Regulation Of HO-1 Expression By Keap1/Nrf2 Signaling Pathway To Explore The Mechanism Of Ginkgo Biloba Extract On Pulmonary Fibrosis In Rats

Objective: In order to further study the effect of Ginkgo biloba extract on the intervention of bomycin induced pulmonary fibrosis in rats,observe its effect on the expression of Nrf2 and HO-1 protein,and explore the mechanism of pulmonary fibrosis delay and antioxidant action of ginkgo biloba extract,and provide some experimental basis for the clinical treatment of pulmonary fibrosis treated by ginkgo biloba extract.Methods: According to random number method,60 healthy male SPF SD rats were equally divided into 4 groups: blank control group,bleomycin group(model group),pirfenidone group and GBE group(Ginkgo biloba extract group),with 15 rats in each group.After 7 days of adaptive feeding,all60 rats were modeled.The blank control group was given a single intratracheal injection of 0.9% sodium chloride solution(Na Cl solution)(5mg/kg),and the other groups were given a single intratracheal injection of bleomycin(BLM)(5mg/kg).The modeling day was the 0 day of recording,and on the 14 th day after modeling,one rat was randomly selected for death in each group,and lung tissue was removed to confirm whether the modeling was successful.On the 15 th day of modeling,drug intragastric administration was started,0.9% sodium chloride solution(Na Cl solution)was administered to blank control group and model group,and pirfenidone group was administered intragastric administration.Ginkgo biloba group was given ginkgo biloba extract by gavage once a day for 28 days.On the 14 th and 28 th day after intragastric administration,6 mice were selected from each group and sacrificed after fasting for 24 hours.After the lung tissue was removed,the right lung of the rats was fixed and embedded,and hematoxylin-eosin(HE)and Masson staining were performed to observe the lung tissue of the rats and evaluate the pulmonary fibrosis of the lung tissue of the rats,and the degree of pulmonary fibrosis of the rats in each group was evaluated by Szapiel score.After the left lung was treated,the expression of Nrf2,HO-1 and other proteins in lung tissues was detected by Western Blotting(Westernblot).Results: 1.General situation of rats: Compared with blank control group,the other three groups of mice had different degrees of mental malaise.During the whole experiment,the rats in the blank control group had good mental state,smooth skin and hair,bright color,stable breathing,good appetite,sensitive to external stimuli,obvious weight gain compared with other groups,and no death in the whole experiment.In the model group,the mental state of rats was poor,the skin and hair color was dull,the breathing was short of breath,the appetite was poor,the external stimulation was dull,the claws were purple and dark,and the body weight was decreased obviously.Pirfenidone group and GBE group: after modeling,the spirit,fur color,respiration,appetite and body reaction of rats in the two groups were similar to those in the model group,and the above conditions were slightly improved on the 14 th day after the administration of corresponding drugs.On the 28 th day,the condition was significantly improved compared with the 14 th day,and all aspects were better improved,but compared with the blank control group,all aspects were slightly inferior.A total of 5 rats died during the whole experiment,including 1 in the blank control group,2 in the pirfenidone group and 1 in the GBE group.2.HE staining results: In the blank control group,the lung tissue structure was clear and no obvious abnormality was observed on the 14 th and 28 th days after modeling,and no inflammation was observed in the alveolar.In the model group,on the 14 th day,collagen deposition,structural destruction,alveolar wall thinning,alveolar space thickening,and inflammatory cell infiltration were observed in the lung tissues of rats.On day 28,the lung tissue of rats showed obvious collagen deposition,severe structural destruction,significant thickening of alveolar septum and extensive infiltration of inflammatory cells in the alveolar cavity.Pirfenidone group and GBE group:on day 14 and 28,the collagen deposition,the thickness of alveolar wall interval and the degree of inflammatory cell infiltration in lung tissue were smaller than those in the model group.Compared with blank