Effect Of Fritillaria Cirrhosa D.Don On The Expression Of NLRP3 Inflammasome In Lung Of Mice With Acute Lung Injury

Objective: To observe effect of Fritillaria cirrhosa D.Don pretreatment and posttreatment on the lung tissue damage induced by lipopolysaccharide(LPS)in mice with acute lung injury(ALI),and investigate effect on the expression of NOD-like receptor protein 3(NOD-like receptor protein 3,NLRP3)inflammasome and interleukin-1β(IL-1β)in lung tissues.Methods:Part one: To investigate the effect of Fritillaria cirrhosa D.Don treated for different time on LPS-induced ALI in mice.1.Fritillaria cirrhosa D.Don pretreatment: 72 SPF male Kunming mice at 6-8 weeks were randomly divided into control group(CON),LPS group and LPS+ Fritillaria cirrhosa D.Don group(LPS+FA),24 mice in each group.The ALI model was established by intraperitoneal injection of LPS.Mice in the LPS+FA group were given225 mg/kg Fritillaria cirrhosa D.Don micro-powder for continuous intragastric administration,once a day,and the other two groups were not treated.Continuously observe changes in the general condition of mice.Two hours after the end of intragastric administration on the 3rd,5 and 7th days,8 mice were taken from each group.Except for the CON group,4 mg/kg LPS was injected intraperitoneally,and the mice were sacrificed 24 hours later,the lung tissues were collected.The histological changes were determined using H&E staining,while NLRP3 and IL-1β level were determined using real-time PCR.2.Post-treatment: The experimental animals and their groups are the same as the pretreatment,namely the control group(CON,n=24),the LPS group(n=24)and the LPS+ Fritillaria cirrhosa D.Don group(LPS+FB,n=24).LPS+FB group and LPS group were injected intraperitoneally with LPS to establish ALI model.Two hours after modeling,LPS+FB group was given Fritillaria cirrhosa D.Don micro-powder(225mg/kg)by gavage,once a day,the other two groups were not treated.The mice were sacrificed on the 3rd,5th and 7th day,the lung tissues were taken to evaluate histological damage after H&E staining,and the expression of NLRP3 and IL-1β m RNA was determined by q PCR.Part two: To explore the effect of different doses of Fritillaria cirrhosa D.Don on LPSinduced ALI in mice.1.Pretreatment: Male Kunming mice grade 6-8 weeks were randomly divided into control group(CON),LPS group and LPS+ Fritillaria cirrhosa D.Don low,medium and high dose groups(LPS+ Fa-L,LPS+ Fa-M,LPS+ Fa-H group),8 animals in each group.LPS+ Fa-L,LPS+ Fa-M,LPS+ Fa-H group were given 56.25 mg/kg,112.5mg/kg,225 mg/kg Fritillaria cirrhosa D.Don micro-powder by intragastric administration,once a day,for 5 consecutive days,the other two groups were not treated.The general conditions of mice were continuously observed,and 2 hours after the end of intragastric administration on the 5th day,4 mg/kg LPS were injected intraperitoneally,24 hours later,the lung tissues were sacrificed to evaluate the histological damage by H&E staining.The expression of NLRP3 and IL-1β m RNA was detected by q PCR,and the protein expression of NLRP3 and IL-1β was detected by Western blot.2.Post-administration: Experimental animals and groupings are the same as pretreatment,namely control group(CON,n=8),LPS group(n=8)and low,medium and high dose treatment groups(LPS+ Fb-L,LPS+ Fb-M,LPS+ Fb-H,n=8).The difference is that in the LPS+ Fb treatment group,2 hours after intraperitoneal injection of LPS,then mice were given 56.25 mg/kg,112.5 mg/kg,and 225 mg/kg Fritillaria cirrhosa D.Don micro-powder to the stomach,once a day,for 5 consecutive days.The general conditions of mice were observed.The mice are sacrificed following 24 hours after the end of gastric administration on the 5th day and lung tissues were taken to evaluate the histological injury by H&E staining.q PCR was used to detect the expression of NLRP3 and IL-1β m RNA,and Western blot was used to detect the NLRP3,IL-1β protein.Results:Part one: To investigate the effect of Fritillaria cirrhosa D.Don treated for different time on LPS-induced ALI in mice.1.Pretreatment:(1)Compared with the CON group,Fritillaria cirrhosa D.Don micropowder 225 mg/kg was intragastrically administered for 3,5 and 7 days.There was no death and other toxic symptoms,such as weight loss,hypoactivity,tremor,convulsions,and diarrhea.