Protective Effect Of Fasudil On Myocardial Injury In Rats With Acute Organophosphorus Pesticides Poisoning

Objective:To investigate the performance of myocardial injury and its mechanism in rats with acute organophosphate poisoning. To clarify the protection and damage mechanism on myocardial from Fasudil-a Rho/ROCK signaling pathway inhibitor.Methods:The acute organophosphorus toxicity model was established by peritoneal injection of dichlorvos. Rats were assigned to the following groups (n=8 each):the control group, the poisoned group, the high-dose fasudil pretreated group and the low-dose fasudil pretreated group. Four groups were all supported with breathing machine connected with the tube from incision of trachea. The control group was injected saline (0.4ml). The poisoned group was injected dichlorvos (60mg/kg 2LD50). The pretreated group was peritoneal injected fasudil (10mg/kg, 1mg/kg) 30 min before anesthesia. Record the electrocardiogram of LeadⅡ. Serum level of cardiac troponin T (cTnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured 6 hours later. Left ventricular cardiac tissue was observed though HE stain. Apoptotic cells were observed by TUNEL assay. The value of Bax and bcl-2 was determined by immunohistochemistry. The expressions of RhoA mRNA and ROCKI mRNA were analyzed by RT-PCR. Phosphorylated MYPT1 (myosin phosphatase target subunit 1) was analyzed by Western blot.Results:1. General conditionAll the rats receiving acute 60 mg kg-1 dose of dichlorvos develop dcholinergic signs (excess of secretions, tremor, fasciculations, respiratory distress, urinary and fecal incontinence). These findings began from the 5th-7th minitue and continued approximately 30-60 min after dichlorvos administration in rats. There were two deaths in the poisoned group and one death in the low-dose fasudil pretreated group. The symptom was lessened by the pretreatment of fasudil. There was no death in the control and the high-dose fasudil pretreated group. 2. Serum biochemical analysis①Serum level of cTnT (ng·ml-1)Cardiac troponin T obviously increased in poisoned group (0.12±0.04) more than the control group (0.01±0.00, P<0.05). High-dose fasudil pretreated group (0.04±0.02) significantly reduced the level of cTnT in serum compared with the control group (P<0.05). The change in the Low-dose fasudil pretreated group (0.13±0.07) was not obvious (P>0.05).②Serum level of NT-proBNP (pg·ml-1)Serum level of NT-proBNP obviously increased in the poisoned group (119.92±41.84) more than the control group (55.94±4.80, P<0.05). Two fasudil pretreated group significantly reduced the level of NT-proBNP compared with the control group (P<0.05). There was no difference between the High-dose (51.86±7.94) and the Low-dose pretreated group (60.35±5.58, P>0.05).3. Pathological changes of left ventricularHE staining of biopsy in rats showed that the control group was normal in myocardial. Poisoning of myocardial tissue was seen congestive heart tissue, inflammatory cell infiltration and necrosis. The myocardial tissue injury of the H-Fas group was obversed with a lesser extent. The structural of myocardial cells was integrity with a small amount of inflammatory cell infiltration and without large areas of congestion and necrosis. The extent of the L-Fas group’s lesion was between the two groups.4. Determination staining for cardiac apoptosis with TUNEL method in rat left heartThe AI value was more significantly increased in the poisoned group (25.60±3.83%) more than the control group (5.20±0.52%, P<0.05). The AI value was significantly decreased in the H-Fas group (15.14±2.61%) compared with the poisoned group (P<0.05). There was no difference between the Low-dose group (22.57±4.73%) and the poisoned group (P>0.05).5. Determination of left ventricular myocardial bax and bcl-2 valueIt was statistically significant (P<0.05) that the expression of bax and bcl-2 and bax/bcl-2 ratio were higher in the control group than those in the poisoned group (36.65±8.13 vs 17.97±2.91,20.27±5.93 vs 12.83±1.64,2.07±1.05 vs 1.43±0.38).The difference was also obviously between the pretreated groups and the poisoned group (P<0.05). The expression of bax and bax/bcl-2 ratio both in the H-Fas group (15.57±3.63 and 0.48±0.16) and L-Fas group (23.31±9.60 and 0.79±0.13) were lower than it in the poisoned group (36.65±8.13 and 2.07±1.05). While, the bcl-2 expression in the H-Fas group (34.18±6.88) and L-Fas (24.31±9.96) group were strongly higher than it in the poisoning group (20.27±5.93). The bax/bcl-2 ratio in H-Fas group was decreased more than in the L-Fas group, but the difference was not statistically significant (P>0.05).6. RT-PCR to detect RhoA mRNA and ROCKI mRNA①The expression of RhoA mRNAThe expression of RhoA mRNA in the poisoned group (1.48±0.25 vs 0.97±0.04) was significantly higher than it in the control group (P<0.05). The expression in the H-Fas group and L-Fas group (1.06±0.17 vsl.48±0.25,1.29±0.11 vs 1.48±0.25) were significantly lower than it in the poisoned group (P<0.05). It was stronger in the H-Fas group than in the L-Fas group (P<0.05).②The expression of ROCK ImRNACompared with the poisoned group, ROCK ImRNA expression in H-Fas group (1.14±0.19 vs 1.56±0.24) and L-Fas group (1.35±0.10 vs 1.56±0.24) were significantly inhibited (P<0.05), both of which in the H-Fas group is stronger than its inhibition of L-Fas group (P<0.05).7. Westem blot analysis of left heartPhosphorylated MYPT1 was measured to indicate the expression of Rho-Kinase. The P-MYPT1 protein electrophoretic bands with internal reference gray value of the ratio was detected. The expression was increased in the poisoned group (0.12±0.00) more than the control gr oup (0.09±0.00). Compared with poisoned group, the expression was not only decreased in the H-Fas group (0.09±0.01) but also in the L-Fas group (0.10±0.01). The degree of the H-Fas group was more significant. These differences were all statistically significant (P<0.05). CONCLUSIONS:1. Acute organophosphorus pesticides poisoning could cause myocardial injury on rats.2. The Rho/ROCK pathway may play an important role on myocardial injury in rats with acute organophosphorus pesticides poisoning. Fasudil may produce protective effect on myocardium with inhibit cardiac apoptosis therapy by reducing the ratio of bax/bcl-2 in AOPP rats.