| The major causes of acute renal failure (ARF) are acute renal ischemia-reperfusion injury due to hemorrhage, severe injury, shock, or kidney transplantation. The mortality associated with ARF in critically ill patients remains high even though application of intensive care and blood purification has provided advanced treatment and a better prognosis for ARF patients. Various agents, including dopamine, atrial natriuretic peptide, insulin-like growth factor, and endothelial receptor antagonists have been found to be effective in animals but ineffective in clinical studies. It is necessary to explore and find more efficient and practical methods for timely prevention and cur. ARF is a complex disorder resulting from heterogeneous pathogenic factors. In the past ten years the pathogenesis of ARF is mainly the renal ischemia reperfusion damage theory, cell metabolic disorder of renal injury and renal hemodynamic changes theory. The ischemia reperfusion injury is a common pathogenesis of acute kindy injury (AKI).Decreased reperfusion leads to hypoxia. The kidney is impaired with the development of ischemia hypoxia time and beyond the renal hypoxia tolerance ability of hypoxia damage. kidney cell are degenerated, necrosis especial the epithelial cells of the proximal tubule in the renal cortex. Reperfusion induced acute inflammation in the renal epithelial cells eventually led to the occurrence of ARF. Renal ischemia reperfusion injury similar to renal tubular epithelial cells with hypoxia reoxygenation..Recent studies have shown that reactive oxygen species (ROS), inflammatory mediators such as intracellular adhesion molecule (ICAM-1) and monocyte chemotactic protein-1(MCP-1) as well as infiltration of inflammatory cells are implicated in renal ischemia-reperfusion injury. Acute inflammation is characterized by the expression of inflammatory factors such as inflammatory cells, cytokines, chemokines and adhesion molecules ect. Recent studies have shown that decreased the level of oxidative stress, inhibition of nuclear transcription factor (NF-kB nuclear factor kappa,) inflammatory pathway, reduced the expression of intercellular adhesion molecule-1(ICAM-1) and monocyte chemoattractant protein-1(MCP-1) can relieve the ischemic reperfusion renal injury.Traditional Chinese medicine has accumulated rich experience in the treatment of renal diseases, It focus on the overall adjustment and emphasizes the multi link function and has the unique superiority. Animal study found that the effects of Astragalus-Angelica Mixture in ischemia-reperfusion models of renal protection have been related to reduce creatininelevel and significantly reduce therenal pathological changes.Astragalus and Angelica are used commonly. Astragalus and Angelica are wide source and low price. If it can be applied to clinical application, will bring good social benefit.The effects of statins in ischemia-reperfusion models of renal protection have been related to their anti-inflammatory, antioxidant, and vascular actions. Although these findings suggest an association between statin use and preserved renal function in ARF scenarios such as suprarenal aortic clamping, kidney transplantation, and critically ill patients in the intensive care unit, they carry little significance in patients with unpredictable ARF caused by hemorrhage, severe injury, or shock. In these studies, statin intervention has been several days before ischemia-reperfusion injury. This situation is the same as traditional Chinese medicine. However, the effects of Astragalus-Angelica Mixture and statin intervention after ischemia-reperfusion injury are not fully understood and it is of significance to the clinical treatment of ARF. In view of Astragalus and Angelica mixture (AAM) and statins have similar effect of antioxidant and anti-inflammatory, therefore, the present study was undertaken to examine the effects and mechanisms of astragalus-angelica mixture and