Study On The Anti-inflammatory Mechanism Of Hoec And Huang-lian-jie-du-tang

Mechanism study of drug action is an essential step in drug discovery,and it is the basis for the development of new drugs.It is particularly important to identify potential target and then clarify the mechanism of drug action through exploring new approaches for target identification of natural products,using the theory and new technology of chemical biology,pharmacology,molecular biology and modern pharmacognosy.Chemical biology is a scientific discipline spanning the fields of chemistry and biology.It involves the application of small-molecule compounds as chemical probes to study and manipulation of biological systems.It fundamentally provides the theoretical basis and new visions for drug discovery and mechanism study for natural products.Based on the diversity and complexity of the natural product,it is a new direction in drug research to develop theoretical and applied research of chemical biology.Inflammation,the fundamental mechanism in reactions to internal and external injuries or pathogens and can be either acute or chronic depending on duration of the disease,is the most common and complex pathological processes.Most of inflammatory mediators are generated by metabolism of arachidonic acid(AA).5-Lipoxygenase(5-LOX)is a focus on inflammation research because it is an important rate-limiting enzyme for leukotriene B4(LTB4)synthesis in the AA pathway.Therefore,searching for new 5-LOX inhibitors is of great clinical significance for the treatment of many types of acute or chronic inflammatory diseases.Part I Study on the anti-inflammatory mechanism of HOECThe whole plant Incarvillea mairei var.grandiflora(Wehrhahn)Grierson has long been used as folk medicines in the south and southwest of China.It has been reported to have traditional efficacy such as heat-clearing,detoxification and wind dampness-dispelling.So far,there have been no reports of chemistry,pharmacology,chemical biology and pharmacognosy study of the original plant.In order to study the efficacy of the traditional Chinese medicine and material basis of the biological activity for this plant,thus to develop an anti-inflammatory drug of high efficacy and low toxicity,an in-depth reaearch has been doing to study the natural chemistry,chemical biology and pharmacology of this plant in our laboratory.Preliminary studies showed that(+)-2-(1-Hydroxyl-4oxocyclohexyl)ethyl caffeate(HOEC),a caffeic ester which was isolated as a major constituent from I.mairei var.granditlora,was found to possess potent anti-inflammatory activity,and deserves further development and investigation.Based on the results of the preliminary studies,our research group employed a reliable chemical biology strategy to investigate the anti-inflammatory effect and mechanism of HOEC.The main achievements of this study were as follows:1.For the first time,an ex vivo affinity chromatography technique was performed to investigate the potential target proteins of HOEC.The results revealed that HOEC can specifically capture 5-lipoxygenase(5-LOX)from rat peritoneal macrophage lysates.Then,to monitor the biomolecular interaction of HOEC with 5-LOX,a biosensor-based affinity analysis was performed using the BioLayer interferometry(BLI)technique,and the results clearly determined that 5-LOX was specifically bound to HOEC with a concentration-dependent binding profile.As 5-LOX is a key target in inflammation research because it is an important rate-limiting enzyme for LTB4 synthesis in AA pathway,it was indentified as the target protein of HOEC,which laid a good foundation for further study.2.By using an enzyme-linked immunosorbent assay(ELISA),effects of HOEC on the enzyme activities of 5-LOX,15-Lipoxygenase(15-LOX),Leukotriene A4-hydrolase(LTA4H)Cyclooxygenase-1(COX-1)and Cyclooxygenase-2(COX-2)were systematically determined at pure enzyme level.The results revealed that HOEC inhibited 5-LOX activity in vitro in a concentration-dependent manner with IC50 of 34.6 μM.Moreover,HOEC strongly inhibited 15-LOX in a concentration-dependent manner with IC50 of 7.8 μM,whereas it showed no inhibition on LTA4H,COX-1 or COX-2.3.To further validate the effects of HOEC on AA metabolic pathway,a single cell model of inflammation was established by stimulating rat peritoneal macrophages with exogenous AA and calcium ionophore A23187.An Agilent 6410A LC-MS triplequad mass spectrometer was applied to determine the effects of HOEC on AA metabolites systematically in rat peritoneal macrophages.AA can be converted to bioactive eicosanoids through two pathways:lipoxygenase(LOX)or cyclooxygenase(COX)pathway.Firstly,LOX-derived LTB4,5-Hydroxyeicosatetraenoic acid(5-HETE),8-Hydroxyeicosatetraenoic acid(8-HETE),11-Hydroxyeicosatetraenoic acid(11-HETE),12-Hydroxyeicosatetraenoic acid(12-HETE),and 15-Hydroxyeicosatetraenoic acid(15-HETE)were determined.HOEC was found to markedly inhibited the biosynthesis of LOX-derived LTB4 and 5-HETE in a concentration-dependent manner(IC50=8.0 μM and IC50=9.7 μM,respectively),but it presented no significant inhibition on 8-HETE,11-HETE or 15-HETE.Moreover,COX-2-derived prostaglandin E2(PGE2),thromboxane B2(TXB2),and 12-Hydroxyheptadecatrienoic acid(12-HHT)were determined and no inhibition on these COX-derived eicosanoids was observed.These results revealed that HOEC exerted anti-inflammatory effect through inhibiting 5-LOX derived LTB4 formation in macrophages.4.Effects of HOEC on AA metabolites were systematically determined in human whole blood by ELISA,which is a more reliable evaluation system and composed of erythrocytes,platelets and various leukocytes.The data revealed that HOEC was a more potent inhibitor of LTB4 production(IC50=2.8 μM)in whole blood than in rat macrophages.In contrast,no decrease in the PGE2 level was observed with HOEC.In summary,extensive studies at the molecular and cellular level demonstrated that 5-LOX was a direct anti-inflammatory target of HOEC.5.For the first time,a computer-aided dynamic simulation was established to dynamically simulate the activity of HOEC on the AA metabolic network.Concentration curves of the six metabolites(LTB4,5-HETE,12-HETE,15-HETE,PGE2 and TXB2)in the model were fitted and compared to the experimental curves from macrophages,and the corresponding activity of HOEC on each of the enzymes was calculated based on the best-fitted concentration curves.The results indicated that the corresponding activity of the five enzymes(5-LOX,15-LOX,LTA4H,COX-1 and COX-2)were consistent with the data from in vitro enzyme activity.The simulated results integrated the data from pure enzymatic assays with those from cells that had a complicated AA network,which furthered the understanding of the pharmacologic effects of HOEC,and implemented new thinking of drug discovery from a biological network perspective.6.To further validate the anti-inflammatory effect of HOEC,three different types of animal models of inflammation,AA-induced mouse acute ear edema,OVA-induced mouse asthma and collagen-induced rat rheumatoid arthritis(CIA)were used to further validate the anti-inflammatory effect of HOEC,which thoroughly validated the potency of HOEC and confirmed the key role of 5-LOX in various inflammation in vivo.(1)AA-induced mouse acute ear edema model was used to determine the effect of HOEC on acute inflammation which was directly caused by AA.Topical treatment with HOEC markedly inhibited the extent of oedema(66.5%,p<0.001)and protein permeability(66.5%,p<0.001).Importantly,LTB4 production in ear mouse was also drastically suppressed by HOEC and reached a maximum inhibition rate of 82.3%(p<0.001).These results indicated that topical administration of HOEC exerted protective effects on AA-induced acute inflammation through controlling the excessive production of LTB4.(2)O VA-induced mouse asthma model was used to determine the effect of HOEC on chronic allergic asthma.HOEC treatment significantly reduced the elevated BALF total cell counts by 48.9.The elevated LTB4,an important AA metabolite in asthma,was significantly suppressed by HOEC with an inhibition of 94.7%at 1 mg/kg and 79.2%at 0.2 mg/kg.In addition,HOEC was observed to ameliorate OVA-induced lumen narrowing and bronchial thickness by H&E staining.However,no significant decrease in plasma IgE level was observed with HOEC treatment.These findings demonstrated the anti-inflammatory effect of HOEC in chronic asthma.