CN110141563A - A kind of drug and its animal model and construction method improving acne rosacea symptom - Google Patents
A kind of drug and its animal model and construction method improving acne rosacea symptom Download PDFInfo
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- CN110141563A CN110141563A CN201910379249.0A CN201910379249A CN110141563A CN 110141563 A CN110141563 A CN 110141563A CN 201910379249 A CN201910379249 A CN 201910379249A CN 110141563 A CN110141563 A CN 110141563A
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Abstract
The invention belongs to skin diseases pharmaceutical techniques fields, a kind of drug and its animal model and construction method for improving acne rosacea symptom is disclosed, uses 7 week old BALB/C mices 2 times/day of LL37 polypeptide injections to be subcutaneously injected 2 days to induce acne rosacea sample skin injury to carry out acne rosacea modeling；Animal model, mouse shift to an earlier date 5 days stomach-filling tranexamic acids, drug control group stomach-filling PBS buffer solution PBS, stomach-filling induction acne rosacea sample skin injury in the 6-7 days.By the present invention in that having evaluated TXA to acne rosacea influence and its molecular mechanism with the mouse model of LL37 induction and HaCaT cell model；Being handled by TXA significant improves acne rosacea sample symptom；In addition, TXA reduces the expression of congenital immunity gene (TLR2, KLK5 and Camp) and neutrophil leucocyte related gene in acne rosacea sample lesion.
Description
Technical field
The invention belongs to skin diseases pharmaceutical techniques field more particularly to it is a kind of improve acne rosacea symptom drug and
Its animal model and construction method.
Background technique
Acne rosacea is a kind of common chronic facial diseases associated with inflammation, and be generally divided into 4 kinds of Main Subtypes: erythema blood vessel expands
Extensional acne rosacea (ETR), papulopustular acne rosacea (PPR), acne rosacea (PHR) and eye (OR) acne rosacea.Rose
The Clinical symptoms of rare acne includes face red spot, papule, warts, telangiectasis and repeatedly flush, although it is not a kind of
Fatal disease, but the quality of life of acne rosacea patient is adversely affected.Further, it has recently been demonstrated that acne rosacea
It is related with dull-witted and pathogenesis of cancer risk increase.These discoveries show that acne rosacea may be the reaction of systemic disease, value
It must pay close attention to.
The practice guidelines of American National acne rosacea association think that acne rosacea should be given according to different subtype and control accordingly
It treats.This technology is mainly for erythematous telangiectatic type.It include at present part for the drug of erythematous telangiectatic type
Treatment, systematic treating and laser therapy.The external drug occurred in Chinese and foreign every country treatment guidelines at present include adrenaline by
Body agonist, azelaic acid etc..Alpha adrenergic receptor agonist, can be by promoting vascular smooth muscle contraction, to shrink blood
Pipe, therefore only have effect to the blood vessel for having smooth muscle layer, and minute blood vessel and capillary for lacking smooth muscle layer are without receipts
Contracting effect.In the application of azelaic acid, though find that papulopustular type patient symptom of erythema after treating is alleviated in clinical research, nothing
The performance of 15% azelaic acid gel on clinical of clinical research confirmation is only that the acne rosacea of erythema has therapeutic effect.It is mentioned in the common recognition of the whole world
Out, can system take orally beta-2 adrenoceptor retarding agent for example Carvedilol mitigate the thin blood vessel dilatation type acne rosacea of erythema arteries and veins battle array
It sends out sexflush and the case where erythema perstans, Carvedilol can reduce myocardial contraction, slow down heart rate, contract skin blood vessel, but this
Kind drug can cause patients' blood to decline, and hypertensive patient should not take.In addition, system takes Doxycycline For The Treatment acne rosacea
Erythema, but its according to be due to can be adjoint during Doxycycline For The Treatment papulopustular type acne rosacea erythema alleviate it is indirect
Evidence, and the evidence of indirect treatment erythematous telangiectatic type acne rosacea.Therefore, all there is disadvantage in present various drugs
End.The study find that oral tranexamic acid also can reduce the erythema perstans symptom of acne rosacea, then it is the treatment of acne rosacea
A new approaches are provided, the patient of existing drug cannot be used to provide new method for those.
