The Effect Of VD Adjuvant Treatment On The Children Atopic Dermatitis And Its Possible Molecular Mechanism

Part I Analysis of Serum Liposoluble Vitamin Levels in children with Atopic DermatitisBackground: A small number of studies have suggested that vitamin D deficiency(VDD)is associated with atopic dermatitis(AD).Vitamin D may play a protective role in the pathogenesis of AD by regulating immunity and affecting skin barrier function.However,few studies have been explored the relationship between AD and other fat-soluble vitamins,especially vitamin A(VA),vitamin E(VE),Which also play an important role in maintaining the normally physiological function of skin.objective: To detect the serum levels of VA,VD and VE in children with AD.To explore the correlation between the levels of VA,VD and VE and the severity of AD,Whether there is any interaction between these fat-soluble vitamins in AD patients.The results will provide more evidence for the clinical treatment of AD.Methods:According to Hanifin-Rajka criteria,AD patients aged 2-12 years were collected from 2013 to 2015 and compared with age-and gender-matched healthy children at the same time.The severity of AD was assessed by SCORAD method.The serum total Ig E level was detected by ELISA.Absolute eosinophil count was detected by Hematology analyzer.Serum levels of VA and VD were determined by high performance liquid chromatography.Serum level of VE was determined by high performance liquid chromatography-mass spectrometry.Results.: 81 patients with AD and 65 healthy controls were included.Their age and male are matched.The average SCORAD in AD patients was 45.48±13.36.33 children had moderate AD(40.7%),while 48 had severe AD(59.3%).The average level of VD in children with AD was significantly lower than that in normal children(33.51±7.45 nmol/L,50.79±15.43 nmol/L,p<0.001).The level of vitamin D has dramtaticl difference beteewn severe AD group and moderate group(p= 0.0087).The mean levels of VA in patients with AD and individuals in the control group were 0.87±0.25 ?mol/L and 0.96±0.24 ?mol/L,respectively,and there was a significant difference between the two groups(P=0.0423).The level of vitamin A has no difference in severe group and moderate group(P=0.0730).The level of vitamin E in AD patients was 19.8±5.61?mol/L,20.59±5.1?mol/L respectively.There has no differce between AD patients and healthy controls.The proportions of children with VD deficiency(VDD)and VA deficiency(VAD)were higher in the AD group.SCORAD scores were significantly higher in AD patients with both VDD and VAD(co-deficiency)than in other AD patients.Both VD and VA levels were negatively correlated with SCORAD(r=-0.3880,P < 0.001 and r=-0.4203,P<0.001 respectively).There was no relationship between serum levels of VE and AD severity.Total serum Ig E levels and eosinophil counts were significantly higher in AD patients than in healthy controls and positively correlated with the severity of the disease.There was no correlation between serum total Ig E levels and either VA or VD levels in AD patients.However,there was a significant inverse correlation between peripheral blood eosinophil counts and serum levels of VA and VD(r=-0.2385 P=0.0367;r=-0.2498 P=0.0264).Conclusion:The levels of vitamin A and D were not adequate in AD patients,what more the level of vitamin A and D were negatively correlated with the severity of the disease.The level of VE was normal.VA and VD co-deficiency might exacerbate AD symptoms in children.VA and VD deficiency may result in disfuction of skin innate immunity and skin barrier,spontaneously increase the activation of eosinophils in vivo,which aggravate the skin inflammation.Par T ? The safety and efficacy about Vitamin D adjuvant treatment of atopic dermatitisBackgroud: More and more studies have shown that VD deficiency is associated with AD,especially in children.Recently two newest meta-analysis indicate that VD supplementation may alleviate the symptoms of AD and could be a new therapeutic option for AD.However,the experimental evidence is inconsistent.Objective: To explore the safety and effectiveness of vitamin D supplementation on children with atopic dermatitis,in order to find out the best way of VD supplementation,the best dose of VD supplementation,the propriative course of VD supplementation and the best goal of VD for atopic dermatitis.Methods: Children with AD who met Hanifin-Rajka criteria and whose serum VD level was less than 50 nnmol/L were randomly divided into four groups.The VD0 group only received basic treatment(Mometasone furoate once a night stopped after 2 weeks,followed by skin moisturizing therapy).The other three groups received different doses of VD by intramuscular injection or oral administration,namely,VD1 group(oral 800 IU VD3 daily),VD2 group(oral 2000 IU per day),VD3 group(intramuscular VD2 100,000 units per month).After supplementation,follow-up was conducted at two time points:1 month and 3 months.The side effects was documented.The liver and kidney function,blood calcium(Ca),blood phosphorus(Pi),alkaline phosphatase(AKP)and parathyroid hormone(PTH)were detected.VD and SCORAD was assessed at each follow-up.Results: No obvious adverse events occurred during the whole supplementation process.When the dosage of vitamin D was 800 units and 2000 units,the effects on serum calcium,serum phosphorus and PTH were slight.There was no significant difference in the changes of serum levels of the above indicators before and after supplementation.Treatment with 100,000 units VD2 has an effect on serum calcium and PTH in vivo.Serum calcium level will increased significantly.Similarly,under this dose,serum level of PTH will decrease significantly after 1month supplementation.But over time,with the self-adjustment,parathyroid hormone and serum calcium can return to the previous state,so that the body was in a steady state again.The levels of VD increased significantly in each group after 1month vitamin D supplementation.Compared with VD0 group,the serum VD