Research Of Shenlingbaizhusan On Regulating Intercellular Lipids And Tight Junction Protein In Epidermis To Repairing Skin Physical Barrier Function

Objective:Our previous study showed that Shenlingbaizhusan could increase skin hydration,decrease TEWL,alleviate pathological epidermis thickening,hyperkeratosis and parakeratosis,increase total lipids in epidermis of skin physical barrier dysfunction mice model induced by organic reagent.It showed an effect on repairing skin physical barrier.In this study,we observe the effect of Shenlingbaizhusan on intercellular lipids FFA,CHOL,Cer and every subclass of Cer in epidermis,especially the effect on the content of ACer,a type of indispensable lipids for skin barrier function,and the protein expression of enzymes in biosynthesis of ACer.Moreover,we observe the effect on the protein expression of tight junction.We hope to elucidate the mechanism from regulating intercellular lipids and tight junction proteins in epidermis,and provide evidences for treating dermatosis with skin barrier dysfunction.Methods:1.Grouping and treatment of experimental animalsThe ICR mice were randomly divided into four time-groups: 1-day modeling group,3-day modeling group,5-day modeling group and 7-day modeling group.In each time-group,the mice were randomly divided into control group,model group and Shenlingbaizhusan(SLBZS)group.From the first day of the experiment,SLBZS groups were given Shenlingbaizhusan decoction(20g/kg)by gavage,and the other groups were given the same volume distilled water once a day for17 consecutive days.On the 10 th day of the experiment,all mice were shaved to expose back skin.On the 11 th day,skin physical barrier dysfunction model was replicated by organic reagent(acetone: ether=1:1)except for control groups.The model was replicated in mice twice a day with8 hours interval.After the model was replicated for 1 day,3 days,5 days and 7 days,the corresponding time-group animals were executed by cervical dislocation,the back skin was taken and cut into square pieces with 1.2 cm side length,and then were refrigerated at-80°C.2.The research of Shenlingbaizhusan on regulating intercellular lipids in epidermis to repairing skin physical barrierfunctionThe content of FFA and CHOL in skin samples of each group was determined by ELISA respectively.The content of total ceramides and each subclass ceramides was determined by high performance liquid chromatography-tandem mass spectrometry.The protein expression of enzymes ELOVL1,ELOVL4,CYP4F22,CERS3 and PNPLA1 in synthesis pathway of ACer was detected by Western Blot.The difference between groups was analyzed.3.The research of Shenlingbaizhusan on regulating protein expression of tight junction proteins to repairing skin physical barrierfunctionThe protein expression of tight junction proteins,Claudin-1 and Occludin,in the skin was detected by Western Blot.The difference between groups were analyzed.Results:1.The research of Shenlingbaizhusan on regulating intercellular lipids in epidermis to repairing skin physical barrierfunction(1)Increasing the intercellular lipid FFAin epidermis of model animalsIn 1-day,3-day,5-day,7-day modeling groups,the FFA content in model group was lower than control groups or showed downward trend compared with control groups(P<0.01,P>0.05,P<0.001,P<0.001).In 3-day,7-day modeling groups,the FFA content in SLBZS groups was showed upward trend compared with model groups(P>0.05).In 5-day modeling group,the FFA content in SLBZS group was higher than model group(P<0.01).(2)Increasing the intercellular lipid CHOLin epidermis of model animalsIn 1-day,3-day,5-day,7-day modeling groups,the CHOL content in model groups was lower than control groups(P<0.001).In 3-day,5-day modeling groups,the CHOL content in SLBZS groups was showed upward trend compared with control groups(P>0.05).In 7-day modeling group,the CHOLcontent in SLBZS group was higher than model group(P<0.01).