Inhibition Of STAT3 Pathway Activation By Ginkgo Biloba Extract On ARDS Mechanistic Studies Of Protective Effects

Acute respiratory distress syndrome（ARDS）is an acute respiratory failure syndrome caused by non-cardiogenic pulmonary edema.Clinically,ARDS is closely related to sepsis,and sepsis caused by negative bacterial infection is an important cause of ARDS.Lipopolysaccharide（LPS）is the main component of endotoxin,which participates in the cell wall of negative bacteria and has the ability to activate immune response.It is a powerful injurious agent to activate immune cells to secrete cytokines.LPS causes the release of various inflammatory cytokines and cell activation of TNF-α,IL-1,and IL-6 by binding to the receptor CDl4 on the membrane of monocytes and macrophages.Of these,TNF-αis the initiator and IL-6 is the proinflammatory factor that also enables signal transducers and activator of transcription（STAT）,the main stimulating factor for activation Recent studies have shown that the STAT3 signaling pathway is closely related to the body’s inflammatory and immune responses when ARDS occurs.By blocking STAT3 signaling pathway,it can inhibit the infiltration and activation of inflammatory cells in ARDS to some extent and can reduce lung tissue injury.Ginkgo biloba extract（EGB）,whose active components are mainly flavonoid glycosides and terpene lactones,has pharmacological effects such as anti-oxidation,scavenging free radicals,and anti-platelet activating factor.Studies have shown that EGB has the effect of inhibiting the IL-6/STAT3 pathway and has a therapeutic effect on inflammatory bowel disease and septic myocardial injury,while the effect and mechanism of action of EGB on ARDS have rarely been reported.This study was to investigate the protective effect of Ginkgo biloba extract inhibiting STAT3pathway activation on ARDS and its related mechanism.Objective To investigate the effect of Ginkgo biloba extract on ARDS patients,and to further study the protective effect of Ginkgo biloba extract on LPS-mediated ARDS mice and its mechanism.Method Part Ⅰ:Forty-four ARDS patients admitted to the emergency ICU of Liuzhou People’s Hospital from December 2019 to December 2020 were selected as the study subjects,i.e.Admit patients to ICU Day 1 served as the starting point for the study,and admission to the emergency ICU 28 was defined as discharge with improvement,discharge from the ICU,or death as the study endpoint.In The patients were randomly divided into two groups:（1）control group（n=22）treated with routine treatment only;（2）observation group（n=22）treated with normal saline 250 m L+Shuxuening（Ginkgo biloba extract）20m L/d ivgtt for 7-14d.Venous blood samples were collected from all patients on the 7th and 14th day after treatment on the first day of admission Blood Clear Tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）Level Blood gas analysis,blood oxygen partial pressure（Pa0₂）,oxygenation index（Pa0₂/Fi0₂）were measured,and SOFA score was performed according to various biochemical parameters of patients.Finally,clinical parameters such as survival rate,incidence of MODS,duration of mechanical ventilation,duration of vasoactive drugs,duration of antibiotic use,and length of ICU stay were compared between the two groups.Part II:Thirty-two mice were randomly divided into 4 groups（8mice in each group）:healthy control group（control group）,LPS modeling group（LPS group）,and EGB alone（EGB Group）,EGB intervention group（LPS+EGB group）.The wet-to-dry mass ratio of lung tissues in each group was compared,and the histopathological changes of lung tissues in each group were observed and pathologically scored using hematoxylin-eosin staining（HE）and light microscopy.Serum TNF-αand IL-6 levels were measured by enzyme-linked immunosorbent assay（ELISA）in each group of mice.Real-time quantitative polymerase chain reaction（RT-PCR）was used to detect the expression of TNF-αand IL-6 m RNA in the lung tissue of mice in each group.Immunohistochemistry（IHC）was used to detect the expression of STAT3 and p STAT3 protein in the lung tissues of mice in each group.The protein levels of STAT3 and p-STAT3 in the lung tissues of mice in each group were detected by Western blot.Results Part Ⅰ:There was no significant difference in venous serum TNF-αand IL-6 before treatment between the two groups（P>0.05）;after 1 week of treatment,venous serum TNF-αand IL-6 in the observation group were significantly lower than those before treatment,and the difference was significant（P<0.05）.Venous serum TNF-αand IL-6 in the observation group were lower than those in the control group,and the differences were significant（P<0.05）.After 2 weeks of treatment,the venous serum TNF-αand IL-6 in the control group were significantly lower than those