| Objective:In this study,the BuYang HuanWu Secoction Lyophilized powder(BYHWD-LP)and BuYang HuanWu Decoction Containing Serum(BYHWD-CS)was used as the test drug.Human non-small cell lung cancer cells(A549)and human lung fibroblasts(HFL1)were used as test cells.To investigate the molecular mechanism of intervening HMGB1-induced pulmonary fibrosis with BYHWD-LP and to compare the effect of BYHWD-CS and BYHWD-LP on HMGB1-induced pulmonary fibrosis.Methods:1.To detect the effect of different concentrations of BYHWD-LP on the proliferation of HFL1 and A549 cells with MTT assayFirstly,BYHWD-LP was Prepared using a freezing technique and sealed at-20℃.Reconstituted with ultrapure water and stored at℃after high temperature sterilization while used.MTT was dissolved in PBS to a concentration of 5 mg/ml under light conditions,filtered and stored at-20℃until use.A549 cells andHFL1 cells were divided into six groups:blank control group and BYHWD-LP group(the final volume of BYHWD-LP was 5%,10%,15%,20%,25%).The cells were seeded in96-well P lates at 37℃,5%CO2,saturated humidity.When cells fusion reached80%-90%,they were grouped according to the loading table(three replicates per group).Add serum-free medium to the 96-well plate to make the final volume of each well 200ul and culture for 24 hours.Protect from light by adding MTT solution with10ul per well,then culture for 4 hours according to the above conditions.Carefully discard the supernatant when the time comes.Add 100μl DMSO to each well for 10minutes under light protection.The absorbance was measured with a microplate reader at a wavelength of 492 nm after mixing.2.To detect the effect of BYHWD-LP and BYHWD-CS on CDc42 in HFL1 and A549 cells stimulated by HMGB1 with Western blotA549 cells andHFL1 cells were seeded in a 6-well plate at a density of 1 x 10~5/ml with each volume being 1 ml.The cells were divided into eight groups:blank control group,HMGB1inductiongroup(100ng/ml),positivecontrolML141group(hereinafter referred to ML141group),BYHWD-LP pre-culture and then HMGB1 stimulation group(the final volume of BYHWD-LP was 5%,10%,15%),20%BYHWD-CS,20%blank serum group.When the degree of cell fusion in a 6-well plate was observed to be 80%to 90%,cells were dealed with groups and three replicates were used in each group.Add serum-free medium to each well so that the final volume of each well is 15%when we have completed the processes.Remove each group of cells to be extracted from the incubator after 24 hours and remove the culture solution and wash twice with pre-cooled PBS.Add the Cell lysate(RIPA)to the cell pellet with 200 ul per well,and lysed on ice for 5 minutes.Collect cells in centrifuge tubes with cell scraper then centrifuge.To detect the effect of BYHWD-LP and BYHWD-CS on CDc42 in HFL1 and A549 cells stimulated by HMGB1 with Western blot.3.To detect the effect of BYHWD-LP and BYHWD-CS onRAGE and TNF-αin HFL1 and A549 cells stimulated by HMGB1 with ELISACell culture and grouping are the same as Western blot.The cells were seeded in a 6-well plate at a density of 1×10~5/ml.Add BYHWD-LP,BYHWD-CS,HMGB1,blank serum,and culture medium were in groups after the cell fusion reached 80%and culture 24 hours.Remove the 6-well plate from the incubator and discard the supernatant.Assemble it in the centrifuge tube and centrifuge.To detect the effect of BYHWD-LP and BYHWD-CS on RAGE and TNF-αin HFL1 and A549 cells stimulated by HMGB1 with ELISA.Result:1.The effects of different concentration of BYHWD-LP on the proliferation of HFL1 and A549 CellsCalculated by the formula according ro the results of MTT,we knowledged the IC50 of BYHWD-LP on HFL1 and A549 cells were 11%.Compared with the blank control group,20%and 25%significantly inhibited the proliferation of both cells(P<0.01).While the 5%,10%,15%BYHWD-LP group’s inhibition was unobvious(P>0.05).2.The effects of BYHWD-LP and BYHWD-CS on CDc42 in HFL1and A549 Cells Induced by HMGB12.1 The effects of BYHWD-LP on CDc42 in HFL1 and A549 Cells Induced by HMGB1A549:Compared with the blank control group,the expression of CDc42 was up-regulated in the HMGB1 group(P<0.05),while the expression of CDc42 was significantly down-regulated in the ML141 group(P<0.01).Compared with HMGB1group,the expression of CDC42 decreased in BYHWD-LP groups,5%,10%P<0.05;15%P<0.01.Compared with ML141 group,there was no significant difference in CDc42 expression in BYHWD-LP groups.HFL1:Compared with the blank control group,the expression of CDc42 was up-regulated in the HMGB1 group(P<0.01).Compared with HMGB1 group,the expression of CDC42 decreased in BYHWD-LPgroups,5%BYHWD-LP group P<0.01;10%BYHWD-LPgroup P<0.05.Compared with ML141 group,there was no significant difference in CDc42expression in BYHWD-LP groups.2.2 The effects of BYHWD-LP and BYHWD-CS on CDc42 in HFL1and A549 Cells Induced by HMGB1A549:Compared with the blank control group,the expression of CDc42 was up-regulated in the 20%blank serum group;Compared with the 20%blank serum group,the expression of CDc42 was down-regulated in the 20%BYHWD-CS group;Compared with HMGB1 group,the expression of CDC42 decreased in 20%BYHWD-CS group;Compared with the 20%BYHWD-CS group,the expression of CDC42 decreased in BYHWD-LP groups,and 15%BYHWD-LP group(P<0.05)。