control group,the scores of alveolar inflammation in model group,pirfenidone group and GBE group at day 14 and day 28 were significantly increased(P < 0.05);Compared with model group,the scores of pulmonary alveolitis in pirfenidone group and GBE group on day 14 and 28 were significantly decreased(P<0.05);Compared with pirfenidone group,GBE group scores were higher on day 14 and 28,with no statistical significance(P>0.05).3.Masson staining results: On the 14 th and 28 th days after modeling,there was no collagen fiber hyperplasia in the lung tissue of the blank control group,and the lung tissue structure was normal,with no obvious blue staining.On the 14 th day of the model group,there was no significant collagen fiber hyperplasia,no obvious damage to lung parenchyma structure,no significant cyanosis,and small area.On the 28 th day,pulmonary tissue fibrosis was serious,lung tissue structure was disordered,the blue area was large,the staining was obvious;In pirfenidone group and GBE group,there was a little collagen fiber hyperplasia on day 14,and the damage of lung parenchymal structure was not obvious.On day 28,blue collagen fibers and structural damage were visible in lung tissue,and the blue-staining area and color depth were smaller than those in the same period model group.Compared with blank control group,pulmonary fibrosis scores in model groups,pirfenidone groups and GBE groups were increased at day 14 and day 28(P<0.05);Comparison to the model group,the scores of pulmonary alveolitis in pirfenidone group and GBE group on day 14 and 28 were decreased(P<0.05);Compared with pirfenidone group,GBE group scores were lower on day 14 and 28,and the difference was statistically significant(P<0.05).4.westernblot was used to detect:(1)Nrf2 protein expression: compared with blank control group,Nrf2 protein expression level in model group,pirfenidone group and GBE group was significantly increased on day 14 and day 28(P<0.0001),but there was no statistical significance between pirfenidone group and GBE group on day 14(P>0.05).Compared with model group,Nrf2 protein expression level in pirfenidone group and GBE group was significantly increased on day 14 and day 28(P<0.0001);Compared with pirfenidone group,there was no statistical significance in GBE group at the 14days(P>0.05),and Nrf2 protein expression level was higher at the 28 days(P<0.05).(2)HO-1 protein expression:Compared with blank control group,HO-1 protein expression level in model group,pirfenidone group and GBE group on day 14 and day 28 was significantly increased(P<0.0001),and the difference between pirfenidone group and GBE group on day 14 was statistically significant(P<0.0001).Comparison to the model group,HO-1protein expression in pirfenidone group and GBE group was significantly increased on day 14 and day 28(P<0.0001);Compared with pirfenidone group,there was no statistical significance in GBE group on day 28(P>0.05),and HO-1 protein expression was increased on day 28(P<0.0001).(3).Fibrosis indexes: Comparison to the blank control group,the protein expression level of CollageⅠin model group increased on day 14 and day 28(P<0.01),but there was no statistical difference between pirfenidone group and GBE group on day 14 and day 28(P>0.05).Compared with model group,the expression level of Coll I protein in pirfenidone group and GBE group was significantly decreased on day 14 and day 28(P<0.001);Compared with pirfenidone group,there was no significant difference in GBE group on day 14 and day 28(P>0.05).Collagen III:Compared with blank control group,the protein expression level of Collagen III in model group and GBE group was significantly increased on day 14 and day 28(P<0.01),but there was no statistical difference in pirfenidone group on day 28(P>0.05).Compared with model group,the protein expression level of CollagenⅢ in pirfenidone group and GBE group on day 14 and day 28 was significantly decreased(P<0.01).Comparison to the pirfenidone group,there was no statistical significance in GBE group at day 14 and day 28(P>0.05).Conclusion(s): 1.Ginkgo biloba extract can improve the pulmonary fibrosis induced by bleomycin in rats,thus playing an anti-fibrosis role;2.By activating Nrf2/HO-1 signaling pathway,Ginkgo biloba extract can improve the antioxidant capacity of the body,reduce the level of lung oxidative stress,and alleviate lung injury in PF model mice.