| Purpose:Evaluate the effects of Sea buckthorn(SBT)oil and total flavonoids of Hippophae rhamnoides L.(TFH)on atopic dermatitis mice and the possible immune mechanism.Further to provide scientific basis for seeking complementary candidate for AD treatment from natural meterials.Material and method:Part 1.Female healthy specific pathogen-free BALB/c mice were randomly divided into 4 groups: the normal control group,AD model group,AD model group treated with SBT oil(5 ml/kg)and AD model group treated with SBT oil(10 ml/kg),each group with 6 mice.The dorsal skin of each mouse was shaved following DNCB(200 ?l,1%)sensitization three times in total at day 1?4?7 and the skin of left ear was challenged by DNCB(20 ?l,0.5%)seven times in total at day 14?17?19?22?24?27?29.SBT oil(5 ml/kg,10 ml/kg)was applied for AD model group treated with SBT oil by intragastric administration once a day from day15 to day 29,olive oil with the same volume was given for the normal control group and AD model group.At the same time the thickness of left ear was measured every 3 days.All animals were sacrificed at day 30 and samples including blood,left ear tissues and submaxillary lymph node were collected.Left ear tissues were fixed in 4% paraformaldehyde,part of lymph node was made into single cell suspension and remaining was frozen at-80oC.HE and TB staining were employed both to observe pathological features of left ear skin in all mice and to count mast cell number.Serum Ig E level was detected by ELISA.Protein expression level of IL-4?IFN-??TNF-? and TSLP in ear tisse was assessed by quantitive immunohistochemical staining.m RNA expression level of IL-4?IFN-? and TNF-? in lymph node was assessed by real-time polymerase chain reaction(q PCR).Percentage of CD207/CD326?CD86?OX40 and MHC? positive cells in single cell suspension of submaxillary lymph node were determined by flow cytometry.Part 2.Female healthy specific pathogen-free C57BL/6 mice were randomly divided into 4 groups: normal control group,AD model group,cream base treatment group and TFH treatment group,each group with 6 mice.Left ear skin of mice in AD model group,cream base treatment group and TFH treatment group were applied by 2nmol MC903(20 ?l,dissolved in ethanol)once a day from day 1 to 14 to induce AD-like lesion.Mice in normal control group were applied by alcohol at the same time.1% TFH cream(20 ?g)and cream base(20 ?g)were applied topically to ears of mice once a day from day 7 to 14.Thickness of left ear was measured at day 7?10?12?15.All animals were sacrificed at day 15 and samples including blood,left ear tissues and submaxillary lymph node were collected.Lymph node and part of left ear tissue were fixed in 4% paraformaldehyde,remaining left ear tissue was frozen at-80oC.HE and TB staining were employed both to observe pathological features of left ear skin in all mice and to count mast cell number.Expressions of filaggrin(FLG)and loricrin(LOR)were evaluated immunohistochemically.q PCR was performed to assess the m RNA levels of thymic stromal TSLP,IL-4,IFN-?,TNF-? in skin lesions.Ha Ca T cells were cultured in DMEM(with 4.5 g/L glucose)medium,which was supplemented with 10% fetal bovine serum,penicillin(100U/m L),and streptomycin(100?g/m L).Ha Ca T cells kept attached state at 37? in a humidified 5% CO2 incubator.Cell passage was done every other day in 1:2 proportion.Cell was divided into necessary groups according to research purpose and correspongding experiments were carried out.(1)Cell viability was measured by CCK8 assay to identify the effect of TFH with different concentration on Ha Ca T cells.(2)Cytokine antibody arrays were carried out to screen cytokines in IFN-?/TNF-? activated Ha Ca T cells.(3)Results of cytokine antibody arrays were checked by ELISA.(4)Western-blotting was employed to measure the expression level of p-NF-?B,p-ERK and p-P38 in all groups to explore the effects of TFH on NF-?B and MAPK signaling pathway in IFN-?/TNF-? activated Ha Ca T cells.Results: Part 1.1.Morphology examination indicates: No obvious inflammation was appeared during the entire experiment process in mice of normal control group.Left ear skin of mice in AD model group became reddening,swelling,rough and hard since day 7,dryness and itching appears since day 14.The inflammation manifestation became more and more serve along with the increase of chanllenge,even hemorrhage and festering arise in some mice.Since treatment was applied the inflammation manifestation remit gradually of mice in SBT oil treatment groups.Compared with normal control group,dermatitis scores of mice in AD model group was inclined significantly(p<0.01).Compared with AD model group,dermatitis scores of mice in SBT oil treatment groups was declined significantly(p<0.01).2.HE staining indicates: The structure of epidermal and the morphology of cells in left ear skin