Although the pathophysiological mechanism of acne rosacea not yet illustrates completely, current evidence shows that acne rosacea is more
It is always immunized over year, blood vessel and the reason of neurological dysfunction.Studies have shown that abnormal innate immune response is in rose always
Key effect is played in the progress of acne.Toll-like receptor 2 (TLR2), antibacterial peptide (Camp) expression increases, in acne rosacea skin
It is also observed kallikrein 5 (KLK5) in skin damage, and erythema and mound in acne rosacea is improved to the inhibition of KLK5
Rash.LL37 is a kind of important polypeptide found in acne rosacea, it was reported that it can induce mouse and acne rosacea sample skin occurs
Inflammation.Also observe that innate immune cells infiltrate in acne rosacea, including macrophage, neutrophil leucocyte and mast cell,
Adaptive immunity also plays certain effect, including CD4+T cell in acne rosacea.In addition, as blood vessel and nervous function
The skin disease of obstacle, the Histopathological Characteristics of acne rosacea are also embodied by blood vessel dilatation, angiogenesis and nerve fibre it is light
Micro- increase.Genome and qPCR vascular endothelial growth factor (VEGF) and neurogenic inflammation gene (TRP) as the result is shown, vanilla
Aldehyde receptor 1 (TRPV1) increases in acne rosacea.The acne rosacea clinical medicine of U.S. FDA approval includes Doxycycline and α-
Adrenoceptor agonists, the clinical efficacy of these medicaments are attributed to their anti-inflammatory or anti-angiogenesis characteristic.
Tranexamic acid (TXA) is a kind of lysine derivative, is typically used as antifibrinolysis agent, to reduce blood friend
The risk of excessive bleeding in disease, menorrhalgia and clinical surgery operation.In addition, research shows that TXA is in openheart surgery, acute lung
There is potential anti-inflammatory effect in damage.Recently, TXA is used as the therapeutic choice of acne rosacea, but it treats acne rosacea
Specific mechanism is still unclear.It can only know that tranexamic acid can have effect by previous research report, but since the cause of disease is multiple
It is miscellaneous, specifically which link played how to act on it is unclear.
In conclusion problem of the existing technology is: tranexamic acid is used as the therapeutic choice of acne rosacea, but it is controlled
The potential molecular mechanism of therapeutic effect is unclear.
Solve the difficulty of above-mentioned technical problem:
Since the potential molecular mechanism of therapeutic effect is unclear, the immune ring on a large scale for may relate to is needed
Section, corresponding molecule are screened.If determination is related to which kind of cell such as: T cell, B cell, neutrophil leucocyte, macrophage, leaching
Bar cell, NK cell etc..The relevant protein of these cells, cell factor are detected by various technological means.It grinds again later
Study carefully the mechanism of action of these molecules, target cell etc..Due to being the process inversely studied, early stage needs to spend a large amount of
Manpower and material resources carry out big screening operation
Solve the meaning of above-mentioned technical problem:
The present invention provides a kind of new means for the treatment of acne rosacea.And due to the mechanism of action of tranexamic acid with
The mechanism of action of current drug is different, can be used to treat the patient for having taboo to existing drug.
The cause and onset of disease mechanism of acne rosacea further is disclosed, preferably research acne rosacea provides for after
With reference to.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of drug for improving acne rosacea symptom and its animals
Model and construction method.
The invention is realized in this way a kind of verifying improves the animal model of the medicine effect of acne rosacea symptom, it is described
The animal model for improving the drug of acne rosacea symptom is 7 week old BALB/C mices, is treated with tranexamic acid, daily stomach-filling control
The tranexamic acid of buffer solution every kilogram of mouse weight of PBS and 130mg, continuous seven days, in last 2 days subcutaneous injection LL37 polypeptides
To induce acne rosacea sample skin injury.