atorvastatin (ATO) after renal ischemia-reperfusion injury in rats.MEDHODSThe models are acute renal ischemia-reperfusion injury rat model an hypoxia-reoxygenation model of renal tubular epithelial cell (HK2).The methods are followed up to observe the protective effects of AAM and ATO to the acute renal ischemia-reperfusion injury.1.Makeing the acute renal ischemia-reperfusion injury rat model and hypoxia-reoxygenation model of renal tubular epithelial cell:1.1Animal model:choose the health male SD rats,200～250g, anesthetized by3%napental (30mg/kg),through the median abdomianal incision to ablate right kidney, free the left renal AtV, occluse the left renal artery for60minutes,after that reperfuse for24h.1.2Cell model:take the3-4gengration HK2cells,adjust the cell concentration to1×106/ml, plant in6hole culture plate,the condition is37℃、95%O2、5%CO2, for24h, then transfer to Hank,s Liq, put the culture plate in the95%N2、5%CO2incubation vessels for60minutes. Then transfer to the RPMI1640clture fluid which has no serum cultivate for60minutes under the condition of95%O2、5%CO2.2. Animal grouping and administering (1) sham operation group;(2) I/R operation group(simply ischemia(60minutes)-reperfusion(24h));(3)5g/ml Astragalus-Angelica mixture+I/R group;(4) lmmol/L Atorvastatin+I/R group;(5)5g/ml Astragalus-Angelica mixture combine1mmol/L Atorvastatin+I/R group.The success rate of the model is92%, feeding24h free diet under normal conditions.3. Cell grouping:(1)normal control group;(2)H/R group;(3)100μg/ml Astragalus-Angelica mixture+H/R group;(4)10μmol/L Atorvastatin+H/R group;(5)100μg/ml Astragalus-Angelica mixture combine lOμmol/L Atorvastatin+H/R group.4. The effect of AAM and ATO to the renal function of acute renal ischemia-reperfusion injury rats:4.1Take the sample of the blood and urine on schedule, detect the creatinine in the blood and the urine with the Toshiba automatic biochemistry analyzer,calculate the creatinine clearance rate(Ccr).4.2Collect the sample of the renal tissue on schedule, fix it with25%glutaralbuffer phosphate and10%neutral formalin fixation fluid, make the conventional tissue slice, observe the change of the renal tissue under light microscope. The renal tubular score by the reforming method of Megyesi J.5. The mechanism of the protective effect of AAM and ATO to the renal function of acute renal ischemia-reperfusion injury rats.5.1Detect the adanced oxidation protein products (AOPPs) in serum: Take the sample of the blood, centrifuge to keep, detect the AOPPs in the serum with the colorimetric method.5.2Detect the malonaldehyde (MDA) in serum:Take the sample of the blood, centrifuge to keep serum, detect the MDA in the serum with the method of thiobarbituric action.5.3Observe the present of ICAM-1、MCP-1in the renal tissue:make the5μm slice after fixing renal tissue, Observe the present of ICAM-1、MCP-1with the method of histochemical.5.4Detection of ICAM-1and MCP-1mRNA in renal tissue was undertaken. Briefly, total RNA of the renal tissue samples was extracted with an RNA Extraction Agent (TaKaRa Bio, Shiga, Japan) following manufacturer instructions. cDNA was synthesized using a TaKaRa Bio Reverse Transcription kit following manufacturer instructions. RT-PCR was performed using the SYBR Green kit of the TaKaRa Bio Reaction System according to the manufacturer’s instructions. The primers for ICAM-1were:sense,5’-GCGACCACGGAGCCAATTTCTCAT-3’; anti-sense,5’-TCAGGACCCTAGTCGGAAGATCGAA-3’. The primers for MCP-1were:sense,5’-ACGCTTCTGGGCCTGTTGTTCA-3’;anti-sense,5’-TGGGGCATTAACTGCATC TGGCT-3’; glyceraldehyde3-phosphate dehydrogenase was used as the internal control. The result (Ct) from ABI7500Fast system was analyzed and compared using the2-ΔΔCt method.6. The effect as well as the mechanism of the AAM and ATO to the hypoxia-reoxygenation renal tubular epithelial cell.6.1Detect the level of the reactive oxygen species (ROS): collect the cell on schedule. Labeled with the fluorescent probe