Rock Inhibitor Fasudil In Neonatal Rats With Hyperoxia-induced Lung Fibrosis And The Mechanism Research

PART1The establishment of hyperoxia-induced lung injury inneonatal rats modelObjective: Establish an animal model of hyperoxia-induced lunginjury in neonatal rats，to lay the foundation for the subsequent Rockinhibitor fasudil(FAS) intervention experiments and mechanism research.Method: According to the random number tables, forty-eightSprague-Dawley neonatal rats were randomly divided into air group andhyperoxia group. Hyperoxia group rats were placed in the animal oxygenchamber, so that the oxygen concentration>95%, CO2concentration <5%.Put the rats of air group in the same indoor air, the oxygen concentration is21%. Respectively, take the neonatal rats of two groups for lunghistopathology observation, radial alveolar count (RAC) and lunghydroxyproline (HYP) content by sample hydrolysis method at3d,7d,14dand21d.Results:①After hyperoxia-exposed3d and7d, pathological examination showed acute inflammatory reaction in the lung tissue ofhyperoxia group,it mainly manifested as pulmonary capillary dilatation,interstitial edema, inflammatory cell infiltration. After hyperoxia-exposed14d and21d,the inflammation of lung tissue is reduced and obvious fibrosis,it mainly for lung structural disorder, alveolar collapse fusion, septalthickening, a large number of collagen-like material deposition.②The RACwas significantly reduced at7d,14d and21d of Hyperoxia+FAS groupcompared with Hyperoxia+NS group.③The content of hydroxyproline inlung tissue was increased markedly of hyperoxia group compared with theair group at14d and21d，it was more obvious in21d.Conclusion: High concentration of95%oxygen leading to pulmonaryfibrosis and lung development stagnation in neonatal rats,it successfullyestablished a animal model of hyperoxia-induced lung injury in neonatal rats,to lay the foundation for the subsequent FAS intervention experiments. PART2Rock inhibitor fasudil and Rho/Rock signaling pathway inneonatal rats with hyperoxia-induced lung fibrosis and its mechanismObjective: To investigate the Rho kinase inhibitor fasudil(FAS) andRho/Rock signaling pathway in neonatal rats with hyperoxia-induced lungfibrosis and the mechanism research.Method: According to the random number tables, twenty-fourSprague-Dawley neonatal rats were randomly divided into4groups:air+NS(normal saline) group，air+FAS group,hyperoxia+NS group andhyperoxia+FAS group. The rats of two hyperoxia groups were placed in theanimal oxygen chamber, so that the oxygen concentration>95%, CO2concentration <5%. Put the rats of two air groups in the same indoor air, theoxygen concentration is21%. FAS intervention groups from the first day ofthe experiment, intraperitoneal injection of fasudil20mg/（kg·d）, once aday,non-FAS intervention groups injected with the same amount of normalsaline.After21d, the subjects were sacrificed. The changes of lunghistomorphology and radial alveolar count (RAC) were observed;hydroxyproline in the lung was detected by sample hydrolysis method; thedistribution of α-smooth muscle actin(α-SMA) protein was determined byimmunohistochemistry method; the expression of α-SMA，tissue growthfactor-β1（TGF-β1）and RockI,p-MYPT1in Rho/Rock signaling pathwaywere detected by Western blot. Results:①Pathological examination showed structural disorder andobvious fibrosis in lung tissue of Hyperoxia+NS group,and the sings ofimprovement in lung tissue of Hyperoxia+FAS group at21d.②The RAC ofhyperoxia+NS group was decreased obviously compared with air+NSgroup(P <0.05), the RAC of hyperoxia+FAS group was increased comparedwith hyperoxia+NS group after FAS intervention,but no significantdifference.③The content of hydroxyproline in lung tissue was increasedmarkedly of hyperoxia+NS group compared with the air+NS group, theywere significantly reduced of hyperoxia+FAS group compared withhyperoxia+NS group(P <0.05).④Compared with air+NS group, α-SMA andTGF-β1protein expression of hyperoxia+NS group wereup-regulated,however they were significantly down-regulated ofhyperoxia+FAS group compared with hyperoxia+NS group(P<0.05).⑤Compared with air+NS group, RockI protein showed a tendency toincrease in hyperoxia+NS group（P＞0.05）, p-MYPT1protein expressionwas significantly increased(P <0.05)；after FAS intervention, RockI proteinreduced a little（P＞0.05）,p-MYPT1protein expression of hyperoxia+FAS group were significantly reduced compared with hyperoxia+NSgroup（P＜0.05）.Conclusion: Rho kinase inhibitor Fasudil reducing the expression ofpro-fibrotic cytokine TGF-β1, inhibit the generation and accumulation ofmyofibroblast, it could have obvious therapeutic effects on hyperoxia-induced lung fibrosis, which may be related to the inhibition ofRho/Rock signaling pathway activation.