paraquart group (B), and melatonin group (C). The rats in group B and group C were treated with paraquart (25 mg/kg) intraperitoneally, the rats in group A were treated with the same dose of normal saline. In 15 minutes, the rats in group C were given melatonin intraperitoneally at a dose of 10 mg/(kg·d) and the rats in group A and B were treated intraperitoneally with the same dose of normal saline. Six rats in each group were randomly sacrificed at one, three, 12 hours and one, tou, three, five days respectively. Renal histopathological changes were observed under light microscope by HE staining. The protein and mRNA expressions of HO1 were evaluated by immunohistochemical staining and RTPCR respectively. Results ①In group B, there were obvious lesions in the renal tubule of cortical part, including edema, congestion and vacuolar degeneration. These pathologic changes gradually reached the peak on the first day and did not relieved till the end of this study, the pathologic injury score was 3.30±0.31, and there was a statistical significance between group B and group A (Plt;0.05). The aforementioned pathological lesion was more palliative in group C, the pathologic injury score was 2.70±0.26 at the first day; Compared with group B, there was a statistical significance. ②In group A, there was no or weak expression of HO1 and HO1 mRNA. At the third hour, the expression of HO1 in group B was observed in the membrane and cytoplasm of renal tubular epithelial cell of cortical part. Immunohistochemistry score (IHS) was 3.33±1.75 and the expression of HO1 mRNA increased, there was a statistical significance between group B and group A (Plt;0.05). It reached the peak on the first day, IHS was 7.00±2.00, but there was no statistical difference between group B and group A on the fifth day (Pgt;0.05); Compared with group B, the expression in group C was enhanced obviously, IHS were higher obviously on the six hour till to the third day (Plt;0.05), but there were no statistical differences on the fifth day (Pgt;0.05). Conclusion The expression of HO1 in the paraquartdamaged kidney increases and melatonin surely has an protective effect by increasing the expression of HO1, which suggests that oxidative injury might be the main mechanism of paraquartinduced renal injury.
目的:观察百草枯中毒大鼠血浆和肺组织匀浆中的内皮素-1的动态变化,探讨大鼠PQ中毒肺损伤的可能机制。方法:将雄性Wistar大鼠随机分成百草枯中毒组和生理盐水对照组,分别给予16mg/kg百草枯或等体积生理盐水腹腔内注射,并在给药后6、12、24和72 h处死大鼠,观察其一般表现、肺的病理变化。测定肺系数、肺组织匀浆和血浆中内皮素-1的含量。结果: 百草枯中毒后大鼠出现呼吸困难,活动能力下降,肺系数增加,并随时间变化,光镜下表现为肺出血、水肿等不同程度的肺损伤改变。血浆和组织匀浆中内皮素-1在中毒后与对照组大鼠比较明显升高,并随时间变化(Plt;0.01)。结论:在百草枯中毒肺损伤中,内皮素-1可能发挥了重要作用。
ObjectiveTo investigate the changes of inflammatory cytokines in bronchoalveolar lavage fluid (BALF) in rats exposed to paraquat (PQ). MethodsAdult healthy SD rats were randomly divided into a control group (n=8) and three experimental groups (PQ in low dosage of 15 mg/kg,medium dosage of 30 mg/kg,and high dosage of 60 mg/kg,n=24 in each group). The rats in three experimental groups were intragastrically administered with PQ,and the rats in the control group were treated with saline by gavage. Two rats in the control group and six rats in three experimental groups were sacrificed on 1st,7th,14th,and 21st day after exposure respectively. BALF was collected for measurement of interleukin-1(IL-1),IL-6,macrophage inflammatory protein-2(MIP-2),monocyte chemoattractant protein-1(MCP-1),and biopterin by ELISA. ResultsThe levels of cytokines in all experimental groups were higher than those in the control group at any time point. In the exposure day 1 to day 14, IL-1 and biopterin levels in BALF increased significantly with the increase in PQ dose. On 14th and 21st day,IL-6 level in BALF increased significantly with the increase in PQ dosage. The levels of IL-1,IL-6,and biopterin in the experimental groups reached the peak on 14th day. On 14th day,the MIP-2 level in BALF of high-dosage group was significantly higher than that of low-dosage and medium-dosage groups (all P<0.05). The level of MCP-1 in the low-dosage group was lower than that in the medium-dosage and high-dosage groups at any time point (P<0.05). ConclusionIL-1,IL-6,MIP-2,MCP-1,and biopterin may play important roles in the development and progression of PQ-induce lung inflammation.
