Dysregulated renin-angiotensin system contributes to acute lung injury caused by hind-limb ischemia-reperfusion in mice.

The mechanism of acute lung injury (ALI) following limb ischemia-reperfusion (LIR) is not yet clear. We speculate that the unbalanced expression of angiotensin-converting enzymes (ACE and ACE2) and angiotensins [Ang II and Ang-(1-7)] in the renin-angiotensin system (RAS) is a major cause of ALI. To prove this hypothesis, pathological changes, lung edema, and permeability of wild-type mice at different time points within 12 h of reperfusion after 2 h of hind-limb ischemia were first detected by morphological method, measurements of wet-to-dry weight ratio, and bronchoalveolar lavage fluid. Meanwhile, the changes of lung ACE/ACE2 mRNA and protein expression were surveyed by the methods of real-time reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. Angiotensin II/Ang-(1-7) levels in the blood serum and lung tissue were measured by enzyme-linked immunosorbent assay. Then the effects of ACE2 gene insertion and deletion on the previously mentioned parameters were investigated in the mice being exposed to hind-limb 2-h ischemia and 4-h reperfusion. The results revealed that lung injuries in the wild-type mice were gradually aggravated, and the expression of ACE in lung tissue was progressively increased, whereas that of ACE2 decreased within 12 h after LIR. Unexpectedly, both Ang II and Ang-(1-7) in the lung tissue were obviously increased after LIR, showing Ang-(1-7) higher than Ang II in the early stage of reperfusion but lower than Ang II at the late stage of reperfusion. Unlike local Ang II/Ang-(1-7) changes, circulating Ang-(1-7) became greatly descending, and Ang II was markedly ascending from the start of reperfusion, corresponding to local ACE/ACE2 unbalanced expression. ACE2 transgenosis improved the imbalance of ACE/ACE2 and Ang II/Ang-(1-7) expression and alleviated lung injuries, whereas ACE2 knockout further aggravated the imbalance of ACE/ACE2 and Ang II/Ang-(1-7) expression and made lung injuries more serious in the post-LIR mice. The results indicate that the dysregulation of local and circulating RAS with increased expression of ACE/Ang II and decreased expression of ACE2/Ang-(1-7) contribute to ALI caused by LIR in mice. Maintaining RAS homeostasis through upregulating ACE2 expression may lessen lung injury, which provides a new idea for the treatment of posttraumatic ALI.

[Preventive effects of Salvia miltiorrhiza on multiple organ edema in the rats of limb ischemia/reperfusion].

OBJECTIVE: To investigate the preventive effects of Salvia miltiorrhiza (SM) on multiple organ edema in the rats which suffered from hind limb ischemia/reperfusion( LI/R). METHODS: Twenty four Wistar rats were randomly divided into 3 groups (n = 8): control group (C group), ischemia/reperfusion group (I/R group ), Salvia miltiorrhiza group (SM group). Referring to Tourniquet method, the model rats which underwent 4 hours ischemia and 4 hours reperfusion of hind limbs were made. Thirty minutes before reperfusion, SM was given to the rats in SM group by tail vein injection at the dose of 5 mL/kg. Accurately weighed one gram of heart, liver, kidney, lung, brain, intestine and skeletal muscle from every animals, weigh these specimens after baking (60 degrees C, 55 hours), calculated the ratio of wet and dry (Wet/Dry,W/D). The levels of interleukin-1 (IL-1) ,interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) in plasma and the contents of Superoxide dismutase (SOD) and malonaldehyde (MDA) were measured. The morphologic changes of skeletal muscle were observed with microscope. RESULTS: It was found that after suffering from ischemia/reperfusion, the W/D of every specimens increased in different degree (P < 0.05, P < 0.01). In plasma, the values of SOD decreased but MDA increased obviously (P < 0.05, P < 0.01). The levels of IL-1, IL-6 ,TNF-alpha-a in plasma increased (P < 0.05, P <0.01). After LI/R, infiltration of inflammatory cells, broaden interstitial around muscle fiber and disordered arrangement of muscle fibers could be seen under microscope. However, Compared with LI/R group, W/D and levels of serum inflammatory factors in SM group were all lower, the values of SOD in plasma increased but MDA in plasma failed down. Pathological changes in skeletal muscle were improved. CONCLUSION: Limb ischemia/reperfusion can lead to multiple organ edema, Salvia miltiorrhiza can prevent the edema in some degree by anti-oxidation and anti-inflammation.

