CN116270729A

CN116270729A - Application of porous nano rod-shaped cerium oxide in preparation of medicine for treating atopic dermatitis

Info

Publication number: CN116270729A
Application number: CN202211397416.2A
Authority: CN
Inventors: 郑焱; 田志敏; 瞿永泉; 白瑞敏; 王玉倩; 梁琼文
Original assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Current assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Priority date: 2022-11-09
Filing date: 2022-11-09
Publication date: 2023-06-23

Abstract

The invention relates to the technical field of biological medicine, in particular to porous nano rod-shaped cerium oxide (PN-CeO) ₂ ) Application of PN-CeO in preparation of medicine for treating atopic dermatitis ₂ Treatment of atopic dermatitis by blocking oxidative stress of atopic dermatitis, in particular, it can reduce Reactive Oxygen Species (ROS) level, down regulate inflammatory factors, up regulate skin barrier repair molecule level, and use PN-CeO with proper concentration ₂ Can be used for treating mice model of DNFB-induced atopic dermatitis, and can improve skin injury, epidermis thickening, mast cell infiltration, spleen swelling, and reduce scratching times.

Description

Application of porous nano rod-shaped cerium oxide in preparation of medicine for treating atopic dermatitis

Technical Field

The invention relates to the technical field of biological medicine, in particular to porous nano rod-shaped cerium oxide (PN-CeO) ₂ ) The application of the composition in preparing medicines for treating atopic dermatitis is provided.

Background

Atopic dermatitis (Atopic Dermatitis, AD) is one of the most common inflammatory skin diseases. It generally develops during childhood and may last to adulthood. The disease is characterized by recurrent, itchy, localized eczema, often with seasonal fluctuations. Many patients also suffer from allergic asthma, allergic rhinoconjunctivitis, food allergies, and other immediate hypersensitivity (type 1) allergies. The prevalence and incidence of atopic dermatitis has increased over the past few decades. Global disease burden studies showed that the prevalence of children is 15% to 20% and that of adults is as high as 10%, making atopic dermatitis the 15 th most common non-lethal disease and the highest disease burden dermatological disease. Its pathogenesis includes genetic and environmental factors, skin barrier dysfunction, microbial imbalance such as staphylococcus aureus colonization, immune imbalance, etc. