| ObjectiveThis article is to observe the anti-inflammatory effects of Bushen-Qiangdu-Zhilv Decoction and its effective chemical components on LPS(lipopolysaccharide)induced RAW264.7(murine mononuclear macrophage)inflammatory cell model and their effect on the inflammatory axis of IL-23/Th17.MethodsThe HPLC(High Performance Liquid Chromatography)was used to separate the drug components of the water extract and the powder ultrasound solution of Bushen-Qiangdu-Zhilv Decoction.The chromatographic peak was preliminarily obtained.By comparing with the retention time of the standard peak,the components of the standard compounds were identified.Using LPS to act on RAW264.7 cells,induce inflammatory cell model;Effect of Bushen-Qiangdu-Zhilv Decoction and its chemical compositions on RAW264.7cell proliferation was observed by MTT;Observation of NO expression by Griess method;The mRNA expression level of IL-17,IL-23,ROR?t,TNF-a,IL-6 of every group was detected by RT-PCR(Reverse Transcription Polymerase Chain Reaction);Immunofluorescence intensity of IL-17 protein was detected by IF(Immunofluorescence).Results1.Three compounds of Ligustrazine,paeoniflorin and naringin were recognized in the water extract of Bushen-Qiangdu-Zhilv Decoction,and Bushen-Qiangdu-Zhilv Decoction powder ultrasonic liquid identify five compounds: osthole and ursolic acid,except Ligustrazine,paeoniflorin and naringin.2.The prescription of Bushen-Qiangdu-Zhilv Decoction(0-400ug/ml)interfered with 12 h and 24 h in RAW264.7 cells,and had no effect on cell proliferation,and there was no significant difference from that of blank group(Control)(P>0.05);Celecoxib inhibited the proliferation of RAW264.7 cells.The inhibition rate of 25uM[12h:(4.545 ± 7.872)%(P>0.05),24 h inhibition rate:(7.794 ± 6.677)%(P>0.05)] demarcation,with the increase of concentration,celecoxib showed a rapid increase in inhibition rate,to50uM[12h inhibition rate:(71.500 ±2.189)%(P<0.001),the inhibition rate of 24h(78.579 ± 0.088)%(P<0.001)],and the inhibition rate is over50%.Ursolic acid inhibited the proliferation of RAW264.7 cells.The inhibition rate was related to drug concentration and intervention time;The inhibition rate of ursolic acid was 2uM[12h inhibition rate:(30.207±6.053)%,24 h inhibition rate:(61.017±5.136)%] as boundary,with the increase of drug concentration and the prolongation of intervention time,the inhibition rate was increasing.Tetramethylpyrazine interfered with 12 h and 24 h in RAW264.7cells,and the drug concentration(0-10uM)had no inhibitory effect on cell growth,and there was no statistical difference compared with that of the control group(P>0.05);Osthole had inhibitory effect on the proliferation of RAW264.7 cells.At the time point of 12 h and 24 h,the concentration(0-7uM)had no inhibitory effect on cell growth.Compared with the control group,there was no statistical difference.But the inhibition rate of 8uM[12h inhibition rate:(47.579±1.327)%,24 h inhibition rate:(56.428±3.933)%] and above concentration to RAW264.7 cell proliferation was significantly inhibited.Compared with control group,the difference was statistically significant(P<0.001).Naringin had inhibitory effect on the proliferation of RAW264.7cells,the inhibition rate of 8uM[12h inhibition rate:(47.579 ±0.959)%,24 h inhibition rate:(40.803 ±0.926)%] and above concentration inhibited the proliferation of RAW264.7 cells,and the concentration of 8uM at time point12 h,compared with the Control group,the difference was statistically significant(P<0.05)and at the time point 24 h,The difference was statistically significant compared with that in Control group(P<0.01);Paeoniflorin(0-10uM)interfered with 12 h and 24 h in RAW264.7 cells,and had no effect on cell proliferation.Compared with Control group,the difference was not statistically significant(P>0.05).3.Compared with the blank group,the expression of NO in the cellsupernatant increased significantly(P<0.001)by LPS induced RAW264.7 cells,and the mRNA expression of IL-17,IL-23,ROR?t,TNF-?,and IL-6 increased significantly(P<0.001),and the immunofluorescent expression of the cell protein was significantly enhanced(P<0.001).4.Compared with the model group,the expression level of NO in cell supernatant was significantly decreased in high?medium and low dose group of Bushen-Qiangdu-Zhilv Decoction(P<0.001);Bushen-Qiangdu-Zhilv Decoction high?medium and low dose group can significantly reduce the mRNA expression of IL-17,ROR?t,TNF-? and IL-6,Compared with the model group,the difference has statistical significance(P<0.001),but for the expression of IL-23 mRNA,BSF low dose has a inhibitory effect.Compared with the model group,the difference has statistical significance(P<0.05),middle and high dose had no effect on the IL-23 mRNA expression,there was no significant difference between the two groups(P>0.05);Bushen-Qiangdu-Zhilv Decoction high dose group could obviously inhibit the fluorescence expression of IL-17 protein in the cell.Compared with the model group,the difference was significant(P<0.001),while the low dose group and middle dose group had no significantly effect on the expression of IL-17 protein,and there was no statistical significance compared with the model group(P>0.05).5.Compared with the model group,three different gradient concentration of ursolic acid could significantly