levels in each group were significantly higher(P < 0.001).The mean level of vitamin D in VD1 group was 41.61±6.61 nmol/L).There are 2 cases(14.3%)whose vitamin D levels were greater than 50 nmol/L.The average level of vitamin D in VD2 group was 44.01±6.05 nmol/L),3 cases(18.8%)had adequate vitamin D levels.The serum level of vitamin D in VD3 group was 39.37±7.60 nmol/L,and only 1 case had normal vitamin D.There was no difference in VD levels between the groups after 1month VD supplementation.After 3 months of supplementation,vitamin D level increased significantly in all groups(P < 0.001).Only 1 case(4.76%)in the VD0 group exceeded 50 nmol/L.The mean value of vitamin D in each group was: 33.37±5.60 nmol/L,44.32±7.74 nmol/L,60.16±8.05 nmol/L,47.10±12.64 nmol/L respectively,and there were 5 cases(35.7%),14 cases(87.5%)and 8 cases(42.1%)whose vitamin D were normal in each supplementary group.The levels of vitamin D in VD2 group were significantly higher than those in VD1 group and VD3 group after 3 months intervention.There was no significant difference between VD1 group and VD3 group.After 1month vitamin D supplementation,the SCORAD of AD children in the intervention groups were 30.91±8.582,33.03±8.183,37.28 ±8.745,respectively.Compared with the baseline SCORAD,the SCORAD of the intervention groups were significantly decreased,and there was no difference among the groups.There was no significant difference in SCORAD between the groups and VD0 group(P= 0.1129).After vitamin D supplementation for 3 months,the SCORAD of AD children in each group were 27.56±9.224,22.18 ±5.086,26.47 ±5.437,significantlly lower than the baseline,and there was a statistically difference among the group with or without vitamin D supplementation(P < 0.0001).Compared with VD0 group,SCORAD in VD2 group and VD3 group decreased significantly.At the end of the trial,the SCORAD in VD normal group(> 50 nmol/L)and VD insufficient group(< 50 nmol/L)were 22.32±5.454 and 29.14 ±6.801,respectively.The disease severity in VD normal group decreased significantly.Morever deta vitamin D levels were positively correlated with deta SCORAD(P = 0.008,r = 0.377).Conclusion: Vitamin D supplementation is a well-tolerated,safe and effective treatment for children with AD.Duration should not be too short.Oral supplementation with 2000 IU per day was the most effective way.50 nmol/L can be used as a target of VD supplementation in children with AD.Part ? Protective mechanism of vitamin D in atopic dermatitisBackground: TLR-2 and LL-37 play a dual role in skin barrier and innate immunity.Previou studies have shown that actived TLR2 can induce the expression of LL-37 in macrophage via VD/VDR pathway thus directly killing Mycobacterium tuberculosis in vitro.Both previous studies and our results have shown that vitamin D is effective for atopic dermatitis,but there are few studies which focus on the underlying mechanism.The extraosseous effect of vitamin D suggests that it can regulate innate immunity and acquired immunity by gene and non-gene mechanism,affect the expression of structural protein of skin barrier,and make it possible that vitamin D may play a protective role in atopic dermatitis.We hypothesize that vitamin D plays a protective role in supplemtation treatment for atopic dermatitis through VD/VDR-TLR2-LL-37 pathway.Objective:To detect the expression of TLR2 and LL-37 which related to skin barrier and innate immunity before and after VD supplementation in atopic dermatitis.To analyze the relationship between TLR2,LL-37 and vitamin D as well as SCORAD,and to explore the possible mechanism of VD protective effect on atopic dermatitis.Methods: This part includes two sections: the first section is about AD patients.AD patients aged 2-12 years old were enrolled according to Hanifin-Rajka criteria.Age and gender mateched healthy individuals were the control.Real-time PCR was used to detect the expression of VDR,TLR2 and LL-37 in peripheral blood leucocytes.ELISA was used to detect the level of LL-37 in serum.AD patients whose VD level was less than 50 nmol/L were supplemented with 2000 IU vitamin D daily.After 3 months,the levels of VD,SCORAD score,the expression of VDR,TLR2 and LL-37 in peripheral blood leucocytes and the serum level of LL-37 were analysed before and after vitamin D intervention.The second section focuses on the immortal Ha Ca T cell.The expression of TLR2,VDR and LL-37 in Ha Ca T cells was analyzed after stimulating by TLR2 receptor agonist Staphylococcus aureus peptidoglycan(PGN-SA).Secondly,Ha Ca T cells were pretreated with VD and stimulated with PGN-SA,then analyze the expression of TLR2 and LL-37.Results: After 3 months vitamin D supplementation,the level of VD increased significantly and SCORAD decreased dramatically.The expression of VDR,TLR2 and LL-37 in peripheral blood leukocytes of AD children was significantly lower than that of healthy controls.The expression of LL-37 in leukocytes was positively correlated with VDR and TLR2.The expression of LL-37 in leukocytes was positively correlated with serum vitamin D level.The serum level of LL-37 in AD children was significantly lower than that of healthy controls,but not related to the level of vitamin D and SCORAD.After vitamin D supplementation,the expression of TLR2 and LL-37 and the level of serum LL-37 increased significantly.TLR2,VDR and LL-37 of Ha Ca T cell were significantly expressed at 24,48 and 72 hours respectively after PGN-SA stimulated.After VD pretreatment,PGN-SA could induce overexpression of TLR2 and LL-37 in Ha Ca T cells.Conclusion:Continuous oral supplementation of VD 2000 IU per day for 3 months can help alleviate symptoms of atopic dermatitis.The lower expression of TLR2 and LL-37 in children with atopic dermatitis indicate skin barrier and innate immune defet which may be contributed to susceptible to bacterial and virus.Vitamin D may promote the expression of LL-37 by activating VDR via TLR2 pathway,improve the body barrier and innate immune,and play an active protective role in AD.