before treatment,and the differences were significant（P<0.05）.However,there was no significant difference between the two groups（P>0.05）.There was no significant difference in oxygen partial pressure,oxygenation index,and SOFA score between the two groups before treatment（P>0.05）,and after 1 week of treatment,the oxygen partial pressure,oxygenation index,and SOFA score of the two groups were significantly better than those before treatment,and the difference was significant（P<0.05）.The oxygen partial pressure,oxygenation index and SOFA score in the observation group were superior to those in the control group,and the differences were significant（P<0.05）.After 2 weeks of treatment,the oxygen partial pressure,oxygenation index,and SOFA score of the two groups were significantly better than those before treatment,and the differences were significant（P<0.05）.However,there was no significant difference between the two groups（P>0.05）.The survival rate of the observation group was higher than that of the control group,and the mechanical ventilation time,vasoactive drug use time,antibiotic use time,and MODS incidence rate were lower than those of the control group,and the difference was significant（P<0.05）;the ICU length of stay was not significantly different（P>0.05）.Part II:After HE staining of lung tissues from mice in the EGB intervention group by paraffin sections,they were compared with those in the LPS mice Compare,the degree of lung histopathological injury was significantly alleviated compared with mice in the LPS group,the complete destruction of alveolar wall structure was alleviated,the degree of congestion and hemorrhage was alleviated compared with mice in the LPS group,and the infiltration of inflammatory cells in the pulmonary interstitium and alveolar space was reduced.The lung histopathological score in the LPS modeling group was significantly higher than that in the control group（P<0.01）,and compared with the LPS group,the lung histopathological score in the EGB intervention group was significantly lower（P<0.05）,and there was no statistically significant difference in the lung histopathological score between the EGB alone control group（P>0.05）.Compared with the control group,the wet/dry weight ratio was significantly increased in the LPS group（P<0.01）,there was no statistically significant difference compared with the EGB control group（P>0.05）,and the lung tissue W/D was significantly lower in the EGB intervention group than in the LPS group,and the difference was statistically significant（P<0.05）.AND Compared with the control group,the serum TNF-αand IL-6 levels and the m RNA expression level in the lung tissue of mice in the LPS group were significantly increased（P<0.01）.Serum TNF-αand IL-6 levels and m RNA expression levels in lung tissue of mice in the EGB group were not significantly different from those in the control group（P>0.05）.Compared with the LPS group,the serum TNF-αand IL-6 levels and the m RNA expression level in lung tissue were significantly lower in the EGB intervention group,and the differences were statistically significant（P<0.05）.The expression of STAT3and p-STAT3 protein in the lung tissue of mice in each group was detected by immunohistochemistry;the protein levels of STAT3 and p-STAT3 in the lung tissue of mice in each group were detected by Western blotting,and the results showed that there was no significant difference in the expression and protein levels of STAT3 protein in the lung tissue of mice in each group（P>0.05）.Compared with the control group,the expression and protein level of p-STAT3protein in the lung tissue of mice in the LPS group were significantly up-regulated（P<0.01）;compared with the control group treated with EGB alone,the expression and protein level of p-STAT3 protein in the lung tissue of mice were not significantly different（P>0.05）;compared with the LPS group treated with EGB,the expression and protein level of p-STAT3 protein were significantly lower,and the difference was statistically significant（P<0.01）.Conclusion 1.Ginkgo biloba extract can reduce the serum levels of TNF-αand IL-6 in ARDS patients,improve the survival rate of ARDS patients,reduce the incidence of MODS,shorten the duration of mechanical ventilation and vasoactive drug use,and have a certain protective therapeutic effect on ARDS patients.2.EGB can be reduced Mouse ARDS model Serum IL-6 and TNF-αlevels,inhibit the m RNA expression of inflammatory cytokines IL-6 and TNF-αin lung tissue;and improve the degree of pathological lesions in lung tissue.On the other hand,EGB inhibited STAT3 phosphorylation levels in the lung tissue of ARDS in mice,suggesting that EGB alleviates lung injury in a mouse ARDS model related to inhibition of STAT3 signaling pathway activity.