Abstract:
Objective:To investigate the protection mechanism of hydrogenrich saline (HRS) on renal injuries of severe acute pancreatitis (SAP) in rats.
Methods:The experimental study was adopted. Seventytwo Wistar rats were divided into 3 groups by random number table: sham operation group (SO group), SAP model group (SAP group) and HRS treatment group (HRS group) with 24 rats in each group. Acute pancreatitis model was induced by retrograde infusion of 5% sodium taurocholate into the biliopancreatic duct. Rats in the HRS group were administered as a tail intravenously (6 mL/kg) and compensated subcutaneously (20 mL/kg) with HRS at 5 minutes after successful modeling. These rats in the SO group and SAP group were administered with an equivalent volume of normal saline at 5 minutes after sham surgery or successful modeling. All the rats were sacrificed at 3, 12 and 24 hours after the operation (n=8 per group). (1) The levels of serum Chromium (Cr), blood urea nitrogen (BUN), IL1β, IL-6 and renal myeloperoxidase (MPO) were detected. (2) To evaluate the pathological grade of renal injuries. (3) The expressions of 3Nitrotyrosine and NFκB in renal tissues were measured using immunohistochemistry. (4) The expressions of IκB and TNFα were measured using Western blot. Measurement data with normal distribution were presented as
±s. The comparisons of multisample means were analyzed by the repeated measures ANOVA, and comparison between groups was done by the t test. Count data were analyzed using the chisquare test.
Results:(1) The levels of serum Cr in the SO, SAP and HRS groups were respectively (30±6)μmol/L, (51±8)μmol/L , (46±5)μmol/L at postoperative 3 hours and (32± 6)μmol/L, (80±10)μmol/L, (42±7)μmol/L at postoperative 12 hours and (28±5)μmol/L, (100± 11)μmol/L, (58±12)μmol/L at postoperative 24 hours. The levels of serum BUN in the SO, SAP and HRS groups were respectively (5.1±0.6)mmol/L, (9.0±1.1)mmol/L, (8.1±1.3)mmol/L at postoperative 3 hours and (5.0±0.7)mmol/L, (14.9±2.9)mmol/L, (8.4±1.2)mmol/L at postoperative 12 hours and (4.6±1.0)mmol/L, (22.6±1.9)mmol/L, (13.7±2.6)mmol/L at postoperative 24 hours. The levels of serum IL1β in the SO, SAP and HRS groups were respectively (71±9)pg/mL, (192±11)pg/mL, (126± 19)pg/mL at postoperative 3 hours and (67±11)pg/mL, (279±18)pg/mL, (170±16)pg/mL at postoperative 12 hours and (72±12)pg/mL, (322±24)pg/mL, (203±30)pg/mL at postoperative 24 hours. The levels of serum IL-6 in the SO, SAP and HRS groups were respectively (121±9)pg/mL, (249±26)pg/mL, (153± 16)pg/mL at postoperative 3 hours and (114±19)pg/mL, (408±23)pg/mL, (252±21)pg/mL at postoperative 12 hours and (118±10)pg/mL, (452±29)pg/mL, (285±26)pg/mL at postoperative 24 hours. The levels of MPO in renal tissues in the SO, SAP and HRS groups were respectively (0.44±0.04)U/g, (0.79±0.08)U/g, (0.56±0.05)U/g at postoperative 3 hours and (0.49±0.07)U/g, (1.45±0.10)U/g, (0.84±0.06)U/g at postoperative 12 hours and (0.48±0.07)U/g, (1.85±0.20)U/g, (1.21±0.05)U/g at postoperative 24 hours. There were statistically significant differences in the levels of serum Cr, BUN, IL1β, IL-6 and renal MPO among the 3 groups at postoperative 3, 12, 24 hours (F=23.013, 84.858, 109.080, 32.830, 59.338, 205.494, 156.462, 384.787, 231.659, 105.129, 309.131, 413.937, 72.305, 658.698, 237.924, P<0.05). ① There were statistically significant differences in the level of serum IL1β at postoperative 3, 12, 24 hours between the SAP group and SO group (t=24.500, 28.140, 26.150, P<0.05) and between the HRS group and SO group (t=7.399, 15.000, 11.470, P<0.05) and between the HRS group and SAP group (t=8.519, 12.620, 8.570, P<0.05). ② There were statistically significant differences in the level of serum IL-6 at postoperative 3, 12, 24 hours between the SAP group and SO group (t=24.080, 28.425, 26.352, P<0.05) and between the HRS group and SO group (t=4.930, 11.086, 16.956, P<0.05) and between the HRS group and SAP group (t=8.811, 14.370, 12.220, P<0.05). ③ There were statistically significant differences in the level of MPO in renal tissues at postoperative 3, 12, 24 hours between the SAP group and SO group (t=11.070, 22.240, 18.290, P<0.05) and between the HRS group and SO group (t=5.301, 10.738, 24.000, P<0.05) and between the HRS group and SAP group (t=6.895, 14.790, 8.781, P<0.05). (2) The number of rats with pathological grade 0, Ⅰ, Ⅱ and Ⅲ of renal injuries were 8, 0, 0, 0 in the SO group and 0, 0, 3, 5 in the SAP group and 0, 2, 6, 0 in the HRS group, with a statistically significant difference among the 3 groups (χ
2=19.957, P<0.05), and with statistically significant differences between SAP or HRS group and SO group (χ
2=13.472, 13.714, P<0.05) and between HRS group and SAP group (χ
2=7.361, P<0.05). (3) The immunohistochemical results showed that the positive expression of 3Nitrotyrosine of the renal tissues in the SO, SAP and HRS groups were respectively 0.001 0±0.000 3, 0.053 0±0.012 0 and 0.015 0±0.005 0, with a statistically significant difference among the 3 groups (F=128.452, P<0.05), and with statistically significant differences between SAP or HRS group and SO group (t=13.699, 8.838, P<0.05) and between HRS group and SAP group (t=9.244, P<0.05). The positive expression of NFκB of renal tissues in the SO, SAP and HRS groups were respectively 0.003 0±0.001 5, 0.491 0±0.108 0 and 0.170 0±0.042 0, with a significant difference among the 3 groups (F=138.726, P<0.05), and with statistically significant differences between SAP or HRS group and SO group (t=14.367, 12.769, P<0.05) and between HRS group and SAP group (t=8.760, P<0.05). (4) The Western blot results showed that the relative expressions of IκB and TNFα in the SO, SAP and HRS groups were respectively 0.816±0.153, 0.030±0.009, 0.404±0.090 and 0.124±0.033, 0.959±0.113, 0.523±0.094, with statistically significant differences in the relative expressions of IκB and TNFα among the 3 groups (F=44.037, 69.172, P<0.05). There were statistically significant differences in the relative expressions of IκB between SAP or HRS group and SO group (t=8.883, 4.020, P<0.05) and between the HRS group and SAP group (t=7.162, P<0.05). There were statistically significant differences in the relative expressions of TNFα between SAP or HRS group and SO group (t=12.286, 6.937, P<0.05) and between the HRS group and SAP group (t=5.138, P<0.05).
Conclusion:The protection mechanism of HRS may be related to scavenging free radicals, affecting on IκB nitration, inhibiting the activation of NFκB as well as reducing the production of inflammatory cytokines.
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