| Background : Acute respiratory disease syndrome(ARDS)is a serious cause of infection,trauma,shock and other causes of alveolar epithelial cells and capillary endothelial cell damage,leading to acute respiratory failure syndrome.In recent years,the pathogenesis and diagnostic criteria of ARDS have been studied and updated continuously.The incidence rate of ARDS has been lower than that of ARDS.However,clinical investigation shows that the mortality rate is high and still lacks effective treatment.Therefore,it is the focus of the present study to explore the pathogenesis of ARDS and to seek intervention.ARDS pathogenesis is complex,inflammatory response is its essence.Nuclear factor kappaB(NF-κB)is the most important nuclear transcription factor in the cell,plays a vital role in the regulation of transcriptional regulation mediated by various inflammatory stimuli.NF-κB channel activation is closely related to the occurrence and development of ARDS.Pre-B cell colony-enhancing factor(PBEF)is a cytokine with multiple physiological functions,which is involved in cell metabolism,inflammatory response and immune regulation.It is found that PBEF can promote the alveolar epithelial cell injury,pulmonary microvascular endothelial injury,increased alveolar permeability involved in the pathophysiological process of ARDS,and inhibition of PBEF activity can reduce the ARDS inflammatory response.The study shows that PBEF and NF-κB can be used as a target for the study of the pathogenesis of ARDS.In view of the relationship between PBEF and NF-κB,there are some exploratory study on the inflammatory response of umbilical vein endothelial cells and pulmonary microvascular endothelial cells.It is unclear whether PBEF and NF-κB have the specific relationship in alveolar epithelial cell inflammatory response.Tetramethylpyrazine(Tetramethylpyrazine,TMP)is an active component extracted from Chuanxiong root of traditional Chinese medicine.It has the role of calcium channel antagonist and is widely used in cardiovascular and cerebrovascular diseases.Studies have found that TMP can inhibit alveolar macrophages NF-κB activation of rabbit hemorrhagic shock combined with endotoxin-induced acute lung injury;our previous experiments found that oleic acid-induced rat ARDS model,TMP can reduce the PBEF,etc.Inflammatory factor expression,play a role in lung protection.However,the mechanism of TMP in ARDS inflammatory response is not clear at prensent.Purpose:(1)To culture the human type II alveolar epithelial cells(A549 cells,derived from human lung adenocarcinoma)in vitro,the effects of LPS and TMP on the proliferation of A549 cells were observed.The LPS and TMP concentrations selected in the next experiment were screened.(2)To investigate the changes of PBEF and other inflammatory factors after LPS stimulation in A549 cells.(3)To investigate the association of PBEF with other inflammatory factors and the association with NF-κB.(4)To investigate the protective effect and mechanism of TMP on LPS-induced inflammation of human type II alveolar epithelial cells.Method:(1)To culture the human type II alveolar epithelial cells(A549 cells,derived from human lung adenocarcinoma)in vitro,MTT assay was used to detect the effect of LPS and TMP on the proliferation of A549 cells,The optimal concentration of LPS and the safe concentration range of TMP Acting on A549 cells was screened.(2)To set up the normal control group(Control),LPS group,LPS + TMP group and LPS + FK866 group.LPS(final concentration of 5mg / L)stimulated in A549 cells 6h,12 h and 24 h after the establishment of inflammatory model,respectively,by adding TMP(final concentration of 10 mg / L)and PBEF inhibitor FK866(final concentration of 10 nmol / L)intervention,q-PCR and(IL-1β),interleukin-8(IL-8),interleukin-8(IL-8)and interleukin-8(IL-8),PBEF mRNA and protein expression levels were detected by Western Blot.