ObjectiveTo investigate the endoplasmic reticulum stress associated apoptosis gene Caspase-12 expression in paraquat-induced pulmonary fibrosis. Methods30 adult healthy Sprague-Dawley(SD) rats were randomly divided into a nomal control group,two pulmonary fibrosis model groups (intragastrically administered paraquat for 14 days and 28 days,respectively).The model of pulmonary fibrosis was established through intragastrically administering paraquat at the dose of 30 mg/kg.RT-PCR was used to determine the mRNA expression of Caspase-12.Immunohistochemistry was used to determine the protein expression of Caspase-12.HE staining and Masson staining were used to determine the degree of alveolitis and pulmonary fibrosis. ResultsHE staining and Masson staining of lung tissues proved that pulmonary fibrosis model was successfully constructed.The degree of alveolitis and pulmonary fibrosis in the model group was significantly more serious than that in the control group(P<0.01).RT-PCR and Immunohistochemistry results showed that the expression of Caspase-12 were remarkably increased in the pulmonary fibrosis model group(14 d group)(P<0.01),even more elevated in 28 d group compared with the 14 d group. ConclusionThe results demonstrate that the expression of Caspase-12 in paraquat poisoned rats is up-regulated,suggesting endoplasmic reticulum stress plays an important role in paraquat induced-pulmonary fibrosis.
目的 探讨急性百草枯(PQ)中毒鼠肺组织病理损伤和肺组织血红素氧合酶-1（HO-1）的表达及三七总皂甙（PNS）的保护作用。 方法 150只SD雄性鼠分为正常对照组（C组）30只、PQ中毒组（PQ组）60只及PNS组60只。PQ组和PNS组一次性灌胃PQ 25 mg/kg染毒，C组给予等体积生理盐水灌胃。其中PNS组于染毒前15 min以PNS 50 mg/kg阴茎静脉注射保护，以后1次/d给药直至处死前；PQ组、C组分别在同时间点给予等体积生理盐水。观察各组大鼠在中毒后6、12 h，1、3、5、7 d肺组织病理改变，采用蛋白质印迹法分析肺组织HO-1蛋白表达和反转录-聚合酶链反应方法测定鼠肺组织HO-1 mRNA的表达。 结果 C组HO-1蛋白和HO-1 mRNA绝大多数标本有弱表达，个别标本不表达；与C组相比PQ组及PNS组HO-1蛋白和HO-1 mRNA表达增强，差异有统计学意义（P＜0.05）；PQ组HO-1蛋白和HO-1 mRNA的表达在1 d达高峰之后下降，第3天基本恢复到C组水平；PNS组与PQ组相似，但在6 h、12 h、1 d及3 d高于PQ组，差异有统计学意义（P＜0.05），至第5天和第7天二者相比差异无统计学意义（P＜0.05）。PQ组肺组织病理损伤评分在6、12 h，1、3 、5、7 d各亚组均高于PNS相应组，差异有统计学意义（P＜0.05），C组肺组织病理大致正常，与PQ组及PNS组相比，差异有统计学意义（P＜0.001）。 结论 HO-1参与PQ中毒所致急性肺损伤，PNS对PQ中毒所致急性肺损伤有保护作用。
目的:探讨贯叶连翘提取物对百草枯诱导的大鼠肺纤维化模型的干预作用及机制。方法:20只Sprague-dawly大鼠随机等分为4组:百草枯组,贯叶连翘提取物组,百草枯+贯叶连翘提取物组和对照组。百草枯液按80mg/kg一次性灌胃，贯叶连翘提取物按400mg/kg灌胃，连用3d,对照组仅用生理盐水。于21d处死后取肺脏行HE染色鉴定,并用碱水解法测羟脯氨酸含量,同时以八木国夫荧光法测定肺组织脂质过氧化产物,盐酸羟胺法测定组织总超氧化物歧化酶的酶活力。结果:百草枯染毒动物可致肺纤维化,羟脯氨酸和丙二醛含量明显增高。百草枯+贯叶连翘提取物组与百草枯组比较,第21天时肺纤维化减轻,羟脯氨酸和丙二醛含量减少(Plt;0.05),与对照组相比总超氧化物歧化酶无明显变化(Pgt;0.05)。结论:贯叶连翘提取物对百草枯诱导的大鼠肺纤维化模型有抑制作用,其机制可能与抑制脂质过氧化有关。
Objective To investigate the effects and mechanisms of hydroxychloroquine sulfate (HCQ) on pulmonary fibrosis through the PI3K/AKt/mTOR signalling pathway. Methods Paraquat intraperitoneal injection was used to establish a mouse model of pulmonary fibrosis. Thirty-six SPF C57BL/6J female mice were randomly divided into a blank group, a paraquat group (20 mg/kg) and a HCQ intervention group. The HCQ intervention group was divided into two subgroups (10 mg/kg and 30 mg/kg) according to different doses. The general condition and body weight changes of mice were observed. twenty-one days later, lung tissues were stained with hematoxylin-eosin and Masson’s pathological staining, and the content of inflammatory factors (IL-1β, IL-6, TNF-α) and hydroxyproline (HYP) were detected by ELISA. Alpha-smooth muscle actin (α-SMA), E-cadherin (E-cad), the expression levels of PI3K/Akt/mTOR pathway-related proteins, phosphatidylinositol 3 kinase (PI3K) and protein kinase