Role of angiotensin-converting enzyme (ACE and ACE2) imbalance on tourniquet-induced remote kidney injury in a mouse hindlimb ischemia-reperfusion model.

In this study, the relationship between the local imbalance of angiotensin converting enzymes ACE and ACE2 as well as Ang II and Ang (1-7) and renal injury was observed in the different genotypes mice subjected to tourniquet-induced ischemia-reperfusion on hind limbs. In wild-type mice, renal ACE expression increased while renal ACE2 expression decreased significantly after reperfusion, accompanied by elevated serum angiotensin II (Ang II) level and lowered serum angiotensin (1-7) (Ang (1-7)) level. However, renal Ang (1-7) also increased markedly while renal Ang II was elevated. Renal injury became evident after limb reperfusion, with increased malondialdehyde (MDA), decreased super-oxide dismutase (SOD) activity and increased serum blood urea nitrogen (BUN) and creatinine (Cr), compared to control mice. These mice also developed severe renal pathology including infiltration of inflammatory cells in the renal interstitium and degeneration of tubule epithelial cells. In ACE2 knock-out mice with ACE up-regulation, tourniquet-induced renal injury was significantly aggravated as shown by increased levels of MDA, BUN and Cr, decreased SOD activity, more severe renal pathology, and decreased survival rate, compared with tourniquet-treated wild-type mice. Conversely, ACE2 transgenic mice with normal ACE expression were more resistant to tourniquet challenge as evidenced by decreased levels of MDA, BUN and Cr, increased SOD activity, attenuated renal pathological changes and increased survival rate. Our results suggest that the deregulation of ACE and ACE2 plays an important role in tourniquet-induced renal injury and that ACE2 up-regulation to restore the proper ACE/ACE2 balance is a potential therapeutic strategy for kidney injury.

[Effects of taurine on TNF-alpha and NF-kappaB expression of liver injury after limbs ischemia/reperfusion in rats].

AIM: To investigate the effect of pretreatment with taurine on liver injury changes and the change of tumor necrosis factor alpha and NFkappaB expression following rats limbs ischemia/reperfusion. METHODS: The model of limbs ischemia/reperfusion injury on rats was adopted in the experiment. Wistar rats were randomized into 4 groups (n = 10): Control group, T group, I/R group and TR group. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and MDA in the plasma, MDA, MPO, calcium in liver tissues were measured by colorimetric method. The content of TNF-alpha in plasma and liver tissues was determined by radioimmunoassay. The morphologic changes were observed with HE staining. The expressions of NF-kappaBp65 in liver tissues were tested by immuno-histochemistry method. RESULTS: It was found that against the control group, the test values of ALT, AST, et al. and expressions of TNF-alpha, NF-kappaB increased in I/R group and TR group, but values of those in TR group were lower than in I/R group. CONCLUSION: Taurine can decrease the levels of TNF-alpha and NF-kappaB. It can mitigate the liver injury after limb ischemia/reperfusion injury in rats.

[Effect of ischemic preconditioning on lung injury induced by ischemia/reperfusion in the hind limbs of rats].

AIM: To study the effect of ischemic preconditioning on lung injury following ischemia/reperfusion (I/R) in the hind limbs of rats. METHODS: Wistar rats were randomly divided into four groups (n=8): control group,limbs ischemia/reperfusion (LI/R) group, ischemia preconditioning (IPC) group and L-NAME group. At the end of the experiment, blood/gas analysis and the contents of serum MDA, NO, ET and lung tissue MDA, NO, ET, MPO were measured. Meanwhile, lung index and W/D) of lung were measured. RESULTS: After the rats' hind limbs suffered ischemia/reperfusion, the level of PaO2 decreased and the values of W/D, LI, MPO of the lung issure and MDA, NO, ET of plasma and lung all increased significantly in the LI/R group; but the ratio of NO/ET decreased. Compared with LI/R group, the contents of NO and ratio of NO/ET increased but other parameters decreased in the IPC group. Compared with IPC group, the contents of NO and ratio of NO/ET decreased, but other parameters increased in the L-NAME group. CONCLUSION: The IPC can attenuate lung injury following IR in the hind limbs of rats, which may correlated with the increase of NO.

[Effect of MK801 on apoptosis in the development of brain injury after hind limbs ischemia/reperfusion in rats].