tissue of mice in normal control group was excellent without damage.The hyperkeratosis or poor keratinization of epidermal in left ear skin tissue of mice arised in AD model group.Compared with normal control group,epidermal thickness of mice in AD model group was increased significantly(p<0.01).Compared with AD model group,epidermal thickness of mice in SBT oil treatment groups was declined significantly(p<0.01).3.TB staining indicates: Compared with normal control group,mast cell number of mice in AD model group and SBT oil treatment groups were increased significantly(p<0.01).Compared with AD model group,mast cell number of mice in SBT oil treatment groups was declined significantly(p<0.01).4.Serum Ig E level of mice in all groups indicates: Compared with normal control group,serum Ig E level of mice in AD model group and SBT oil treatment groups was increased significantly(p<0.01 or p<0.05).Compared with AD model group,serum Ig E level of mice in SBT oil treatment groups was declined significantly(p<0.01).5.Weight of submaxillary lymph node of mice in all groups indicates: Compared with normal control group,weight of submaxillary lymph node of mice in AD model group and SBT oil treatment groups was increased significantly(p<0.01 or p<0.05).Compared with AD model group,weight of submaxillary lymph node of mice in SBT oil treatment groups was declined significantly(p<0.01 or p<0.05).6.Protein expression of IL-4?IFN-??TNF-? and TSLP in ear tissue according to immunohistochemical staining indicates: Compared with normal control group,protein expression of IL-4?IFN-??TNF-? and TSLP in ear tissue of mice in AD model group and SBT oil treatment groups was increased significantly(p<0.01).Compared with AD model group,protein expression of IL-4?IFN-??TNF-? and TSLP in ear tissue of mice in SBT oil treatment groups was declined significantly(p<0.01).7.m RNA expression of IL-4?IFN-? and TNF-? in submaxillary lymph node of mice in all groups indicates: Compared with normal control group,m RNA expression of IL-4?IFN-? and TNF-? in submaxillary lymph node of mice in AD model group and SBT oil treatment groups was increased significantly(p<0.01 or p<0.05).Compared with AD model group,m RNA expression of IL-4?IFN-? and TNF-? in submaxillary lymph node of mice in SBT oil treatment group(10ml/kg)was decreased(p<0.01 or p<0.05).Compared with AD model group,m RNA expression of IFN-? and TNF-? in submaxillary lymph node of mice in SBT oil treatment group(5ml/kg)was decreased(p<0.05).8.Percentage of CD207/CD326?CD86?OX40 and MHC? positive cells in single cell suspension of submaxillary lymph node indicates: Compared with normal control group,percentage of CD207/CD326?CD86?OX40 and MHC? positive cells in single cell suspension of submaxillary lymph node of mice in AD model group and SBT oil treatment groups was increased significantly(p<0.01).Compared with AD model group,percentage of CD207/CD326?CD86?OX40 and MHC? positive cells in single cell suspension of submaxillary lymph node of mice in SBT oil treatment groups was decreased significantly(p<0.01).Part 2.1.Morphology examination indicates: No obvious inflammation was appeared during the entire experiment process in mice of normal control group.Left ear skin of mice in AD model group became reddening,swelling,rough and hard since day 7.The inflammation manifestation became more and more serve along with the increase of MC903 applying,even hemorrhage and festering arise in some mice.No obvious difference was found between AD model group and cream base treatment group.Inflammation manifestation remit gradually of mice in TFH treatment groups.Compared with normal control group,dermatitis scores of mice in AD model group was inclined significantly(p<0.01).Compared with AD model group,dermatitis scores of mice in TFH treatment group was declined significantly(p<0.01).2.Left ear thickness of mice in all groups indicates: At day 7,compared with normal control group,dermatitis scores of mice in AD model group?cream base treatment group and TFH treatment group was inclined significantly(p<0.01).At day 10,compared with normal control group,dermatitis scores of mice in AD model groups?cream base treatment group and TFH treatment group was inclined significantly(p<0.01);compared with AD model group,dermatitis scores of mice in TFH treatment group was decreased(p<0.05).At day 12 and 15,compared with normal control group,dermatitis scores of mice in AD model group?cream base treatment group and TFH treatment group was increased significantly(p<0.01),compared with AD model group,dermatitis scores of mice in TFH treatment group was decreased significantly(p<0.01).At day 7?10?12 and 15,compared with AD model group,no difference was found in cream base treatment group(p>0.05).3.HE staining indicates: The structure of epidermal and the morphology of cells in left