Another object of the present invention is to provide the cells that a kind of verifying improves the medicine effect of acne rosacea symptom
The construction method of model, the construction method that the verifying improves the cell model of the medicine effect of acne rosacea symptom include:
The first step, by HaCaT cell culture in cultivating without calcium basis containing 10% fetal calf serum and penicillin/streptomycin
In base, cultivated in 37 DEG C, the incubator of 5% carbon dioxide.After HaCaT cell reaches 90% degrees of fusion, calcium training has been used instead
Support and cultivated in base, according to experiment purpose, be divided into blank group --- without any processing, (4 μM) of LL37 group --- LL37 stimulations
12h, TXA group --- TXA (120mg/L) stimulate 12h, TXA+LL37 group --- TXA (120mg/L) to stimulate for 24 hours, in the 12nd hour
LL37 (4 μM) are added stimulates 12h simultaneously.Then RNA is collected, is stored in -80 DEG C until analysis；
Second step, Human umbilical vein endothelial cells (HUVEC) are containing 10% fetal calf serum and penicillin/streptomycin (50U/
Ml it is cultivated in 1640), is placed in 37 DEG C, in the incubator of 5%CO2；Cell uses serum-free basal medium overnight starvation first,
It is similar to first step processing afterwards, replacement training base, and packet transaction.Blank group --- without any processing, LL37 group --- LL37
(4 μM) stimulations 12h, TXA group --- TXA (120mg/L) stimulate 12h, TXA+LL37 group --- TXA (120mg/L) to stimulate for 24 hours,
In the 12nd hour addition LL37 (4 μM) while stimulating 12h.After collect RNA, -80 DEG C save until analysis.
Further, the verifying improves the construction method of the animal model of the medicine effect of acne rosacea symptom further include:
(1) histologic analysis fixes whole skins of acne rosacea sample lesion with paraffin, is cut into 4 μ m-thicks；Slice uses bush
Essence and eosin stains, and tectology is observed under standard light microscope；
(2) immunofluorescence fixes 8mm frozen section 15 minutes with paraformaldehyde at room temperature, and with 5% normal donkey serum
It is closed at room temperature with 0.2%TritonX and continues 1 hour；Skin biopsy is handled at 4 DEG C with anti-CD4 and 1 antibody of AntiCD3 McAb
Overnight, 1H is dyed with the anti goat igg antibody room temperature that Alexa Fluor 488 is marked；It is captured using ZeissAxio ScopeA1
Image；
(3) real-time PCR analysis separates total serum IgE from cell or skin injury using TRIzol reagent；It uses
2 μ gRNA are transcribed into cDNA by PrimeScript RT kit, and are used on 7500 machine of Applied Biosystems
ITaqUniversal SYBR GREEN Supermix carries out qPCR；
(4) it statisticallys analyze, analyzes all data with GraphPad Prism6, and indicate with average value ± SEM；
Student's t is examined for the comparing difference between two groups.
It is described to change another object of the present invention is to provide a kind of drug for improving acne rosacea symptom using the verifying
The drug of kind acne rosacea symptom is tranexamic acid.Tranexamic acid is compared with the drug of systemic treatment acne rosacea, effect
Mechanism and side effect are as follows:
In summary: advantages of the present invention and good effect are as follows: by the present invention in that with LL37 induction mouse model and
The molecular mechanism that HaCaT cell model has evaluated TXA and its influences on acne rosacea.It is handled by TXA and significantly improves rose
Acne sample symptom, including erythema and tissue pathologies change, and increase proinflammatory cytokine (IL-6 and TNF α) and
MMP9 expression.In addition, TXA also reduces congenital immunity gene (TLR2, KLK5 and Camp) and neutrophil leucocyte related gene
Expression in acne rosacea sample lesion.For adaptive immunity, CD4+T cellular infiltration and Th cell factor and chemotactic because
The gene expression of son is adjusted by the TXA in skin injury.In addition, in the anti-inflammatory effect of TXA and the HaCaT cell of LL37 activation
TLR2, proinflammatory cytokine (IL-6 and TNF α) are related with the inhibition that chemotactic factor (CF) (CCL10) is expressed.Finally, TXA passes through reduction
The quantity of CD31+ cell inhibits angiogenesis with the expression for lowering VEGF in acne rosacea.In short, experiment shows TXA
Mainly improve acne rosacea symptom by adjusting immune response and angiogenesis.