(3)CIA model was applied to further investigate the effects of HOEC on autoimmune inflammation.Representative pictures of rat legs also showed that HOEC treatment ameliorated the extent of swelling in CIA.HOEC treatment(1 mg/kg,i.p.)moderately decreased the clinical scoring and incidence of CIA,indicating that HOEC moderately decreased the severity and incidence of CIA.Biochemical detection revealed that HOEC(1 mg/kg,i.p.)profoundly reduced the elevated LTB4 levels in the plasma and joint with inhibition rates of 45.8%and 50.7%,respectively.In contrast,PGE2 levels were not affected by HOEC.Real-time PCR and Western blotting analysis demonstrated that 5-LOX mRNA and protein expression in the joints were moderately reduced by HOEC treatment.Moreover,no significant inhibition on anti-CⅡ antibodies in the plasma was observed after HOEC treatment.Furthermore,H&E staining and micro-CT analysis revealed that HOEC can ameliorate cartilage damage,synovium proliferation,bone erosion and bone destruction,which were caused by CIA treatment.These findings confirmed the anti-5-LOX activity of HOEC and demonstrated its amelioration of the disease symptoms of CIA.In vivo studies of AA-induced ear oedema,OVA-induced asthma and collagen-induced arthritis demonstrated that HOEC exerted potent anti-inflammatory effects through inhibiting 5-LOX-derived LTB4 generation.Moreover,the results validated the key role of 5-LOX in various inflammation.7.Toxicity studies on HOEC were determined by MTT assay and flow cytometry.The results revealed that no apparent induction of apoptosis or necrosis in macrophages was observed for HOEC even at concentrations of up to 100 μM,indicating that HOEC didn’t exhibit any significant side effects on RAW 246.7 cell line.8.The present study demonstrated that HOEC presented anti-inflammatory effect by in vitro,cellular,and in vivo assays.Mechanism study revealed that HOEC presented anti-inflammatory effect through controlling the excessive production of LTB4,which was attributed to its inhibition on 5-LOX.HOEC is a natural anti-inflammatory agent small molecule and can be used as lead compound in the development of new natural anti-inflammatory 5-LOX inhibitor,which is worthy of further investigation.The present study provided a promising and rational paradigm for target identification and mechanism of action research for small molecules in drug research.Part II Study on the anti-inflammatory mechanism of HOEC Huang-Lian-Jie-Du-TangHuang-Lian-Jie-Du-Tang(HLJDT)is a classic traditional Chinese prescription to clear"heat" and "poison",which is an aqueous extract of four herbal materials:Coptidis Rhizoma,Radix Scutellariae,Cortex Phellodendri,and Fructus Gardeniae.Modern pharmacological studies have demonstrated that HLJDT have anti-inflammatory,anti-bacterial,anti-cerebral ischemia,anti-oxidation and anti-endotoxin effects.HLJDT has been used to treat Rheumatoid arthritis,ulcers,viral myocarditis,septicemia,meningococcal meningitis and hepatitis clinically.Preliminary studies showed that the main 13 pure components in HLJDT were baicalin,baicalein,wogonoside,wogonin,berberine,coptisine,geniposide,magnoflorine,phellodendrine,palmatine,jateorhizine,crocin,and chlorogenic acid.In order to lay a foundation for the development of modern Chinese medicine from HLJDT,and provide a scientific basis for its clinical application,an in-depth study was performed for the mechanism of the anti-inflammatory effect of HLJDT.The main achievements of this study were as follows:1.The effects of HLJDT,component substances and the components of HLJDT on cell viability were determined by MTT.HLJDT,Radix scutellariae,and Fructus gardeniae did not show cytotoxicity,but Rhizoma coptidis,Cortex phellodendri exhibited moderate cytotoxicity.Among the 13 component substances of HLJDT,berberine and coptisine exhibited moderate cytotoxicity.Palmatine,phellodendrine,and jateorrhizine showed weak cytotoxicity.Baicalin,baicalein,wogonoside,wogonin,magnoflorine,crocin,chlorogenic acid or geniposide did not show cytotoxicity at concentration of up to 100 μM.2.In order to find out the contributory components and their precise targets,an HPLC assay was applied to determine the effects of HLJDT prescription,four single component herbs and 13 pure components(baicalin,baicalein,wogonoside,wogonin,berberine,magnoflorine,phellodendrine,coptisine,palmatine,jateorrhizine,crocin,chlorogenic acid,and geniposide)of HLJDT on LTB4,5-HETE,and 12-HHT generation in rat elicited peritoneal macrophages.HLJDT showed significant inhibition on LTB4 biosynthesis with an inhibition ratio of 62.8%at 50 μg/ml.Among the four herbs,Radix scutellariae and Rhizoma coptidis significantly