B (AKt), and mammalian target of rapamycin (mTOR) were detected by Western blot. The gene expression levels of α-SMA and E-cad were detected by q-PCR. Results Compared with the blank group, the mice in the paraquat group had lower body weight, worse general condition, higher serum levels of inflammatory factors, increased lung structure destruction and collagen deposition, significantly increased HYP content, and higher expression level of PI3K/AKt/mTOR signaling pathway related proteins (all P<0.05). The expression levels of E-cad protein and gene decreased, α-SMA protein and gene increased (all P<0.05). While the HCQ intervention group improved the degree of pulmonary fibrosis in different degrees, and the relevant indexes of PI3K/AKt/mTOR signaling pathway decreased compared with the paraquat group (all P<0.05). Conclusion HCQ can ameliorate paraquat-induced pulmonary fibrosis by inhibiting the PI3K/AKt/mTOR signaling pathway.
Objective To investigate the mechanismof lung injury caused by paraquat poisoning by observing the changes of fibrogenic cytokines in acute paraquat poisoned rats and the effects of pyrrolidine dithiocarbamate ( PDTC) . Methods Sprague-Dawley rats were randomly divided into three groups, ie. acontrol group ( n =6) , a PDTC group ( n =36) , a paraquat group ( n = 36) , and a paraquat + PDTC group( n =36) . The rats in the PDTC group, the paraquat group, and the paraquat + PDTC group were subdivided into 6 subgroups sacrificed respectively on 1st, 3rd,7th,14th, 28th and 56th day after the treatment. The levels of transforming growth factor-β1( TGF-β1 ) , platelet-derived growth factor ( PDGF) , insulin-like growthfactor-1 ( IGF-1) in serum were measured. Meanwhile the expression of connective tissue growth factor ( CTGF) and hydroxyproline in lung tissues were detected. The relationship of above cytokines with hydroxyproline was analyzed. Results The destructive phase in early ( 1 ～7 d) was characterized by hemorrhage, alveolar edema, and inflammatory cell infiltration. The proliferous phase in later stage ( 14 ～56 d) was characterized by diffused alveolar collapse with fibroblast proliferation and patchy distribution of collagen fibers. Compared with the control group, the level of TGF-β1 on all time points, the level of PDGF from7th to 56th day, the level of IGF-1 from3rd to 56th day in the paraquat group all significantly increased ( P lt;0. 01) . Immunohistochemistry results showed CTGF positive cells mainly located in aleolar epithelialcells, endothelial cells,macrophages in early stage, and fibroblasts were main positive cells on the 28th and the 56th day. The expression of CTGF in the paraquat group increased gradually compared with the control group on different time points ( P lt; 0. 05 or P lt; 0. 01) . Meanwhile, the levels of above cytokines were positively correlated with the level of hydroxyproline. Noteworthy, PDTC treatment led to significant decreases of above cytokines compared with the paraquat group in corresponding time points ( P lt;0. 05 or P lt;0. 01) .Conclusions Over expressions of IGF-1, TGF-β1 , PDGF, IGF-1 and CTGF may play important roles in lung fibrosis of paraquat poisoned rats. PDTC, as a b NF-κB inhibitor, may inhibits NF-κB activity and further significantly decreases expressions of cytokines, leading to significantly attenuated pulmonary inflammation and fibrosis. However, the mechanisms of PDTC intervention still remain to be explored.