Detailed description of the invention
Fig. 1 is the building of the animal model for the medicine effect that verifying provided in an embodiment of the present invention improves acne rosacea symptom
Method flow diagram.
Fig. 2 is that TXA treatment provided in an embodiment of the present invention improves acne rosacea sample phenotype schematic diagram；
In figure: after (a) going hair removal, LL37 intracutaneous injection being induced acne rosacea sample phenotype into skin of back, is led to
Cross administration by gavage pretreatment TXA；It is scored based in redness；(b) and area；(c) severity of acne rosacea sample phenotype is assessed；
(d) H&E is used for the histologic analysis of acne rosacea sample skin；(e) pass through qPCR analysis detection IL-6, the table of TNF-α and MMP9
Up to level.As a result three independent experiments are represented.Data represent average value ± SEM, and P < 0.01 P < 0.05, * * * and * * * P <
0.001。
Fig. 3 is the congenital immunity schematic diagram that the present invention implements that the TXA provided inhibits acne rosacea sample mouse；
In figure: (a) TLR2, KLK5, Camp, IL-6, the expression of TNF-α and MMP9 in acne rosacea sample mouse；?
The expression of mast cell (b) macrophage (c) and neutrophil leucocyte (d) related gene in acne rosacea sample mouse；Data
Represent the average value ± SEM of independent experiment three times；* P < 0.01 P < 0.05, * * and P < 0.001 * * *.
Fig. 4 is the immune response signal that CD4 positive cell is adjusted in acne rosacea sample mouse that the present invention implements to provide
Figure；
In figure: the expression of CD4 in the skin (a) shown by immunofluorescence；Green indicates CD4+T cell.Blue indicates
DAPI；Scale bar, 50 μm；The expression of Th1 (b), Th17 (c), Th2 (d) and Treg (e) cell relating gene-1 is small in acne rosacea sample
Significant adjusting in mouse.Data represent average value ± SEM；* P < 0.01 P < 0.05, * * and P < 0.001 * * *.
Fig. 5 is the inflammatory cytokine and chemokine expression that the present invention implements that the TXA in HaCaT cell provided is reversed
Schematic diagram.
Fig. 6 is the angiogenesis signal that the present invention implements that the TXA provided reduces the acne rosacea sample mouse of LL-37 induction
Figure；
In figure: the expression of CD31 in the skin (a) shown by immunofluorescence.Green indicates CD31 cell；Blue indicates
DAPI.Scale bar, 50 μm；(b) vegf expression in acne rosacea sample mouse；(c) the TRPV1 table in acne rosacea sample mouse
It reaches；(d) LL37 and TXA is to ET1 in HUVEC, the influence of TNF α and vegf expression level.Data represent average value ± SEM.*P<
0.05, * P < 0.01 * and P < 0.001 * * *.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
It is used as the therapeutic choice of acne rosacea for tranexamic acid, but the potential molecular mechanism of therapeutic effect is unclear
The problem of, by the present invention in that having evaluated TXA with the mouse model of LL37 induction and cell model and its being influenced on acne rosacea
Molecular mechanism.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
The drug provided in an embodiment of the present invention for improving acne rosacea symptom is tranexamic acid.
As shown in Figure 1, verifying provided in an embodiment of the present invention improves the cell model of the medicine effect of acne rosacea symptom
Construction method the following steps are included:
S101: by HaCaT cell culture containing 10% fetal calf serum and penicillin/streptomycin without calcium basal medium
In, it is cultivated in 37 DEG C, the incubator of 5% carbon dioxide.After HaCaT cell reaches 90% degrees of fusion, turn having calcium culture
Cultivated in base, according to experiment purpose, be divided into blank group --- without any processing, (4 μM) stimulation 12h of LL37 group --- LL37,
TXA group --- TXA (120mg/L) stimulates 12h, TXA+LL37 group --- TXA (120mg/L) to stimulate for 24 hours, is added in the 12nd hour
LL37 (4 μM) stimulates 12h simultaneously.Then RNA is collected, is stored in -80 DEG C until analysis；
S102: Human umbilical vein endothelial cells (HUVEC) are containing 10% fetal calf serum and penicillin/streptomycin (50U/ml)
1640 in cultivate, 37 DEG C are placed in, in the incubator of 5%CO2；Cell passes through serum-free basal medium first and is starved overnight,
Replacement training base afterwards, and packet transaction.Blank group --- without any processing, LL37 group --- LL37 (4 μM) stimulations 12h, TXA
Group --- TXA (120mg/L) stimulates 12h, TXA+LL37 group --- TXA (120mg/L) to stimulate for 24 hours, is added in the 12nd hour
LL37 (4 μM) stimulates 12h simultaneously.After collect RNA, -80 DEG C save until analysis.