(3)Effect on intercellularlipid Cerin epidermis of model animalsIn 1-day,3-day,5-day,7-day modeling groups,Cer content in model groups showed downward trend compared with control groups(P>0.05).In 1-day,5-day modeling groups,Cer content in SLBZS groups was higher than model groups(P<0.01,P<0.05).8 subclasses Cer were detected in mice epidermis,they were Cer-NS,Cer-ADS,Cer-NH,Cer-NP,Cer-AH,Cer-AP,Cer-EOS and Cer-EOH.The last 2 classes belong to ACer,a kind of Cer with very long chain fatty acid.In 1-day modeling group,the content of Cer-NP and Cer-AH in SLBZS group was higher than model group(P<0.05).In 3-day modeling group,Cer-EOH content in model group was lower than control group(P<0.05),and Cer-AH content in model group was higher than control group(P<0.05),Cer-AH content in SLBZS group was lower than model group(P<0.05).In 5-day modeling group,the content of Cer-NS,Cer-ADS,Cer-NP and Cer-AH in SLBZS group was higher than model group(P<0.05).Among the 8 subclass Cer,Cer-EOS and Cer-EOH are ACer with ultra-long chain,which play a key role in the skin physical barrier function.In 3-day modeling group,ACer content in model group was lower than control group(P<0.05).In 5-day modeling group,ACer content in model group showed only downward trend compared with control group(P>0.05),however,ACer content in SLBZS group was higher than model group(P<0.05).(4)Effect on the protein expression of enzymes in ACerbiosynthetic pathwayIn 3-day,5-day,7-day modeling groups,the protein expression of ELOVL1 in model group was lower than control group(P<0.05,P<0.01,P<0.05).In 3-day,5-day modeling groups,the protein expression of ELOVL1 in SLBZS group showed upward trend compared with model group(P>0.05).In 7-day modeling group,the protein expression of ELOVL1 in SLBZS group was higher than model group(P<0.05).In 1-day,3-day,5-day,7-day modeling groups,the protein expression of ELOVL4 in model groups was lower than control groups or showed downward trend compared with control groups(P>0.05,P<0.05,P<0.01,P<0.05).In 1-day,3-day modeling groups,the protein expression of ELOVL4 in SLBZS groups showed upward trend compared with model groups(P>0.05).In5-day,7-day modeling groups,the protein expression of ELOVL4 in SLBZS groups was higher than model groups(P<0.05).In 1-day,3-day,5-day modeling groups,the protein expression of CYP4F22 in model groups was lower than control groups(P<0.05,P<0.01,P<0.01).In 1-day modeling group,the protein expression of CYP4F22 in SLBZS group showed upward trend compared with model group(P>0.05).In 3-day modeling group,the protein expression of CYP4F22 in SLBZS groups was higher than model group(P<0.01).In 5-day,7-day modeling groups,the protein expression of CERS3 in model groups was lower than control groups(P<0.05),and the protein expression of CERS3 in SLBZS groups showed upward trend compared with model groups(P>0.05).In 3-day,5-day,7-day modeling groups,the protein expression of PNPLA1 in model groups was lower than control groups or showed downward trend compared with control groups(P<0.01,P<0.01,P>0.05).In 3-day modeling group,the protein expression of PNPLA1 in SLBZS groups was higher than model group(P<0.05).In 5-day modeling group,the protein expression of PNPLA1 in SLBZS group showed upward trend compared with model group(P>0.05).2.Research of Shenlingbaizhusan on regulating protein expression of tight junction proteins to repairing skin physical barrierfunctionIn 3-day,5-day,7-day modeling groups,the protein expression of Claudin-1 in model groups was lower than control groups(P<0.01),however,in 5-day,7-day modeling groups the protein expression of Claudin-1 in SLBZS groups was higher than model groups(P<0.05).In 5-day,7-day modeling groups,the protein expression of Occludin in model groups was lower than control groups(P<0.01,P<0.05),however in 5-day modeling group the protein expression of Occludin in SLBZS group was higher than model group(P<0.05).Conclusions:Shenlingbaizhusan can increase the content of intercellular lipids FFA,CHOL,Cer,ACer in epidermis,and increase the protein expression of ELOVL1,ELOVL4,CYP4F22 and PNPLA1 the enzymes in biosynthetic pathway of ACer.It may repaire the skin barrier function by increasing epidermal intercellular lipids.Furthermore,Shenlingbaizhusan may repaire skin barrier function by increasing the protein expression of tight junction protein Claudin-1 and Occludin.