| Background:Centellaasiatica is a plant of Umbelliferae,and botanists put it into genus coriander.Its chemical constituents are complex,and the main effective components include Asiaticoside,Asiaticoside a,borrelin and so on.Centellaasiatica has been used in China’s medical history for more than two thousand years,and it is usually used to treat patients with swelling,poison and sores,trauma,bleeding and injuries,etc.At present,great progress has been made in using Centellaasiatica and studying its pharmacology.Asiaticoside,abbreviated AS AS,is a compound proposed from Centellaasiatica that promotes wound healing and is used in the treatment of injuries,surgical wounds and keloids.Recent studies have shown that AS has obvious anti-inflammatory effects,but its effects on mast cell activation,asthma and allergic contact dermatitis have not been reported.Allergic diseases are also called allergic diseases.It is a disease caused by abnormal reaction after allergen entering the body.Induced by the external factors of allergen,it can cause the disease of different organs with the target cells of allergen,such as bronchial asthma,allergic skin diseases and so on.TLR4/NF-κB(Toll like receptors 4/nuclear factor kappa beta)pathway is found to be closely related to anti-inflammatory immune mechanism in recent years.TLR4/NF-κB(Toll-like receptors4/Nuclear factor kappa beta)pathway is a recently developed pathway that is closely related to the anti-inflammatory immune mechanism.There are two pathways of this signaling pathway,one is the dependent pathway myeloid differentiation factor 88,referred to as MYD 88 for short,and the other is the non-dependent pathway MyD 88.These two pathways are interdependent and interact with each other,and the signal transduction process is through them,they cooperate in common.One of the major signaling pathways in cells is the Wnt/β-catenin pathway,which activates the expression of target genes in the nucleus.This pathway leads to the accumulation ofβ-catenin in the cytoplasm,which is eventually transcribed into the nucleus as transcription factors(the LEF family/co-activators of the TCF(T cytokine)).Signaling pathway NRF2/heme oxygenase-1(nuclear factor E2-related factor 2,HO-1)/nuclear factor E2-related factor 2(nuclear factor E2-related factor 2,HO-1)is a multi-organ protective chain relative to the corresponding stress environment.It is a hotspot in the research on the inducement and development mechanism of oxidative stress disease in the world.Because the NR2/HO-1 signaling axis can protect against various kinds of organ oxidative stress damage,this signaling axis is of great significance for gene targeted therapy of related diseases.In this study,Centella glycoside was selected as the research drug to explore its mechanism of action on allergic diseases such as asthma and allergic contact dermatitis.Research objective:In vivo and in vitro,Asiaticoside can alleviate various inflammatory reactions through TLR4/NF-κB,Wnt/β-Catenin pathway and Nrf2/HO-1.Objective to study the effect and mechanism of Asiaticoside on mast cells in allergic inflammation,the effect and mechanism of Asiaticoside on Ova induced inflammation in asthmatic mice,and the effect and mechanism of Asiaticoside on DNCB induced allergic contact dermatitis in mice.These studies provide a new theoretical basis for the relationship between asiatica glycoside and inflammatory response,and provide a new target for the clinical treatment of inflammatory response diseases such as asthma and allergic contact dermatitis.Materials and methods:1.The effect of Asiaticoside on mast cells in allergic inflammation(1)In vivo:100 7-week-old male BALB/c mice(weight 18-22 g).Fifty mice were randomly divided into five experimental groups:model group(i.e.,IgE+Ag group)and normal Control group(i.e.,Control group),as 10 group(asiaticoside low-dose treatment group,dosage of 10 mg/kg),as 20 group(asiaticoside medium dose treatment group,dosage of 20 mg/kg),as 40 group(asiaticoside high-dose treatment group,dosage of 40 mg/kg),with 10 mice in each group.Mice in IgE+Ag group were injected with 50 μl PBS containing 0.5 μg anti DNP IgE to sensitize skin,while mice in control group were injected with 50 μ1 PBS.After 48 hours,each mouse was injected with 1 mg dnp-hsa and 4%Evans blue(1:1)by