Application principle of the invention is further described combined with specific embodiments below.
1. material and method
1.1 compound
TXA is purchased from Selleck Chemicals (California, USA).LL37 is by Shenggong (Chinese Shanghai)
Synthesis has amino acid sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTE, is then purified by HPLC, and
Their identity is confirmed by mass spectrum.
1.2 zoopery
The 7 week old BALB/C mices for experiment are purchased from Shanghai Slac Laboratory Animal Co.Ltd.
(Chinese Shanghai).All zooperies are ratified through the animal welfare committee, Xiangya Hospital, Central-South China Univ..TXA is treated, TXA
(Selleck Chemicals, California, USA) stomach-filling daily compares every kilogram of Mice Body of buffer solution PBS and 130mg
The tranexamic acid of weight, continuous seven days, in last 2 days subcutaneous injection LL37 polypeptides to induce acne rosacea sample skin injury.By
The acne rosacea sample lesion of LL37 induction photographs to record and is based on foregoing red and swollen scoring and assessed with area.
1.3 cell culture and treatment
By HaCaT cell culture containing 10% fetal calf serum and penicillin/streptomycin without in calcium basal medium,
It 37 DEG C, cultivates in the incubator of 5% carbon dioxide.After HaCaT cell reaches 90% degrees of fusion, turn to train in having calcium culture medium
Support, be divided into blank group --- without any processing, LL37 group --- LL37 (4 μM) stimulations 12h, TXA group --- TXA (120mg/
L) stimulate 12h, TXA+LL37 group --- TXA (120mg/L) stimulate for 24 hours, in the 12nd hour addition LL37 (4 μM) stimulate simultaneously
12h.Then RNA is collected, is stored in -80 DEG C until analysis；
Human umbilical vein endothelial cells (HUVEC) are containing 10% fetal calf serum and penicillin/streptomycin (50U/ml)
It is cultivated in 1640, is placed in 37 DEG C, in the incubator of 5%CO2；Cell passes through serum-free basal medium first and is starved overnight, after
Replacement training base, and packet transaction.Blank group --- without any processing, LL37 group --- LL37 (4 μM) stimulations 12h, TXA
Group --- TXA (120mg/L) stimulates 12h, TXA+LL37 group --- TXA (120mg/L) to stimulate for 24 hours, is added in the 12nd hour
LL37 (4 μM) stimulates 12h simultaneously.After collect RNA, -80 DEG C save until analysis.
1.4 histologic analysis
Whole skins that acne rosacea sample lesion is fixed with paraffin, are cut into 4 μ m-thicks.Sections stained with hematoxylin and Yihong (H&E)
Dyeing dyeing, and their tectology is observed at standard light microscope (OLYMPUS, Japan) as previously described.
1.5 immunofluorescence
8mm frozen section is fixed 15 minutes with paraformaldehyde at room temperature, and with 5% normal donkey serum (NDS) (#
017000-121, Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania) and
0.2%TritonX is closed at room temperature continues 1 hour.Then by skin biopsy with anti-CD4 (1:100, ebioscience) and
(1:100, the ebioscience) antibody of AntiCD3 McAb 1 is handled overnight at 4 DEG C, the anti-mountain then marked with Alexa Fluor 488
Sheep IgG antibody dyes (Life technology) room temperature 1H.Image is captured using Zeiss Axio Scope A1 (Zeiss, Germany).
1.6 real-time PCR analysis
Divided from cell or skin injury using TRIzol reagent (Invitrogen Life Technologies, USA)
From total serum IgE.2 μ gRNA are transcribed into cDNA using PrimeScript RT kit (Takara, Shiga, Japan), and
ITaq Universal SYBR GREEN is used on 7500 machine of Applied Biosystems (Life Technologies)
Supermix (Bio-Rad, California, USA) carries out qPCR.Primer is as shown in table 1.