AIM: To evaluate development of brain injury after hind limbs ischemia/reperfusion (LI/R) in rats, and the effect of MK801 on the brain injury following LI/R. METHODS: The limbs ischemia/reperfusion model was established in rats. The MDA contents were evaluated in each group, apoptotic cells were detected with TUNEL, the expression of apoptosis-associated protein, such as bcl-2, cytoC and caspase-3 were determined with immunohistochemistry and Western-blot. RESULTS: The contents of MDA in brain tissue increased significantly following LI/R. The expression of bcl-2, cytoC, Caspase-3 was increased than those in the control group (P < 0.01) following LI/R significantly. The expression of Caspase-3 was increased 24 h after the onset of reperfusion. The expression of Caspase-3, bcl-2 gene was quite obvious in the midbrain red nucleus region. MK801 inhibited the expression of bcl-2, cytoC, Caspase-3 obviously. CONCLUSION: The excessive apoptosis and apoptosis-associated factors could play an important role in the brain injury following LI/R in rat, MK801 might decrease the production of free radical and the excite toxicity of glutamate, inhibit the expression of apoptosis associated protein and reduce the occurrence of apoptosis.

[The affection and significance of NO on the expression of P-selectin in renal injury following hind limb ischemia/reperfusion in rats].

AIM: To probe into the affection and significance of NO on the expression of P-selectin in renal injury following hind limb ischemia/reperfusion in rats. METHODS: In accordance with the conventional approaches of our department, the model rats were prepared after they were made to undergo 4 hours or ischemia followed by 4 hours of reperfusion of hind limbs. The Wistar rats were divided into four groups randomly: Control group, LI/R group, L-Arg group and L-NAME group. And then in those four groups of Wistar rats, a series of values of measurement were determined such as: Plasma concentrations of nitric oxide (NO), blood urea nitrogen (BUN) and creatinine (Cr). Furthermore, biochemically there came to the assessment of the values including myeloperoxidase (MPO), NO, total nitric oxide synthase (tNOS), inducible NOS (iNOS) and constitutive NOS (cNOS) of renal tissue in different groups. By the methods of electrophoresis and biochemistry, the urine protein was mensurated. The immunohistochemical method was used to detect the expression of P-selectin protein. The morphologic changes were observed with a microscope. RESULTS: After hind limbs had suffered from ischemia/reperfusion for 4 hours, there was the occurrence of a series of results such as in the following which were based on the comparison between plasm of LI/R group and control group. The values of NO, BUN and Cr increased significantly, and the trend of indexes such as NO in renal tissue was similar to that in plasma. The values of MPO, tNOS and iNOS in renal tissue all increased significantly after reperfusion, while cNOS decreased distinctly. The urine protein appeared, especially large molecular weight protein. Renal pathology revealed that after LI/R there were edema and infiltration of polymorphonuclear neutrophil (PMN). Immunohistochemically, the expression of P-selectin was upregulated significantly. Compared with LI/R rats, all injury changes were alleviated in L-Arg group. Morphologic changes were mild. Both the content of urine protein and the percentage of apoptosis cell decreased. The expression of P-selectin was downregulated. In L-NAME group, all injury changes got worse. Immunohistochemical results showed strong positive staining of P-selectin. CONCLUSION: The renal injury after LI/R may relate to the strong expression of P-selectin. NO may have protective affection by decreasing the expression of P-selectin and alleviating the adhesion, aggregation and infiltration of neutrophils.

[Expression NOS in acute lung injury following limb ischemia/reperfusion and its significance in rats].

AIM: To investigate the expression and role of inducible NOS (iNOS) and endothelial NOS (eNOS) in acute lung injury following limb ischemia/reperfusion (4h/4h). METHODS: Wistar rats were randomized into four groups: control group, ischemia/reperfusion (I/R) group, L-Arginine (L-Arg) pretreatment group, Aminoguanidine (AG) pretreatment group. The lung tissue of each group was subjected to assay of content of MDA, MPO, W/D and NO2-/NO3-. The expression of iNOS and eNOS was examined with immunohistological staining. The pulmonary morphologic changes were observed under microscope respectively. RESULTS: The acute lung injury existed after limb ischemia/reperfusion. The eNOS downregulation and iNOS upregulation among I/R, L-Arg and AG groups were observed contrasted to the control group. There was no expressional and statistical difference of iNOS between I/R group and L-Arg group. The expression of eNOS was similar between IR and AG but iNOS expression was downregulated in AG. The parameters of MDA, MPO, W/D and NO2-/NO3- in pulmonary tissue were significantly increased in I/R groups compared with those of the control group. The parameters of L-Arg and AG pretreatment groups in comparison with those of the I/R group showed significantly difference. Based on the results of pulmonary pathology, the congestion and infiltration of inflammatory cells existed obviously in IR group. L-Arg played definite role in militating lung injury and AG might make lung injury aggravated. CONCLUSION: The NO definite production from iNOS is possible to play a competitivly protective role in acute lung injury following limb ischemia/reperfusion and antagonist of iNOS may aggravate the lung injury.

