| Objectives : To investigate the effect of Hydrogen-Rich Saline Solution on the expression of P38 MAPK at renal ischemia-reperfusion injury by modeling the I/R injury in rats.Methods:2.1 24 Spargue-Dawley rats(3 month-old)were raised in the light period of constant12-hour lightness and 12-hour darkness,weighing 220 to 250 g,with free access to food and water.They were randomly divided into 4 groups: Sham Operating Group(SO group,n=6),Normal Saline Solution Group(NS group,n=6),Hydrogen-Rich Saline Solution qd Group(HRS qd group,n=6),and Hydrogen-Rich Saline Solution bid group(HRS bid group,n=6).Three days before experimentation,NS group was pretreated with normal saline solution lavage in a dose of 5ml/kg body weight,HRS qd group with hydrogen-rich saline solution qd lavage in ta dose of 5ml/Kg body weight,and HRS bid group with hydrogen-rich saline solution bid lavage in a dose of 5ml/Kg body weight.SO group was subjected to right renal resection and left renal pedicle mobilization to reveal the left kidney for one hour without I/R injury,and blood sampling and left kidney harvesting were done at 24 h after abdomen-closing.NS group,HRS qd group and HRS bid group were subjected to removal of right kidney,clamping of left kidney pedicle for one hour,blood sampling and left kidney harvesting at 24 h after abdomen closing and reperfusion.2.2 The blood urea nitrogen(BUN)and serum creatinine(Scr)were measured with automatic biochemistry analyzer.The serum content of Malondialdehyde(MDA)was determined with TBA methods.Renal morphologic changes were scored with Paller's criterion on hematoxylin and eosin(H&E)stained sections.Renal tubular cell apoptosis were detected with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling procedure(TUNEL).The staining of P38 MAPK and P-P38 MAPK was observedimmunohistochemically.The experimental results were analyzed statistically by means of SPSS17.0 software.All data were expressed by arithmetic average(The summation of all observed values are divided by the number of observation cases)± standard deviation.Results:3.1 Histological examination: After renal ischemia for one hour and reperfusion for24 hours,the renal morphological score was 2.82±1.25 in SO group,4.51±0.46 in HRSqd group,5.18±1.01 in HRSbid group,and 25.36±5.02 in NS group.There was obvious difference between each two groups of SO group,HRSqd group and NS group(P<0.05),and also significant difference between each two groups of SO group,HRSbid group and NS group(P<0.01).In addition,there is no statistically significant difference between HRSqd group and HRSbid group(P>0.05).3.2 Renal function: The serum creatinine level(?mol/L)was 33.25±8.25 in SO group,47.6±10.01 in HRSqd group,and 80.4±10.22 in NS group,with obvious difference between each two groups(P<0.05).Between each two groups of SO group,HRSbid group(50.4±7.27)and NS group,there was also obvious difference(P<0.01).There was no statistically significant difference between HRSsd group and HRSbid group regarding the serum creatinine level(P>0.05).The blood urea nitrogen(BUN)level(mmol/L)was5.92±0.21 in SO group,17.07±2.14 in HRSqd group,and 30.13±4.26 in NS group,with obvious difference between each two groups(P<0.05).There was also obvious difference between each two groups of SO group,HRSbid group(15.82±7.66)and NS group(P<0.01).There was no statistically significant difference between HRSqd group and HRSbid group regarding the blood urea nitrogen(BUN)level(P>0.05).The serum SOD level(?mol/(g.prot))was 40.5±6 in SO group,54.2±12.1 in HRSqd group,and 90.2±12.8in NS group,with obvious difference between each two groups(P<0.05).Between each two groups of SO group,HRSbid group(55±10.5)and NS group,there was also obvious difference(P<0.05).There was no statistically significant difference between HRSqd group and HRSbid group regarding the SOD level(P>0.05).It follows that the hydrogen-rich saline solution can effectively reduce renal dysfunction,and there is no obvious difference between HRSqd and HRSbid.3.3 The contents of MDA in plasma and tubular cell apoptosis: the MDA value in HRSqd group and HRSbid group were 5.84±0.81nmol/ml and 6.02±0.13nmol/ml respectively,significantly different from that in NS group(8.11±1.25nmol/ml)and SOgroup(3.04±0.42nmol/ml)(P<0.01).There was no statistically significant difference between HRSqd group and HRSbid group(P>0.05).At 24 h after renal ischemia reperfusion,the renal apoptosis score in HRSqd group and HRSbid group were6.41±1.26/HPF and 4.69±1.84/HPF respectively,significantly different from that in NS group(16.22±2.17/HPF)and SO group(1.83±0.17/HPF)(P<0.01).There was no statistically significant difference between HRSqd group and HRSbid group(P>0.05).3.4 Expression of P38 MAPK in groups: The expression of P38 MAPK in HRSqd group and HRSbid group were 1.74±0.81 and 6.02±0.13 respectively,significantly different from that in NS group(4.12±0.25)and SO group(0.63±0.42)(P<0.01),indicating that HRS is helpful in reducing the renal ischemia reperfusion injury.There was no statistically significantdifference between HRSqd group and HRSbid group(P>0.05),indicating there was no dose difference in the effect of reducing the renal tissue injury.3.5 Renal pathological scores compared between each two groups(P value): With NS group compared to SO group,P=0.00(<0.05),it indicates significant difference between these two groups,suggesting that NS group had renal tissue injury.With HRS group compared to SO group,P=0.06(>0.05),it indicates that HRS group had no obvious renal tissue injury.With HRS group compared to NS group,P=0.00(<0.05),it indicates that there was significant difference,suggesting that HRS obviously reduced the extent of renal tissue injury.With HRSqd group compared to HRSbid group,P=0.95(>0.05),it indicates that there was no dose difference in the effect of reducing the renal tissue injury.4.Conclusions:1.Renal ischemia/reperfusion injury may result in significant histological damage and renal dysfunction.The pretreatment of Hydrogen-Rich Saline lavage in rats results in lessening the oxidative damage and apoptosis,lowering the level of Scr,BUN and MDA,thereby protecting kidney from ischemia reperfusion injury.2.The pretreatment of Hydrogen-Rich Saline lavage in rats of HRSqd group and HRSbid group regulates and improves renal microenvironment by upregulating the expression of SOD in kidney tissue,and thereby promotes the tubular cell regeneration and remodeling of the injured tubular architecture,but there is no obvious difference in dose regarding the role of HRSqd and HRSbid.3.Normal renal tissue doesn't express P38 MAPK or expresses only small amount.The expression of P38 MAPK in NS group significantly increased in comparison with thatin SO group.It indicates that the ischemia reperfusion caused the activation of P38 MAPK in renal tissue.Both HRSqd and HRSbid can inhibit the over expression of P38 MAPK caused by renal ischemia reperfusion,and there is no obvious relationship between dose and effect of HRSqd and HRSbid.4.The pathological score of kidney tissue showed that the NS group had renal tissue injury,both HRSqd and HRSbid could obviously prevent renal ischemia reperfusion injury,but there was no obvious difference in dose between these two groups.Conclusion: The hydrogen-rich saline has definite protection effect in the renal ischemia reperfusion injury,and it's one of effective treatment methods for preventing and mitigating the renal ischemia reperfusion injury. |
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