ear skin tissue of mice in normal control group was excellent without damage.The hyperkeratosis or poor keratinization of epidermal in left ear skin tissue of mice arised in AD model group.Compared with normal control group,epidermal thickness of mice in AD model group was increased significantly(p<0.01).Compared with AD model group,epidermal thickness of mice in TFH treatment group was declined significantly(p<0.01),and no difference was found in cream base treatment group(p>0.05).4.TB staining indicates: Compared with normal control group,mast cell number of mice in AD model group?cream base treatment group and TFH treatment group were increased significantly(p<0.01).Compared with AD model group,mast cell number of mice in TFH treatment group was decreased significantly(p<0.01),and no difference was found in cream base treatment group(p>0.05).5.Weight of submaxillary lymph node of mice in all groups indicates: Compared with normal control group,weight of submaxillary lymph node of mice in AD model group?cream base treatment group and TFH treatment group were increased significantly(p<0.01).Compared with AD model group,weight of submaxillary lymph node of mice in TFH treatment group was decreased significantly(p<0.01),and no difference was found in cream base treatment group(p>0.05).6.Protein expression of FLG and LOR in ear tissue according to immunohistochemical staining indicates: Compared with normal control group,protein expression of FLG and LOR in ear tissue of mice in AD model group and cream base treatment group were decreased significantly(p<0.01),no difference was found in TFH treatment group(p>0.05).Compared with AD model group,protein expression of FLG and LOR in ear tissue of mice in TFH treatment group was increased significantly(p<0.01),and no difference was found in cream base treatment group(p>0.05).7.m RNA expression of IL-4?IFN-??TNF-? and TSLP in left ear tissue of mice in all groups indicates: Compared with normal control group,m RNA expression of IL-4?IFN-??TNF-? and TSLP in left ear tissue of mice in AD model group?cream base treatment group and TFH treatment group were increased significantly(p<0.01).Compared with AD model group,m RNA expression of IL-4?IFN-??TNF-? and TSLP in left ear tissue of mice in TFH treatment group was decreased significantly(p<0.01),and no difference was found in cream base treatment group(p>0.05).8.Effect of TFH with different concentration on viability Ha Ca T cells indicates: Compared with 0mg·L-1 TFH treatment group,OD value increased significantly when TFH concentration was 2.5 mg·L-1.9.Cytokine antibody arrays results indicates: Compared with blank control group,level of IL-1??IL-1??IL-6?MCP-1?MCP-3?MDC?PDGF-BB and TARC in TNF-?/IFN-? intervened group was increased significantly(p<0.05).Compared with TNF-?/IFN-? intervened group,level of IL-1??IL-1??IL-6?MCP-1?MCP-3?MDC?PDGF-BB and TARC in TNF-?+IFN-?+TFH intervened group was decreased significantly(p<0.05).10.Level of IL-6?MDC and TARC of supernatant by ELISA in all groups indicates: Compared with blank control group,level of IL-6?MDC and TARC in other four groups was increased sifnificantly(p<0.05).Compared with TNF-?/IFN-? intervened group,level of IL-6?MDC and TARC in TFH intervened groups(with 3 different concentration)was decreased significantly(p<0.01)in a dose dependent manner.11.Protein expression of p38?ERK?NF-?B and their phosphates by Western-blot indicates: Compared with blank control group,protein expression of p38?ERK?NF-?B in other four groups was no significant different(p>0.05).Compared with blank control group,protein expression of p38?ERK?NF-?B in TNF-?/IFN-? intervened group was increased significantly(p<0.01).Compared with TNF-?/IFN-? intervened group,protein expression of p-p38?p-ERK?p-NF-?B in TFH intervened groups(with 3 different concentration)was decreased significantly(p<0.01)in a dose dependent manner.Conclusion: 1.SBT oil has the ability to reduce the local inflammatory response of AD mice,the intensity of hypersensitive response led by Ig E,and intensity of immune response of local draining lymph node.2.SBT oil could down-regulate the expression of TSLP in skin lesion of AD mice and inhibite the activation and antigen capture capability of LCs,finally regulate Th1/Th2 balance.3.TFH could reduce the local inflammatory response of AD mice and intensity of immune response of local draining lymph node.4.TFH could up-regulate expression of FLG and LOR in skin lesion of AD mice,ulteriorly repair and protect skin barrier damaged in AD mice.5.TFH could restore Th1/Th2 balance by inhibiting the the expression of TSLP in skin lesion of AD mice 6.TFH(2.5 mg·L-1)could improve Ha Ca T cells proliferation.7.TFH could inhibite the inflammation response of Ha Ca T cells through MAPK-NF-?B signaling pathway. |
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