tail vein.Mice in AS 10 group,AS 20 group and AS 40 group were injected and given corresponding dose of asiatica side by gavage 1 h before DNP-HAS injection.Observation indexes:Determination of dye exudation.Fifty mice were randomly divided into five groups:Control group(normal Control group),IgE+Ag group(model group),as 10 group(asiaticoside low-dose treatment group,dosage of 10 mg/kg),as 20 group(asiaticoside medium dose treatment group,dosage of 20 mg/kg),as 40 group(asiaticoside high-dose treatment group,dosage of 40 mg/kg),with 10 mice in each group.Mice in IgE+Ag group were injected with 20 μ1 PBS containing 200 ng anti DNP IgE subcutaneously,while mice in control group were injected with 20 μ1 PBS subcutaneously.After 24 hours,mice were injected with 0.1 mg dnp-has and 4%Evans blue(1:1)by tail vein.Mice in as 10,as 20 and as 40 groups were given corresponding doses of Asiaticoside by gavage 1 hour before anti DNP IgE injection.One hour after antigen injection,the mice were anesthetized.OUTCOME MEASURES:ear histological staining and mast cell count.(2)In vitro experiment:the two kinds of cells were divided into five groups:IgE+Ag group(model group),as 5 group(asiaticoside low dose treatment group,5 μmol/L),as 10 group(asiaticoside medium dose treatment group,10 μmol/L),as 15 group(asiaticoside high dose treatment group,15 μmol/L).OUTCOME MEASURES:the histamine release level of RPMCs was determined by radioimmunoassay.45Ca uptake of RPMCs was measured by mast cells.Total RNA was collected from RBL-2H3 cells.TNF-α,IL-4,IL-1 β and IL-8 were detected by RT-PCR kit.The supernatant of RBL-2H3 cells treated with drugs was collected by ELISA.According to the instructions,ELISA tests were performed with ELISA kits for TNF-α,IL-4,IL-1 β and IL-8.Western blot was used to detect the expression of p-syk,NF-κ B p65,Nrf2 and HO-1 in RBL-2H3 cells,p-gab2,PLC-γ1,p-PLC-γ1,I κ B α,p-Akt,GAB2,Akt,Lyn,p-p38,Syk,p38,p-lyn,JNK,p-JNK p-I κ B α,p-ERK,ERK.2.Effect of Asiaticoside on Ova induced inflammation in asthmatic mice(1)In vivo:50 male BALB/c mice aged 7 weeks(body weight 18-22 g).Fifty mice were randomly divided into five experimental groups:model group(i.e.OVA group)and normal Control group(i.e.Control group),as 10 group(asiaticoside low-dose treatment group,dosage of 10 mg/kg),as 20 group(asiaticoside medium dose treatment group,dosage of 20 mg/kg),as 40 group(asiaticoside high-dose treatment group,dosage of 40 mg/kg),with 10 mice in each group.The mice were sensitized by intraperitoneal injection of 200 μL mixed alumina hydroxide and OVA on the 1st,7th and 14th days.From day 21,mice in all groups except the Control group were placed in a mouse atomized drug dispense apparatus.After 1%ova solution was atomized,the mice were inhaled to stimulate the mice,30 minutes a day for 3 consecutive days.Mice in the normal group were replaced with the same amount of PBS,and mice in the drug treatment group were treated with the corresponding dose of drug 30 min before each excitation.Specifically,the following indexes were observed:The inflammatory cells contained in BALF,bronchoalveolar lavagefluid(bronchial lavagefluid)were counted in each group by classification.The distribution of goblet cells and inflammatory cell infiltration in lung tissue were observed by histological staining.The levels of IL-4,IL-5,IL-13 and IFN-γ in BALF were detected by ELISA kit.The contents of TLR4,p-p65,Wnt5a,Wnt7a,β-Catenin,Nrf2 and HO-1 in lung tissue were determined by Western blot.The changes of Nrf2 and HO-1 in lung tissues were determined by immunofluorescence method.(2)In vitro experiment:16HBE cells were divided into four groups:model group(LPS group)and normal Control group(Control group),as 10 group(asiaticoside medium dose treatment group,10 μmol/L),as 15 group(asiaticoside high dose treatment group,15 μmol/L).The cells were pretreated with 16HBE inhibitor,pg490(inhibitor of NF-κb)and kya1797k(inhibitor of β-Catenin)for 2 hours,and then treated with Asiaticoside 10 μm and 20 μm for 24 hours.After the intervention,it can be used for testing.OUTCOME MEASURES:16HBE activity was detected by MTT method.The contents of TLR4,p-NF-κbp65,NF-κbp65,p-I κ B α,I κ B α,Wnt5a,Wnt7a,β-Catenin,p-GSK-3 β,GSK-3 β,Nrf2 and HO-1 were determined by Western blot.Changes n of Nrf2 and HO-1 contents in 16HBE