[The protect effect of ischemic preconditioning on the gastric mucosal injury following ischemia/reperfusion of hind limbs of rats].

AIM: To observe the degree of gastric mucosal injury following limb ischemia/reperfusion (LI/R), and to investigate the mechanism of gastric mucosal injury and the protection of ischemic preconditioning (IPC) on gastric mucosal injury. METHODS: The model rats which underwent 4 hours of ischemia and 4 hours of reperfusion of hind limbs were made. Then we respectively observed and determined the histologic lesion score after I/R and IPC + I/R. The gastric barrier mucus in mucus were measured in different groups. The values of MPO, SOD, MDA and XOD in gastric mucosa and the values of MDA, XOD, SOD, LDH in plasma were detected. RESULTS: In the LI/R group, the histologic lesion score increased significantly. The content of gastric barrier mucus in mucus decreased significantly. The value of MPO, MDA, XOD in gastric mucosa and the values of MDA, XOD, LDH in plasma increased remarkably and SOD activity in gastric mucosa and in plasma decreased. However in the IPC group, the histologic lesion score decreased significantly and the content of gastric barrier mucus in mucus increased significantly and the value of MPO MDA XOD LDH in gastric mucosa or in plasma decreased remarkably and the SOD activity increased compared to LI/R group. CONCLUSION: LI/R will lead to the development of stress ulcer, oxygen free radicals play an important role in it. IPC can alleviate the damage of gastric mucosa following ischemia/reperfusion of hind limbs. The decrease of OFR is one of the protection mechanism of IPC.

[Therapeutic effect of zinc sulfate on lung injury during superior mesenteric artery occlusion(SMAO) shock].

AIM: To study preventive and therapeutic effect of zinc sulfate on lung injury during superior mesenteric artery occlusion (SMAO) shock and their mechanism of action. METHODS: Model of rabbit SMAO shock was made. The effect of zinc sulfate on the malondialdehyde (MDA) in erythrocyte membrane and plasma, oxidase (XOD) in plasma, superoxide dismutase (SOD) in erythrocyte and MDA, SOD and pulmonary surfactant (PS) in lung tissues homogenate were observed. RESULTS: The administration of zinc sulfate decreased MDA and XOD, prevented the reduction of SOD and PS, and alleviated lung injury. CONCLUSION: It is suggested that lung is injured during SMAO shock and zinc sulfate possesses preventive and therapeutic effect, through stabilized membrane.

[A cell model of ischemia/reperfusion injury on skeletal muscle].

AIM: To establish a model of ischemia/reperfusion injury on L-6TG cell. METHODS: Cultured L-6TG cells were divided into 2 groups: control group (C), ischemia/reperfusion group (I/R), LDH in culture fluid, SOD, XOD, free calcium in L-6TG cell and mitochondria respiration were evaluated in each group, the micromorphologic changes were observed with microscope. RESULTS: Compared with control group, after L-6TG cell suffered ischemia 4 hours and reperfusion 4 hours, LDH in culture fluid, XOD, free calcium in L-6TG cell all increased significantly, while SOD in L-6TG cell and mitochondrial respiration decreased, structural damage to L-6TG cell was severe. CONCLUSION: Using mimicking ischemic solution and mimicking reperfusion solution can successfully establish a model of ischemia/reperfusion injury on L-6TG cell.

[The effect of NO, ET-1 on brain injury after hind limbs ischemia/reperfusion in rats].

