| Background and Objective: Sepsis is a serious threat to human health,however,the effective prevention and treatment of drug side effects are limited.Rhubarb is a kind of traditional Chinese medicine,with complex components,anti-inflammatory,anti-tumor,immune regulation and other effects.Anthraquinones are recognized as the active substances of rhubarb.At present,there are many studies on the anti-inflammatory effects of various monomers of rhubarb anthraquinone(RA),however,there is few comparative studies on the main anthraquinone components of Rheum officinale,such as free anthraquinone(FA)and combined anthraquinone(CA).In this study,rhubarb from Li County of Gansu province was isolated by systematic isolation,quality control and pharmacodynamic observation.The objective of the assay is to investigate the effects of rhubarb FA and CA extracts on lipopolysaccharide(LPS)-induced inflammatory injury in lung and intestine,and to explore the underlying molecular mechanisms and drug targets,in order to provide a theoretical basis for the development and utilization of rhubarb.Methods: 1.RA components were extracted and separated from rhubarb by solvent extraction,and refined by macroporous adsorption resin to prepare rhubarb FA and CA extracts respectively.The contents of FA and CA extracts from rhubarb were determined by spectrophotometer.The contents of different components(Aloe emodin,Emodin,Rhein,Chrysophanol and Parietin)of rhubarb extract before and after hydrolysis were determined by high-performance liquid chromatography(HPLC)to control the quality of rhubarb extract.2.The inflammation model of mice was established by LPS,and the pharmacodynamics of rhubarb FA and CA extract was observed.The levels of white blood cell(WBC),neutrophil and C-reactive protein(CRP)were measured by hematology analyzer and biochemical analyzer.The levels of procalcitonin(PCT),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin-10(IL-10)in the serum of mice were measured by enzyme-linked immunosorbent assay(ELISA).Permeability changes of lung and ileum epithelial cells were evaluated by wet dry(WD)and Evans Blue(EB)permeation experiments.Hematoxylin-eosin(H&E)staining and transmission electron microscopy were used to observe the changes of lung and small intestine injury.3.The expression levels of ZO-1,occludin and claudin-5 in lung and ileum were detected by immunohistochemistry(IHC)and western blotting(WB),the expression of p-NFκB p65/NFκB p65,p-IKKβ/IKKβ and inflammatory regulator Cyclooxygenase-2(Cox-2),inducible nitric oxide synthase(iNOS),and Ras homolog gene family member A(RhoA)/Rho-associated protein kinase 1(Rock1)signaling pathway key proteins RhoA,Rock1,p-MYPT1/MYPT1 were detected by WB.In addition,RhoA/Rock signaling agonist U46619 was used to further verify the protein expression of TJs and inflammatory regulatory proteins.4.Mouse macrophage RAW264.7 was cultured in vitro.The inhibitory activity of rhubarb extracts FA and CA on RAW264.7macrophages was detected by CCK8 method in order to screen the optimal drug concentration of FA and CA.RAW264.7 macrophage inflammatory model was established by LPS.The changes of IL-1β,TNF-α and IL-10 were detected by ELISA before and after FA and CA treatment.The protein expressions of p-NFκB p65/NFκB P65,p-IKKβ/IKKβ and inflammatory regulators Cox-2 and iNOS were detected by WB method.Results: FA and CA were extracted from Rheum officinale according with quality control requirements.The contents of FA and CA were more than 50%,and the monomer components of the extract accorded with the qualitative characteristics.In vivo,the results of blood cell analysis showed that compared with LPS,the white blood cell level and neutrophil count level in FA group and CA group were significantly higher.The difference was statistically significant(both p < 0.001).The results of biochemical analyzer showed that compared with LPS,the level of CRP in FA group and CA group were significantly lower,and the difference was statistically significant(both p < 0.001).The ELISA results showed that compared with the control group,the levels of TNF-α,IL-1β and PCT in the serum of LPS group mice increased significantly,while IL-10 levels decreased(both p < 0.001);After intervention with FA or CA,the levels of TNF-α,IL-1β and PCT were significantly decreased,while the levels of IL-10 were significantly increased.The results of lung H&E showed that compared with LPS group,the FA and CA group significantly improved the degree of LPS-induced lung injury in mice,and the difference was statistically significant(p < 0.01 and p < 0.05,respectively);Compared with FA group,the degree of improvement of LPS-induced lung injury in CA group was weaker than that in FA group(p < 0.05).The ileal H&E results showed that compared with LPS group,FA or CA group significantly improved the degree of LPS-induced ileal injury in mice,with a statistically significant difference(p < 0.001 and p < 0.05,respectively);Compared with FA group,the improvement of ileal injury in CA group was weaker than that in FA group,and the difference was statistically significant(p <0.01).The results of electron microscopy showed that rhubarb FA or CA extract significantly improved the TJ structure damage of lung and ileum caused by LPS,and the improvement effect of FA was better than that of CA.The W/D experiment indicated that compared with LPS group,FA and CA groups significantly reduced the lung W/D value,with a statistically significant difference(p < 0.001 and p < 0.01,respectively).The results of lung EB test showed that compared with LPS group,the treatment of FA or CA inhibited the lung EB leakage in the presence of LPS,and the difference was statistically significant(p < 0.01 and p < 0.05,respectively);The effect of FA group on improving pulmonary EB leakage was better than that of CA group,and the difference was statistically significant(p < 0.05).The results of ileal EB test were similar to those of lung EB test.The results of lung IHC showed that in the mice pretreated with FA or CA,the expression of ZO-1,occludin and claudin-5 was significantly higher than that of LPS;The results of intestinal IHC showed that compared with LPS group,the expression levels of ZO-1,occludin and claudin-5 in FA and CA groups were significantly increased(all p < 0.001);The results of lung WB showed that compared with LPS group,the expression of ZO-1,occludin and claudin-5 in FA group was significantly up-regulated,and the difference was statistically significant(p < 0.01,p < 0.001 and p < 0.01,respectively).The CA group also had similar results(p < 0.05,p < 0.01 and p < 0.05,respectively).The results of ileal WB were consistent with those of lung WB.In addition,in lung tissue,compared with LPS group,the phosphorylation expression of NFκB p65 and IKKβ was significantly decreased in FA group(both p < 0.001),and similar results were obtained in CA group(p < 0.01 and p < 0.001,respectively);The results of ileum tissue were similar to those of lung tissue.We detected RhoA/Rock1 pathway related proteins in lung tissue,and the results showed that compared with LPS group,the expression of RhoA,Rock1,p-MYPT1/MYPT1,Cox-2 and iNOS were significantly decreased after FA treatment,and the difference between the gray values of each index was statistically significant(p < 0.01,p < 0.01,p < 0.01,p < 0.001 and p < 0.01,respectively),the results of CA group were similar to those of FA group(p < 0.05,p <0.05,p < 0.05,p < 0.05 and p < 0.01);The protein test results of RhoA/Rock1 pathway in ileum tissue are similar to those in lung tissue.After using RhoA/Rock1 pathway agonist,compared with LPS group,the expression level of Cox-2 and iNOS protein in FA group decreased and the expression level of ZO-1,occludin and claudin-5 increased in lung and ileum tissues.However,the protective effect of FA on TJs was partially eliminated under the intervention of U46619.In vitro experiment,the CCK-8 experiment results showed that FA and CA inhibited the proliferation of RAW264.7 macrophages in a dose-dependent manner,and the inhibition effect of FA was slightly better than that of CA.The ELISA results showed that compared with LPS,after FA and CA treatment,the content of TNF-α and IL-1β decreased significantly,while IL-10 increased in RAW264.7 macrophages.The results of WB showed that compared with LPS group,the phosphorylation expression of NFκB p65 and IKKβ was significantly decreased in FA group,the quantitative analysis of gray value ratio showed that the difference was statistically significant(p < 0.001 and p <0.01,respectively).CA group also had similar results(p < 0.01 and p < 0.001,respectively).The expression levels of Cox-2 and iNOS were also consistent with those of p-NFκB p65/NFκB p65 and p-IKKβ/IKKβ.Conclusions: In vivo experiments,we found that RA can effectively reduce LPS-induced inflammatory reaction in mice,reduce the permeability damage of lung and small intestinal epithelial cells,and its mechanism may be related to the down-regulation of RhoA/Rock1 signal pathway and the enhancement of alveolar and small intestinal epithelium tight junction protein.In addition,NF-κB pathway is also involved in the anti-inflammatory effect of RA substances.In vitro experiments further confirmed that macrophages are one of the target cells for RA substances to play an anti-inflammatory role and protect against organ(lung and ileum)permeability damage. |
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