Table 1:
1.7 statistical analysis
All data are analyzed with GraphPad Prism6 (GraphPad Software, La Jolla, CA), and with average
Value ± SEM is indicated.Student's t is examined for the comparing difference between two groups.*, P < 0.05 and * *, P < 0.01 are considered as
With significant difference.
2. result
2.1 TXA mitigate the dermatitis symptom in the acne rosacea sample mouse model of LL-37 induction
Clinical research shows that the symptom of acne rosacea patient can be effectively relieved in TXA.Therefore, using the small of acne rosacea
Mouse model studies the pharmacological Mechanism of TXA.As shown in Figure 2 a, TXA significantly improves the acne rosacea sample skin of LL37 induction
Erythema.The significantly reduced average in redness of TXA and area respectively may be about 60.6% and 54.6% (Fig. 2 b and c).
Histologic analysis is shown, improves acne rosacea sample dermatitis, including inflammatory infiltration and rose Cuo by TXA treatment
The blood vessel hyperplasia (Fig. 2 d) of sore mouse.Proinflammatory cytokine (IL-6, TNF-α) and matrix metalloproteinase (MMP9) are in various inflammation
It is raised in property disease, therefore, IL-6, the expression of TNF-α and MMP9 is had detected by qPCR, and show to pass through TXA
Processing reduces IL-6 in acne rosacea, the up-regulation (Fig. 2 e) of TNF-α and MMP9 expression.In short, these are the result shows that TXA improves
The symptom of acne rosacea in the mouse model of LL37 induction.
2.2 in acne rosacea sample mouse, and TXA reduces the immune disorder of LL37 induction
, by studying the degree of participation of congenital immunity and acquired immunity in acne rosacea sample mouse, thus more preferably
Ground understands the immunological regulation of TXA in acne rosacea.In acne rosacea, congenital immunity reaction is critically important, wherein acne rosacea
The expression of TLR2, KLK5 and Camp increase in the lesion of patient and acne rosacea sample mouse.TXA processing is obviously reduced LL37 induction
TLR2, KLK5 and Camp expression (Fig. 3 a).The recruitment and work of innate immune cells are also observed in acne rosacea patient
Change, including mast cell, macrophage and neutrophil leucocyte.In order to determine whether mast cell participates in acne rosacea, analyze
The mRNA expression of Tpsb1 and CMA1, and find that these mRNA significantly raise (Fig. 3 b) in acne rosacea mouse.In addition,
The participation (Fig. 2 c) of macrophage is supported in the identical up-regulation of macrophage marker (ITGB2 and ITGAM).However, passing through TXA's
Handle the up-regulation (Fig. 3 b and c) without alleviating mast cell and macrophage marker in acne rosacea mouse.Gro-beta-T
QPCR analysis shows that neutrophil recruitment chemotactic factor (CF) (including CXCL1 and CXCL5) significant up-regulation, handled by TXA
Inhibit (Fig. 3 d).These are the result shows that mast cell, macrophage and neutrophil leucocyte are sent out in acne rosacea scytitis
The effect of waving, TXA is by reducing neutrophil leucocyte rather than mast cell and macrophage in the leaching of acne rosacea sample mouse skin lesion
Profit.Generally speaking, these are the result shows that the part TXA improves acne rosacea by inhibition of innate immune reaction.