dichlorofluorescein (DCFH).Labeled cells with2×105/hole96hole plate which was analysised by the system of VICTOR Wallac1420markers system(USA PE) and the excitation light at485±10nm,535±10nm emitting. The dynamic changes of fluorescence was detected by the quantity of oxidized DCFH cells generated under the condition of37℃, which reflects the amount of ROS production.6.2Detect the present of ICAM-1、MCP-1in renal tubular epithelial cells with the method of ELISA:collect the cell on schedule, detect the concentration of the ICAM-1、MCP-1as the instruction, and calculate the total protein of per hole, divide the concentration by the counter total protein of hole to balance the difference of cells number between per hole.6.3Detect the present of ICAM-1、MCP-1mRNA wih RT-PCR: collect the cell on schedule, extract the total RNA, take the RT-PCR action with the MJ PTC-200PCR device under the following condition:92℃degeneration1minute,56crenaturation25seconds,72℃progradation2minutes. ICAM-1promer is Sense Primer5’-ACCTTCCTCACCGTGTACTGGACT-3’, Anti-sense Primer5’-TGCG GCACGAGAAATTGGCT-3’. MCP-1primer is Sense Primer5’-TCAGCCAGAT GCAATCAATGCCC-3’, Anti-sense Primer5’-TGGGTTTGCTTGTCCAGGTG GT-3’. The inner control is β-Actin, the primer is Sense primer5’-AAGGATTCCT ATGTG GGC-3’, Anti-sense primer5’-CATCTCTTGCTCGAAGTC-3’.RESULTSI. The protective effect as well as the mechanism of AAM and ATO to the renal function of acute renal ischemia-reperfusion injury rats.1. AAM and ATO can protect the acute renal ischemia-reperfusion injury dose-dependently. Sixty minutes of ischemia and subsequent reperfusion caused a decline in renal function, and Scr levels were increased significantly, compared with the sham-operated group (270.75±46.84μmol/L vs61.23±8.49μmol/L; p<0.05). Decreases in renal function were ameliorated by AAM and ATO treatment, which demonstrated increases in Scr levels24h after surgery (171.88±40.96μmol/L189.00±31.85μmol/L), compared with sham-operated animals (p<0.05). Right nephrectomy alone (sham operation) did not increase creatinine levels. Ccr levels were decreases in renal function were ameliorated by AAM and ATO treatment, which demonstrated decreases in Ccr levels24h after surgery (0.0026±0.00069ml/min n=80.0030±0.00059ml/min n=7), compared with control-operated animals (0.0015±0.0004ml/min n=8p<0.05). AAM%ATO group (0.0053±0.0014ml/min n=11) more decreases in Ccr levels compared with control-operated animals. Right nephrectomy alone (sham operation) did not decrease Ccr levels.2. The renal tubular cells pathological changes were alleviated in AAM and ATO groups rats ischemia-reperfusion injury caused severe damage to epithelial cells of the proximal tubule in the renal cortex. Loss of the brush border and detachment of epithelial cells from the basement membrane caused tubular obstruction, tubular casts, and necrosis. After AAM and ATO treatment, tubular necrosis was markedly reduced and the pathological changes described above mitigated. Renal tubular score of each treatment groups were significantly lower than the control operation group (p<0.05).3. AAM and ATO can inhibit the level of the oxidative stress in the course of renal ischemia-reperfusion injury dose-dependently.MDA levels were increases in renal function and ameliorated by AAM and ATO treatment, which demonstrated decreases in MDA levels24h after surgery (11.24±1.33μmol/L n=812.04±1.97mol/L n=7), compared with control-operated animals (0.0015±0.0004ml/min n=80p<0.05).AAM&ATO group (8.17±0.86μmol/L n=7) more decreases in MDA levels compared with control-operated animals (p<0.01). Right nephrectomy alone (sham operation) did not decrease MDA levels. AOPPs levels were increases in renal function were ameliorated by AAM and ATO treatment, which demonstrated decreases in AOPPs levels24h after surgery compared with control-operated animals. the AOPPs of administered AAM、ATO、AAM&ATO groups down5%,3%,10%to the control operation group.4. AAM