decrease the expression level of NO in cell supernatant(P<0.001);Ursolic acid high ? medium and low dose can significantly reduce the mRNA expression of IL-17?IL-23?TNF-??IL-6.Compared with the model group,the difference has statistical significance(P<0.001),but the expression of ROR?t,ursolic acid high dose has a inhibitory effect,compared with the model group,the difference has statistical significance(P<0.05);Ursolic acid high?medium and low dose group can significantly inhibit the expression of IL-17 protein fluorescence,compared with the model group,the difference was statistically significant(P<0.001).6.Compared with the model group,the three different level of Ligustrazine significantly decreased the expression level of NO in cell supernatant(P<0.001);The high and low dose group of tetramethylpyrazine could significantly reduce the mRNA expression of IL-17,IL-23,TNF-? and IL-6.Compared with the model group,the difference was statistically significant(P<0.001).The high dose group of tetramethylpyrazine could also reduce theexpression of ROR?t.Compared with the model group,the difference was statistically significant(P<0.001),The mRNA expression of ROR ? t was reduced in the medium dose group of Ligustrazine,compared with the model group,had statistical significance(P<0.01),The low dose group of Ligustrazine had no effect on the expression level of ROR?t,and there was no statistical significance compared with the model group(P>0.05);The high dose group of tetramethylpyrazine significantly inhibited the expression of IL-17 protein in the cell,and compared with the model group,the difference was statistically significant(P<0.05),but the low and medium dose group had no effect on the expression of IL-17 protein in the cell,and there was no statistical significance compared with the model group(P>0.05).7.Compared with the model group,the medium and high dose of osthole could significantly decrease the NO expression level of cell supernatant(P<0.001),and the low dose group had no effect on the expression of NO in the cell supernatant(P>0.05).the three different level of osthole had no effect on the mRNA expression of IL-17,IL-23,ROR?t,TNF-?,IL-6,and the difference was no difference compared with the model group(P>0.05),osthole low?medium and high dose had no effect on the expression of IL-17 protein fluorescence,and the difference was not statistically significant(P>0.05).8.Compared with the model group,the medium and high dose of naringin significantly decreased the NO expression level of cell supernatant(P<0.001),and the low dose group had no effect on the expression of NO in cell supernatant(P>0.05),and the mRNA expression of IL-17?IL-23?ROR?t,TNF-?,IL-6 in the different level group of naringin was no difference,Compared with the model group.and the difference was not statistically significant(P>0.05).9.Compared with the model group,the different level of paeoniflorin had no effect on the NO expression in cell supernatant(P>0.05).the mRNA expression of IL-17?IL-23?ROR?t,TNF-?,IL-6 in the different level group of paeoniflorin was no difference,Compared with the model group.and the difference was not statistically significant(P>0.05).10.Compared with the model group,celecoxib significantly decreased the NO expression level of cell supernatant(P<0.001),and celecoxib could significantly reduce the mRNA expression of IL-17?ROR?t?TNF-??IL-6 in cells.Compared with the model group,the difference was statistically significant(P<0.001),but there was no effect on the expression of IL-23 mRNA(P>0.05);celecoxib group,compared with the model group,the fluorescence expression of IL-17 protein could be suppressed,and the difference was statistically significant(P<0.01).Conclusion1.HPLC experiment showed that there were five compounds: Ligustrazine,paeoniflorin,naringin,osthole and ursolic acid in Bushen-Qiangdu-Zhilv Decoction.2.This study proved that the RAW264.7 inflammation model was constructed successfully.The increased expression of inflammatory factors and the imbalance of the IL-23/Th17 axis in the RAW264.7 model cells,This model cells could be used to study the anti-inflammatory effects of Bushen-Qiangdu-Zhilv Decoction and their chemical components,and the mechanism of target IL-23/Th17 axis.3.Bushen-Qiangdu-Zhilv Decoction can inhibit the expression of NO and inflammatory factors TNF-? and IL-6,and inhibit the expression of IL-17,ROR?t and IL-23,and have no effect on cell proliferation.It shows that Bushen-Qiangdu-Zhilv Decoction has the anti-inflammatory effect and can regulate the IL-23/Th17 inflammatory axis,it has low cytotoxicity.4.The effective components of ursolic acid and ligustrazine in the Bushen-Qiangdu-Zhilv Decoction also can inhibit the expression of NO and TNF-??IL-6?IL-17?ROR?t and IL-23 in the RAW264.7 model cells,indicating that these two compounds have anti-inflammatory effects and can regulate the IL-23/Th17 inflammatory axis.5.Naringin and osthole can inhibit the expression of NO in the RAW264.7model cells,but have no effect on the expression of TNF-??IL-6?IL-17?ROR ? t and IL-23,indicating that naringin and osthole have some anti-inflammatory effects,but have no effect on the IL-23/Th17 inflammatory axis.Paeoniflorin showed no significant anti-inflammatory effect and had no effect on IL-23/Th17 inflammatory axis. |
PDF Full Text Request