Previous studies show that CD4 positive T cell is occupied an leading position in the immunocyte infiltration in acne rosacea.For
Influence of the research TXA to acquired immunity, we are thin by Immunofluorescence test CD4 positive T in acne rosacea sample mouse
Born of the same parents.As a result similar to the result of acne rosacea patient, CD4 positive T is thin in the acne rosacea sample dermatitis that LL37 is induced in mouse
The quantity of born of the same parents greatly increases (Fig. 4 a).TXA processing is obviously reduced the CD4+T cellular infiltration (Fig. 4) of LL37 induction.Due in rose
Observe that Th1/Th17 polarizes in the cutaneous lesions of patients with acne, thus it is speculated that T cell polarization may also assist in the rose of LL37 induction
Acne can be regulated and controled by TXA.Herein, the expression of T cell polarization related gene in acne rosacea sample mouse is had detected.With
Th1 with the Th17 related gene expression raising observed in acne rosacea patient is consistent, Th1 related gene (STAT1, STAT4,
CCR5 and CXCL10) and the expression of Th17 related gene (STAT3, CCR6 and CXCL20) significantly raise.Acne rosacea sample
In mouse (Fig. 4 b and c), TXA treatment considerably reduces the Th1 related gene (STAT1 and CXCL10) and Th17 phase of LL37 induction
Correlation gene (CXCL20).In addition, LL37 significantly induces Th2 related gene (IL4 and CCR3) and Treg related gene (FOXP3)
The expression of (Fig. 4 d and e).TXA processing increases Th2 related gene (the IL4 and CCR3) expression of LL37 induction, but reduces
FOXP3 expresses (Fig. 4 a and c).In short, these are the result shows that TXA inhibits the CD4 positive T cell induced by LL37 infiltration and T thin
Born of the same parents' polarization, this potentially contributes to its anti-inflammatory effect to acne rosacea treatment.
2.3 TXA reduce the expression of cell factor and chemotactic factor (CF) in HaCaT cell
The overexpression of congenital immunity related gene is the key factor in acne rosacea pathogenesis in keratinocyte.
Next the influence of these gene expressions in Human keratinocytes (HaCaT) model that TXA induces LL37 is had checked.?
The significant up-regulation that TLR2 expression is observed in the HaCaT of LL37 induction handles reverse both (Fig. 5) by TXA.For determination
The potential anti-inflammatory mechanisms that TXA influences acne rosacea have detected inflammatory cytokine in the HaCaT cell that TXA activates LL37
With the influence of chemokine expression.The result shows that the significant up-regulation TLR2 of LL37, proinflammatory cytokine (IL-6 and TNF α) and Th17
The expression of chemotactic factor (CF) (CCL10), it is all these that (Fig. 5) is all reversed by TXA processing other than IL-6.Demonstrating TXA can press down
Make TLR2 in the keratinocyte caused by LL37, the generation of cell factor and chemotactic factor (CF).
2.4 TXA reduce angiogenesis and inhibit vegf expression
In order to confirm TXA to the inhibiting effect of acne rosacea sample mouse Nerve blood vessel disturbance, to CD31 (vascular markers) and
The expression of VEGF and TRPV1 mRNA has carried out immunostaining.It is consistent with previous result, LL37 with CD31 quantity increase
Significant induction of vascular generates capilary and vegf expression (Fig. 6 a).The angiogenesis of LL37 in corium is sharply eliminated with TXA treatment
It acts on (Fig. 6 a and b).In addition, although the expression of LL37 up-regulation TRPV1, TXA processing does not significantly change TRPV1 expression (figure
6c).These are the result shows that TXA inhibits by the angiogenesis of the LL37 induction in acne rosacea sample mouse.Next, attempting to detect
Influence of the LL37 and TXA to HUVEC.As shown in fig 6d, TXA processing significantly inhibits the expression water of endothelin -1 (ET1) and TNF α
It is flat, however, LL37 processing and LL37+TXA processing do not influence the expression of ET1 and TNF α.In addition, TXA, LL37 and LL37+
TXA processing does not influence the expression of VEGF.As a result illustrate that TXA reduces angiogenesis and ET-1 is inhibited to express.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (4)
1. a kind of drug for improving acne rosacea symptom, which is characterized in that the drug for improving acne rosacea symptom is ammonia first
Naphthenic acid.
2. a kind of animal model of the medicine effect of improvement acne rosacea symptom described in verifying claim 1, which is characterized in that institute
It is 7 week old BALB/C mices that stating, which improves the animal model of the drug of acne rosacea symptom, is treated with tranexamic acid, daily stomach-filling pair
It is continuous seven days, more in last 2 days subcutaneous injection LL37 according to the tranexamic acid of buffer solution every kilogram of mouse weight of PBS and 130mg
2 times/day of peptide, to induce acne rosacea sample skin injury.