cells with immunofluorescence.3.Effect of Centellaasiatica glycoside on allergic contact dermatitis in mice(1)In vivo experiment:50 male C57 mice aged 7 weeks(weight 18-22 g).Fifty mice were randomly divided into five groups:model group(ACD group),normal Control group(Control group),as 25 group(asiaticoside medium dose treatment group,dosage of 25 mg/kg),as 50 group(asiaticoside high dose treatment group,dosage of 50 mg/kg),DEX group(dexamethasone group,dosage of 1 mg/kg),10 mice in each group.ACD was induced by 2,4-dinitrochlorobenzene(DNCB)in model group,low-dose cryptotanshinone group and high-dose cryptotanshinone group.Cryptotanshinone low-dose group and cryptotanshinone high-dose group were administered after sensitization.Observation indexes:morphological and histopathological changes of skin after challenge.Spleen index of mice.Changes of IL-4 and IFN-γ in serum of mice.The expressions of TLR4,p-p65,Wnt5a,Wnt7a,β-Catenin,Nrf2 and HO-1 in skin tissue were detected.(2)In vitro experiment:HaCaT cells were divided into four groups:model group(TNF-α group),normal control group(control group),as 10 group(asiaticoside medium dose treatment group,10 μmol/L),as 20 group(asiaticoside high dose treatment group,20 μmol/L).21/5000.The changes of IL-4 and IFN-γ in HaCaT were measured by flow cytometry.The contents of TLR4,NF-κBp65,Wnt5a,Wnt7a,β-Catenin,Nrf2 and HO-1 were determined by Western blot.The content of Wnt5a and Wnt7a in immunofluorescent HaCaT cells changed.Results:Part 1.Effects of Centellaasiatica glycoside on mast cells in allergic inflammation and its mechanism(1)Compared with the IgE+Ag group,asiatica glycosides treated with different concentrations could significantly inhibit the dye exudatio(p<0.05),and Asiaticoside had a certain anti allergic effect on antigen-mediated PCA reaction;(2)Compared with IgE+Ag group,Asiaticoside treatment group could alleviate ear swelling induced by anti DNP IgE and significantly reduce ear thickness;(3)Compared with IgE+Ag group,Asiaticoside could not inhibit the increase of mast cells(p<0.05);(4)The cell viability of RPMC or RBL-2H3 was not significantly affected by different concentration of asiatica glycosides;(5)Compared with IgE+Ag group,Asiaticoside pre incubation significantly inhibited the degranulation of RPMC,but the cells were slightly larger than normal RPMC cells;(6)Compared with IgE+Ag group,Asiaticoside inhibited the histamine release induced by anti DNP IgE in RPMC in a concentration dependent manner(p<0.05,p<0.01);(7)Compared with IgE+Ag group,Asiaticoside at concentrations of 10 and 20 μm significantly inhibited the calcium uptake(p<0.05,p<0.01);(8)Compared with IgE+Ag group,Asiaticoside could inhibit the expression of TNF-α,IL-1 β,IL-4 and IL-8 in a dose-dependent manner(p<0.05);(9)Compared with IgE+Ag group,Asiaticoside could inhibit the phosphorylation of Syk,Lyn and GAB2(p<0.05).In addition,it also inhibited the phosphorylation of PLC-γ1(p<0.05).(10)Compared with the IgE+Ag group,Asiaticoside significantly inhibited the expression of TLR4 and MyD88 protein(p<0.05);(11)Compared with IgE+Ag group,Asiaticoside significantly decreased the level of NF-κB p65 in the nucleus(p<0.05),and increased the level of NF-κB p65 in the cytoplasm.Moreover,Asiaticoside significantly inhibited antigen induced phosphorylation of I κ B α(p<0.05);(12)Compared with the IgE+Ag group,Asiaticoside significantly inhibited the expression of Wnt5a,Wnt7a and β-Catenin protein(p<0.05);(13)Compared with the IgE+Ag group,Asiaticoside can significantly activate Nrf2 and promote the expression of HO-1 in antigen-induced mast cells(p<0.05)Part 2.Effect of asiatica glycoside on the inflammatory response induced by OVA in mice with asthma and its mechanism(1)Compared with OVA group,the number of eosinophils,lymphocytes and neutrophils in BALF decreased significantly after Asiaticoside treatment(p<0.05);(2)Compared with OVA group,Asiaticoside could inhibit the expression of TNF-α,IL-1 β,IL-4 and IL-8 in BALF in a dose-dependent manner(p<0.05);(3)Compared with Ova Group,the infiltration of inflammatory cells in Asiaticoside group was significantly decreased(p<0.05).Moreover,the degree of fibrosis around the airway of lung tissue in the asiaticoside administration group was significantly less than that in the OVA group;(4)compared