HFL1:Compared with the blank control group,the expression of CDc42 wasup-regulated in the 20%blank serum group;Compared with the 20%blank serum group,the expression of CDc42 was down-regulated in the 20%BYHWD-CS group;Compared with HMGB1 group,the expression of CDC42 decreased in 20%BYHWD-CS group;Compared with the 20%BYHWD-CS group,the expression of CDC42 decreased in BYHWD-LP groups,and 5%BYHWD-LP group(P<0.05)3.The effects of BYHWD-LP and BYHWD-CS on TNF-α,RAGE in HFL1 and A549 Cells Induced by HMGB13.1 The effects of BYHWD-LP on TNF-αin HFL1 and A549 Cells Induced by HMGB1A549:Compared with the blank control group,the expression of TNF-αwas up-regulated in the HMGB1 group(P<0.01),While the expression of TNF-αwas significantly down-regulated in the ML141 group(P<0.01);Compared with HMGB1group,the expression of TNF-αdecreased obviously in 10%,15%BYHWD-LP groups(P<0.01);Compared with ML141 group,10%,15%BYHWD-LP groups can indibit the expression of TNF-α,and 15%BYHWD-LP group(P<0.05).HFL1:Compared with the blank control group,the expression of TNF-αwas down-regulated in the HMGB1 group and in the ML141 group,The ML141 group P<0.01;Compared with HMGB1 group,the expression of TNF-αdecreased obviously in5%,10%,15%BYHWD-LPgroups(P<0.01)ComparedwithML141group,BYHWD-LP groups can indibit the expression of TNF-α(P<0.01).3.2 The effects of BYHWD-LP on RAGE in HFL1 and A549 Cells Induced by HMGB1.A549:Compared with the blank control group,the expression of RAGE in HMGB1grouPand BYHWD-CS grouP s was increased obviously(P<0.01);Compared with the HMGB1 group,the expression of RAGE was decreased obviously,5%BYHWD-CS group(P<0.05);10%,15%BYHWD-CS groups P<0.01.Compared with ML141 group,the expression of RAGE has no obvious difference in 5%,10%,15%BYHWD-CS groups.HFL1:Compared with the blank control group,the expression of RAGE in HMGB1 group and BYHWD-CS groups was increased obviously(P<0.01).Compared with the HMGB1 group,the expression of RAGE was decreased obviously,5%BYHWD-CS group(P<0.05).Compared with ML141group,theexpressionofRAGEhasnoobviousdifferencein5%,10%,15%BYHWD-CS groups.3.3 The effects of BYHWD-LP and BYHWD-CS on extracellular TNF-αof HFL1and A549 cells stimulated by HMGB1.A549:Compared with the blank control group,the expression of TNF-αover-expression in 20%blank serum group.Compared with the 20%blank serum group,the expression of TNF-αin 20%BYHWD-CS groups has decreased(P<0.05).ComparedwiththeHMGB1group,theexpressionofTNF-αin20%BYHWD-CS groups has decreased(P<0.05).Compared with the 20%BYHWD-CS,the expression of TNF-αhas decreased obviously(P<0.01)HFL1:Compared with the blank control group,the expression of TNF-αOver-expression in20%blank serum group.Compared with the 20%blank serum group,the expression of TNF-αin 20%BYHWD-CS has decreased(P<0.05).Compared with the HMGB1group,the expression of TNF-αin 20%BYHWD-CS groups has increased;Compared with the 20%BYHWD-CS,the expression of TNF-αin BYHWD-LP groups has decreased obviously(P<0.01).3.4 The effects of BYHWD-LP and BYHWD-CS on extracellular RAGE of HFL1 and A549 cells stimulated by HMGB1.A549:Compared with the blank control group,the expression of RAGE over-expression in 20%blank serum group;Compared with the 20%blank serum group,the expression of RAGE in 20%BYHWD-CS groups has decreased(P<0.05);Compared with the HMGB1 group,the expression of RAGE in 20%BYHWD-CS group has decreased,but P>0.05;Compared with the 20%BYHWD-CS group,theexpressionofRAGEinBYHWD-LPgroupshas decreased,10%,15%BYHWD-LPgroupsdecreasedRAGEobviously(P<0.05).HFL1:Compared with the blank control group,the expression of RAGE Over-expression in 20%blank serum group.Compared with the 20%blank serum group,the expression of RAGE in 20%BYHWD-CS group has no statistical significance due to decreased(P>0.05).Compared with the HMGB1 group,the expression of RAGE in 20%BYHWD-CS group has no statistical significance due toincreased.Compared with the 20%BYHWD-CS group,the expression of RAGE in 5%,10%BYHWD-LP groups has decreased and 5%BYHWD-LP P<0.05.Conclusion:1.BYHWD-LP can inhibit the develoP ment of IP F by inhibiting the HMGB1/RAGE/CDc42 signaling P athway in HFL1 and A549 cells.2.BYHWD-LP anti-P ulmonary fibrosis effect overall better than BYHWD-CS. |
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