inhibited the LTB4 biosynthesis,which were responsible for the suppressive effect of HLJDT prescription on eicosanoid generation.Radix scutellariae showed greater inhibition(IC50=5.8μg/ml)than Rhizoma coptidis(IC50=84.3 μg/ml)did.Similar inhibitions on 5-HETE were also observed for HLJDT.Among the 13 pure components of HLJDT,baicalein and baicalin markedly inhibited the biosynthesis of LTB4 and 5-HETE.Meanwhile,wogonin,wogonoside,and coptisine also show weak inhibitory effect on LTB4.None of other components showed significant inhibitory activity on LTB4 and 5-HETE.Whereas,as for 12-HHT,no significant effect was observed for HLJDT and the components.3.To identify the targets and clarify their mechanism,for the first time,the efficacies of 13 pure compounds were evaluated on cell-free purified enzymes:LTA4H,5-LOX,15-LOX,COX-1 and COX-2.In LOX inhibitor screening assay,baicalein showed potent inhibitory activity on 5-LOX(IC50=13.1 μM)and 15-LOX(IC50=12.6 μM).In comparison,baicalin showed a weak inhibitory activity.However,no effect was observed for other 11 components.In LTA4H assay,only coptisine showed a moderate inhibition on LTA4H in a concentration dependent manner,with IC50 of 18.1 μM.In vitro COX enzyme inhibition studies showed that none of the 13 components exhibited inhibitory activity on COX-1 or COX-2.4.To observe the effects of HLJDT and its 4 representative components(baicalin,baicalein,berberine and geniposide)on COX-2-catalyzed PGE2 or 5-LOX-catalyzed LTB4 generation,the topical anti-inflammatory activities and inhibition of eicosanoid formation of HLJDT and the 4 components were observed in an AA-induced mouse ear edema model.The results revealed that topical treatment of HLJDT significantly inhibited AA-induced mouse ear edema and reduced release of PGE2 and LTB4 in the edematous ears,the inhibition were 62.3%,59.3%and 35.1%at the dose of 4 mg/ear,respectively.Among the four representative components,baicalein,and berberine also ameliorated the symptoms and suppressed eicosanoid generation with varying efficacies.Baicalein dramatically inhibited the extent of edema,the biosynthesis of PGE2 and LTB4 with inhibition ratios 64.3%,61.2%and 44.2%at 2 mg/ear.Berberine(2 mg/ear)present weaker effects on ear edema and PGE2 formation(12.3%and 39.0%reduction).However,berberine(2 mg/ear)caused a marked decrease in LTB4 formation(72.4%reduction).In addition,baicalin and geniposide,failed to present any significant effect on LTB4 and PGE2 biosynthesis.5.To distinguish the effects of HLJDT on enzyme activity from those on enzyme expression,macrophage-based systems were established to further study the effects of HLJDT and its 4 representative components(baicalin,baicalein,berberine and geniposide)on enzyme activity and enzyme expression of COX-2 and 5-LOX.Cell-based assays showed that,(1)HLJDT and baicalein inhibited the PGE2 levels by decreasing COX-2 enzyme expression without affecting COX-2 enzyme activity in RAW 246.7 cells.(2)HLJDT and baicalein inhibited the LTB4 levels mainly by decreasing 5-LOX enzyme activity and expression in rat elicited peritoneal macrophages.6.Further real-time PCR and western blotting experiments were carried out to confirm these effects at the mRNA and protein levels.The data demonstrated that HLJDT and baicalein reduced COX-2 expression at the mRNA and protein levels in RAW 246.7 cells,whereas no inhibition on 5-LOX expression was observed in RAW 246.7 cells or rat elicited peritoneal macrophages.7.The present study,HLJDT prescription,the component herbs and pure components of HLJDT were systematically determined at in vitro,cellular,and in vivo levels to identify the targets and clarify their mechanism for the first time.Radix scutellariae and Radix scutellariae were demonstrated as the active component herbs of HLJDT.As well,baicalein was demonstrated as the main active component.Furthermore,our study showed that HLJDT can suppress eicosanoid generation via both the COX and LOX pathways,which definitely contributes to its topical anti-inflammatory activity.We have confirmed that its dual inhibition on the COX and LOX pathways resulted mainly from the downregulation of COX-2 expression and direct inhibition of 5-LOX activity,respectively.These findings made the mechanism of HLJDT clearer and provided a scientific basis for the development and clinical applications of new drugs.