with OVA group,the amount of lung 4 was significantly decreased in NF-Bp65 and Asiaticoside group,while the expression of Cytosol(NF-Bp65)was increased(p<0.05);(5)Compared with OVA group,Wnt5a,Wnt7a and β-Catenin in Asiaticoside group were significantly decreased(p<0.05);(6)Compared with OVA group,Asiaticoside could significantly activate Nrf2 and promote HO-1 expression in lung(p<0.05);(7)Compared with LPS group,Asiaticoside significantly down regulated the expression of TLR4 and NF-κ bp65 in 16HBE cells(p<0.05);(8)Compared with LPS group,Asiaticoside inhibited the expression of Wnt5a,Wnt7a and β-Catenin in 16HBE cells(p<0.05);(9)Compared with the LPS group,Asiaticoside can significantly activate the expression of Nrf2 and HO-1 in 16HBE cells(p<0.05);(10)Compared with the LPS group,Asiaticoside was able to inhibit the expression levels of p-NF-κBp65,NF-κBp65(nuclear)and p-IκBα after treatment with PG490,an inhibitor of NF-κB,in 16HBE cells(P<0.05).Increase the expression of NF-KBp65(cytosol)and IκBα(p<0.05);(11)Compared with LPS group,Asiaticoside could inhibit the expression of β-Catenin and p-gsk-3 β(p<0.05),but increase the expression of GSK-3 β(p<0.05);(12)Compared with sicontrol group,the expression level of NF-κB decreased(p<0.05),while the expression level of β-Catenin increased(p<0.05).Compared with sicontrol group,the expression level of β-Catenin was decreased(p<0.05),while the expression level of NF-κB was increased(p<0.05).Part 3.Effect of asiatica glycoside on DNCB induced allergic contact dermatitis in mice and its mechanism(1)Compared with ACD group,the ACD response of Asiaticoside group and dexamethasone group were significantly improved;(2)In dexamethasone group,the skin healed well and inflammatory cell infiltration decreased.In the low-dose Asiaticoside group,the skin healing was slightly poor,the epidermis was damaged,it was difficult to distinguish the dermis and epidermis,but it also improved significantly;(3)Compared with ACD group,the spleen index of mice in each dosage of Asiaticoside was higher than that in the blank control group(p<0.05).compared with ACD group,The spleen index of dexamethasone treated mice decreased(p<0.05);(4)Compared with ACD group,dexamethasone group and Asiaticoside low-dose and high-dose groups could significantly reduce serum IL-4 and IFN-γ(p<0.05);(5)Compared with ACD group,the contents of TLR4 and NF-κ bp65(nuclear)in dexamethasone group and Asiaticoside group were significantly decreased,while the expression of NF-κ bp65(cytosol)was increased(p<0.05);(6)Compared with the ACD group,the skin Wnt5a,Wnt7a and β-Catenin protein levels in the dexamethasone and Asiaticoside groups were significantly reduced(p<0.05)(7)Compared with the ACD group,the amount of HO-1 and Nrf2 protein in the skin of the Asiaticoside and dexamethasone administration group increased significantly(p<0.05)(8)Compared with the TNF-α group,Asiaticoside significantly down-regulated the expression of TLR4 and NF-κBp65 proteins in keratinocytes(p<0.05);(9)Compared with the TNF-α group,Asiaticoside significantly down-regulated the expression of Wnt5a,Wnt7a and β-Catenin protein in keratinocytes(p<0.05);(10)Compared with the TNF-α group,asiaticoside increased the expression of Nrf2 and HO-1 protein in keratinocytes;(11)Compared with the TNF-α group,the Asiaticoside low and high dose groups can significantly reduce the expression of IFN-y and increase the expression of IL-4 in keratinocytes(p<0.05).Conclusion:(1)Asiaticoside has a certain inhibitory effect on mast cell allergic inflammation in vivo and in vitro,and its mechanism may be related to the inhibition of TLR4/NF-κB and Wnt/β-Catenin signaling pathways and activation of Nrf2/HO-1 Signal pathway related;Asiaticoside can alleviate the inflammatory response of asthma by inhibiting the activity of TLR4/NF-κB and Wnt/β-Catenin signaling pathways;(2)Asiaticoside may alleviate airway inflammation in mice with allergic asthma by inhibiting TLR4/NF-κB and Wnt/β-Catenin signaling pathways and activating Nrf2/HO-1 signaling pathways;(3)Asiaticoside may inhibit the TLR4/NF-κB and Wnt/β-Catenin signaling pathway and activate the Nrf2/HO-1 signaling pathway to alleviate the inflammatory response of allergic contact dermatitis in mice. |
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