3. a kind of construction method of the cell model for the medicine effect for improving acne rosacea symptom as claimed in claim 2, special
Sign is that the construction method of the cell model of the medicine effect for improving acne rosacea symptom includes:
The first step, by HaCaT cell culture containing 10% fetal calf serum and penicillin/streptomycin without calcium basal medium
In, it is cultivated in 37 DEG C, the incubator of 5% carbon dioxide；After HaCaT cell reaches 90% degrees of fusion, calcium culture has been used instead
It is cultivated in base, according to experiment purpose, is divided into blank group: without any processing, LL37 group: (4 μM) stimulation 12h of LL37；TXA group:
TXA (120mg/L) stimulates 12h；TXA+LL37 group: TXA (120mg/L) stimulate for 24 hours, in the 12nd hour additions LL37 (4 μM) together
When stimulate 12h；RNA is collected, is stored in -80 DEG C until analysis；
Second step, Human umbilical vein endothelial cells are cultivated in 1640 containing 10% fetal calf serum and penicillin/streptomycin, are placed in
37 DEG C, 5%CO2Incubator in；Cell serum-free basal medium overnight starvation, similar to first step processing afterwards, replacement
Pei Ji, and packet transaction；Blank group: without any processing；LL37 group: (4 μM) stimulation 12h of LL37；TXA group: TXA (120mg/
L 12h) is stimulated；TXA+LL37 group: TXA (120mg/L) is stimulated for 24 hours, in the 12nd hour addition LL37 (4 μM) while stimulating 12h,
After collect RNA, -80 DEG C save until analysis.
4. improving the construction method of the animal model of the medicine effect of acne rosacea symptom, feature as claimed in claim 3
It is, the construction method of the animal model of the medicine effect for improving acne rosacea symptom further include:
(1) histologic analysis fixes whole skins of acne rosacea sample lesion with paraffin, is cut into 4 μ m-thicks；Sections stained with hematoxylin and
Eosin stains dyeing, and tectology is observed under standard light microscope；
(2) immunofluorescence fixes 8mm frozen section 15 minutes with paraformaldehyde at room temperature, and with 5% normal donkey serum and
0.2%TritonX is closed at room temperature continues 1 hour；Skin biopsy is processed at 4 DEG C with anti-CD4 and 1 antibody of AntiCD3 McAb
Night dyes 1H with the anti goat igg antibody room temperature that Alexa Fluor 488 is marked,；It is caught using Zeiss Axio Scope A1
Obtain image；
(3) Real-time PCR Analysis separates total serum IgE from cell or skin injury using TRIzol reagent；It uses
2 μ gRNA are transcribed into cDNA by PrimeScript RT kit, and are used on 7500 machine of Applied Biosystems
ITaq Universal SYBR GREEN Supermix carries out qPCR；
(4) it statisticallys analyze, analyzes all data with GraphPad Prism6, and indicate with average value ± SEM；Student's t
It examines for the comparing difference between two groups.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111149767A (en) * | 2020-02-24 | 2020-05-15 | 中南大学湘雅二医院 | Humanized skin type lupus erythematosus mouse model and construction method and application thereof |
| CN111534490A (en) * | 2020-05-11 | 2020-08-14 | 中南大学湘雅医院 | Model construction method for treating acne rosacea by hydroxychloroquine |
| WO2023092768A1 (en) * | 2021-11-24 | 2023-06-01 | 中南大学湘雅医院 | Application of serpinb3/b4 as target in drugs for treating inflammatory skin diseases such as rosacea |
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2019
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111149767A (en) * | 2020-02-24 | 2020-05-15 | 中南大学湘雅二医院 | Humanized skin type lupus erythematosus mouse model and construction method and application thereof |
| CN111534490A (en) * | 2020-05-11 | 2020-08-14 | 中南大学湘雅医院 | Model construction method for treating acne rosacea by hydroxychloroquine |
| WO2023092768A1 (en) * | 2021-11-24 | 2023-06-01 | 中南大学湘雅医院 | Application of serpinb3/b4 as target in drugs for treating inflammatory skin diseases such as rosacea |
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