(3)Western Blot was used to detect the dynamic changes of phosphorylated P65 protein in the nucleus and cytoplasm of three time periods to reflect the activation of NF-κB.Result:(1)Effects of LPS on proliferation of A549 cellsThere was no significant difference in the inhibitory rate(IR)between 5mg/L and 10 mg /L,20mg/L and 50mg/L experimental group at the same time point(P>0.05).There was significant difference between any two groups Apart from these(P<0.05).There was no significant difference in IR between 1mg / L LPS at 6h,12 h and 24h(P>0.05).There was significant difference between the two groups in the other experimental groups were at different time points(P <0.05).(2)Effect of TMP on the proliferation of A549 cellsAt the same time point,10 mg / L and 50 mg / L of TMP on A549 cells(survival rate,SR)difference was not statistically significant(P>0.05),There was no significant difference between 200mg/L and 300mg/L experimental group at 6h(P>0.05),The difference between the other experimental groups was statistically significant at the same time(P <0.05).At different time points,10mg/L and 50mg/L experimental group difference was not statistically significant(P> 0.05),200 mg/L and 300 mg /L in the experimental group at 12 h and 24 h,the difference was not statistically significant(P> 0.05),The difference between the other experimental groups at any time point was statistically significant(P <0.05).(3)Effects of drug intervention on the expression of TNF-a,IL-1β,IL-8 and PBEF mRNAThe expression of TNF-a,IL-1β,IL-8 and PBEF mRNA in LPS-stimulated A549 cells was significantly higher than that in the control group(P<0.001),and the LPS group increased significantly(P<0.001)<0.001).The expression of TNF-a,IL-1β and IL-8 mRNA in FK866 group was lower than that in LPS group(P<0.05).The expression of TNF-a,IL-1β,IL-8 and PBEF mRNA in TPS group was lower than that in LPS group(P <0.05).(4)Effects of drug intervention on the expression of TNF-a,IL-1β,IL-8 and PBEFThe expression of TNF-a,IL-1β,IL-8 and PBEF protein in A549 cells was higher than that in control group(P<0.05).The expression of TNF-a,IL-1β,IL-8 and PBEF increased with the prolongation of time And 24 h and 6 h were statistically significant(P<0.05).The levels of TNF-a,IL-1β and IL-8 protein in the LPS group were significantly lower than those in the control group(P<0.05),and the levels of TNF-a,IL-1β,IL-8 and PBEF The expression of LPS group was lower than that of LPS group(P<0.05).(5)Effects of drug intervention on NF-KB activityThe expression of phosphorylated P65 protein in A549 cells was significantly higher than that in the control group(P<0.001),and the difference between the two groups was statistically significant(P<0.001).The phosphorylated P65 protein in FK866 group was significantly lower than that in LPS group(P<0.001).The above indexes were also lower than those of LPS group(P<0.01)after TMP intervention.The expression of phosphorylated P65 protein in A549 cells was significantly higher than that in the control group(P<0.001).The phosphorylated P65 protein began to decrease with the prolongation of the time,and the difference between the two groups was statistically significant(P<0.05).The difference of P65 protein between FK866 group and FK866 group was significantly lower than that of LPS group(P<0.05).The levels of cytoplasmic phosphorylated P65 protein in TMP group were also lower than those in LPS group at the same time(P<0.05),and there was difference between TMP group at 6h and 24h(P<0.05).Conclusions:(1)LPS inhibited the proliferation of A549 cells in a dose-dependent manner.(2)TMP had no effect on the proliferation of A549 cells at low concentration,and inhibited at 100 ~ 300 mg / L.(3)PBEF can regulate the expression and release of inflammatory cytokines TNF-a,IL-1β and IL-8 induced by LPS in A549 cells by NF-κB(4)TMP can decrease the expression of PBEF and the activity of NF-κB in LPS-induced A549 cells and decrease the expression and release of inflammatory factors TNF-a,IL-1β and IL-8.Therefore,TMP may reduce the expression of PBEF and inhibit the activity of NF-κB,thereby reducing the inflammatory response of type II alveolar epithelial cells and exerting the protection of ARDS. |
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