AIM: To study the roles of nitric oxide (NO) and ET-1 in brain injury after hind limbs ischemia/reperfusion in rats and to investigate the effect of NO/ ET-1 balance on brain injury. METHODS: On a model of the hind limbs ischemia/reperfusion (LI/R) of rats, we used L-Arg(L-arginine, L-Arg), one of the substrates in the process of nitric oxide, aminoguanidine (AG) which inhibits nitric oxide synthase(NOS) and ETA receptor antagonist BQ123, to observe the changes of NO, ET-1, MDA, XOD, SOD, LDH in plasma and tNOS, iNOS, cNOS, NO, ET-1, MDA, XOD, MPO, SOD in brain tissue. RESULTS: Compared with the control group, the content of MDA, XOD, LDH in plasma and MDA, XOD, MPO in brain tissue increased. The activity of SOD decreased (P < 0.01). The content of tNOS, iNOS in brain tissue increased, cNOS decreased (P < 0.01). The content of NO, ET-1 in I/R group in plasma and brain tissue increased, the ratio of NO/ET-1 decreased. The brain injury was deteriorated. After using L-Arg and BQ123, the ratio of NO/ET-1 in plasma and brain tissue increased, the brain injury lightened. Whereas after using AG, the ratio of NO/ET-1 decreased, brain injury became more serious. CONCLUSION: The NO/ET-1 ratio decreased after LI/R, brain injury became more serious.

[Effects of PKC activation on apoptosis during ischemia/reperfusion in L-6TG rat skeletal myoblasts].

AIM: To study the effects of PKC activation on apoptosis during ischemia/reperfusion in L-6TG rat skeletal myoblasts. METHODS: Cultured L-6TG cells were divided into 3 groups: control group (C), ischemia/reperfusion group (I/R), PMA + ischemia/ reperfusion group (PMA), SOD, XOD and free calcium and mitochondrial respiration in L-6TG cell were evaluated in each group. Apoptosis was detected by flow cytometer with PI staining method and agarose gel electrophoresis, the immunohistochemical method was used to determine the expression of caspase-3. RESULTS: Compared with I/R group, in PMA group, XOD , free calcium in L-6TG cell and apoptotic percentage all decreased significantly, while SOD and mitochondrial respiration in L-6TG cell increased. DNA fragmentation analysis of L-6TG cell showed no laddering pattern. The expression of caspase-3 was down regulated significantly. CONCLUSION: Activation of PKC can lessen ischemia/reperfusion injury and apoptosis through lessening oxidative injury and mitochondrial injury, adjusting calcium dyshomeostasis and down expression of caspase-3.

[The protective effect of taurine on lung injury following limbs ischemia/reperfusion of rats].

AIM: On the model of limb ischemia/reperfusion (LIR), the effects of taurine on pulmonary morphological changes in rats were observed. METHODS: Wistar rats were divided into three groups (n=8): control group, ischemia/reperfusion group (IR) and taurine + IR (Tau + IR). Then macroscopic inspection and optical and transmission electron microscopies (TEM) were performed to assess the morphological changes of the lung tissues and their lung index (LI) and lung permeability index (LPI) and reactive oxygen species (ROS), malondialdehyde (MDA) were measured as well. RESULTS: The morphological changes of lung tissue after LIR were characterized by an increase of permeability of the alveoli-capillary membrane and infiltration of inflammatory cells. Under optical microscopy, there were congestion and swelling in pulmonary microvessels with broadened the spaces around the blood vessels. Under TEM, a number of tight-junctional regions between adjacent alveolar epithelial cells and between pulmonary microvessels endothelium were "open". The LI, LPI, MDA and ROS increased. The specimens of Taurine + IR group revealed slight to moderate degrees of damages in the lung tissues. CONCLUSION: Taurine protects lung from LIR in rats and the protective action on tight-junctional regions between cells and anti-oxygen is one of the protective mechanisms of taurine.

[The effect of nitric oxide on acute lung injury following ischemia/reperfusion of hind limbs in the rat].

On a model of reperfusion after ischemia in the hind limbs (LIR) of rats, we used aminoguanidine (AG) which inhibits nitric oxide synthase (NOS) and L-arginine (L-Arg), one of the substrates in the process of nitric oxide synthesis, to observe the changes in NO, NOS, malondialdehyde (MDA), myeloperoxidase (MPO) and wet/dry ratio (W/D) in both skeletal muscles and the lung as well as the changes in phosphatidyl choline (PC) of lung surfactant. The morphologic changes were observed with microscopy. It was observed that the values of NOS, MPO, MDA of the muscle and lung in LIR group increased significantly and the content of PC decreased obviously compared with those of the normal control. Pulmonary observation revealed that after LIR leucocyte assembling and infiltration took place, which was dominated by polymorphocytes with broadened pulmonary interstitial tissue. In LIR+L-Arg group the above changes were reversed, and in LIR+AG group the injuries became more serious. The results obtained suggest that the activity of NOS and the production of NO following ischemia/reperfusion of hind limbs increased significantly, and that the endogenous NO may play a protective role during the early stage of acute lung injury after LIR.