(2)The ALI mouse model was successfully made.Compared with the CON group,the LPS group began to have symptoms of poisoning after 6 hours of intraperitoneal injection of LPS.Such as shortness of breath,unsteady walking,messy hair,reduced activity,and mixed intestinal mucosa after 12 hours Abnormal stools of the membranous material,reddish discharge at the corners of the eyes,shrinkage of eye clefts,and sluggish eyes.Under the microscope,obvious structural damage of lung tissue can be observed.Compared with the control group,the score of pathological damage was significantly increased(P<0.05).(3)After pretreatment with Fritillaria cirrhosa D.Don micro-powder(225 mg/kg)for 3,5and 7 days,there was no significant reduction in lung tissue pathological damage in ALI mice,and the damage score was not significantly reduced(P>0.05).(4)The results of q PCR showed that compared with the control group,the expression of NLRP3 and IL-1β m RNA in the lung tissue of the ALI mouse model was significantly increased(P<0.05).Compared with the LPS group,Except for the 5-day time point,the pretreatment of Fritillaria cirrhosa D.Don micropowder(225 mg/kg)at each time point could not significantly down-regulate NLRP3 and IL-1β m RNA in mouse lung tissues(P>0.05).2.Post-treatment: After ALI model mice were treated with Fritillaria cirrhosa D.Don micro-powder(225 mg/kg)for 3,5 and 7 consecutive days,the damage to the lung tissue structure of the mice was significantly improved,and the improvement in the group of 5 and 7 days was more significant.Compared with the corresponding LPS group,after 5 and 7 days of treatment with Fritillaria cirrhosa D.Don micro-powder(225 mg/kg),the lung tissue pathological damage score of ALI mice was significantly reduced,and the difference was statistically significant(P<0.05),and the decline is most obvious at 5 days.The results of q PCR showed that compared with the corresponding LPS group,the expression of NLRP3 and IL-1β m RNA in the lung tissue of mice with ALI decreased significantly after treatment with Fritillaria cirrhosa D.Don micro-powder(225 mg/kg)for 3 days.Part two: To explore the effect of different doses of Fritillaria cirrhosa D.Don on LPSinduced ALI in mice.1.Pre-treatment: Compared with the CON group,the lung tissue in mice showed obvious alveolar structure destruction after 24 hours of intraperitoneal injection of LPS.There were no significant changes in lung tissue injury degree and no significant difference in lung tissue injury score in ALI mice after pretreatment with high(225mg/kg),medium(112.5 mg/kg)and low(56.25 mg/kg)doses of Fritillaria cirrhosa D.Don micro-powder(P>0.05).q PCR and western blot showed that compared with the LPS group,the NLRP3、IL-1βm RNA in lung tissue of mice was significantly downregulated by each dose of Fritillaria cirrhosa D.Don micro-powder(P<0.05),while the expression of NLRP3 and IL-1β protein did not decrease significantly(P>0.05)2.Post-administration: ALI mice were treated with high(225 mg/kg),medium(112.5mg/kg),and low(56.25 mg/kg)doses of Fritillaria cirrhosa D.Don micro-powder,the pathological damage of lung tissue was obviously alleviated with medium and high dose.At the same time,both middle and high doses of Fritillaria cirrhosa D.Don micropowder treatment could significantly reduce the injury score,and the difference was statistically significant(P<0.05).q PCR and Western blot showed that compared with the corresponding LPS groups,the expressions of NLRP3 and IL-1β m RNA and protein in the lung tissues of mice in each dose of Fritillaria cirrhosa D.Don micro-powder treatment group were significantly down-regulated,and the difference was statistically significant(P<0.05);the expression of IL-1β protein was dose-dependently decreased.Conclusion: Preventive administration of Fritillaria cirrhosa D.Don cannot significantly reduce LPS induced lung pathological injury;the middle and high dose Fritillaria cirrhosa D.Don post-treatment can alleviate the ALI caused by LPS in mice after 5 days of intragastric administration,and its effect may relate to downregulation expression of NLRP3 and IL-1β in the lungs.