and ATO can inhibit the level of the inflammatory reaction in the course of renal ischemia-reperfusion injury dose-dependently.After ischemia-reperfusion injury, the presentation of the ICAM-1and MCP-1is strengthened. After24h AAM、 ATO、AAM&ATO groups of the positive staining of ICAM-1and MCP-1in renal tissue are weaker than the control operation group, and the level of the presentation of ICAM-1and MCP-1mRNA in the renal tissue are als lower than the control operation group. The ICAM-1mRNA of administered AAM、ATO. AAM&ATO groups down6%,13%,16%to the control operation group. The MCP-1mRNA of administered AAM. ATO. AAM&ATO groups down18%,8%,24%to the control operation group.II. The protective effect as well as the mechanism of AAM and ATO to renal tubular epithelial cells which through the hypoxia-reoxygenation course;1. AAM and ATO can inhibit the level of the oxidative stress of HK2cells in the course of hypoxia-reoxygenation dose-dependently. The results of ROS show that the level of the oxidative stress increased significantly after the course of hypoxia-reoxygenation (p<0.05), AMM and ATO can inhibit the level of the oxidative stress of HK2cells. The ROS of administered AAM. ATO、AAM&ATO groups down11%,10%,31%to the I/R group. AAM&ATO group was more significantly decreased the level of ROS and the effect of AAM and ATO mayhave some interaction (p<0.05).2. AMM and ATO can inhibit the level of the ICAM-1of HK2cells in the course of hypoxia-reoxygenation dose-dependently. The results of ELISA show that IR group increase in ICAM-1compared with the normal control group (0.19±0.02) ng/ml, and AAM、ATO. AAM&ATO groups were decreased significantly, compared with the IR group (0.28±0.05ng/ml.0.22±0.03ng/ml.0.21±0.02ng/ml vs0.59±0.06ng/ml; p<0.05). AAM&ATO group was more significantly decreased the level of ICAM-1and the effect of AAM and ATO may have some interaction (p<0.05).3. AMM and ATO can inhibit the level of the ICAM-1mRNA of HK2cells in the course of hypoxia-reoxygenation dose-dependently. The results of RT-PCR show that IR group increase ICAM-1mRNA compared with the normal control group(0.16±0.01)%, and AAM、ATO. AAM&ATO groups were decreased significantly, compared with the IR group ((0.20±0.01)%.(0.18±0.01)%.(0.15±0.01)%vs(0.27 ±0.02)%; p<0.05).4. AMM and ATO can inhibit the level of the MCP-1of HK2cells in the course of hypoxia-reoxygenation dose-dependently. The results of ELISA show that IR group increase in MCP-lcompared with the normal control group (1.92±0.83) ng/ml, and AAM、ATO、AAM&ATO groups were decreased significantly, compared with the IR group (2.04±0.79ng/ml、1.92±0.92ng/ml、1.82±1.04ng/ml vs4.66±0.29ng/ml; p<0.01). AAM&ATO group was more significantly decreased the level of MCP-1and the effect of AAM and ATO may have some interaction (p<0.05).5. AMM and ATO can inhibit the level of the MCP-1mRNA of HK2cells in the course of hypoxia-reoxygenation dose-dependently. The results of RT-PCR show that IR group increase MCP-1mRNA compared with the normal control group(0.14±0.01)%, and AAM、ATO、AAM&ATO groups were decreased significantly, compared with the IR group ((0.14±0.01)%、(0.16±0.01)%、(0.11±0.01)%%vs(0.26±0.02)%; p<0.01).CONCLUSIONS1. AMM and ATO have the protective effect to the renal ischemia-reperfusion injury and the effect of combination therapy significantly.2. The mechanism of the protection to the renal ischemia-reperfusion injury of the AMM and ATO may be relative to its inhibition to the oxidative stress and the inflammatory reation.3. AMM and ATO has the protective effect to the renal tubular epithelial cells which through the hypoxia-reoxygenation course and the effect of combination therapy significantly.4. The mechanism of the protection of AAM and ATO to the renal tubular epithelial cells which through the hypoxia-reoxygenation may be relative to its inhibition to the oxidative stress and the inflammatory reation. |
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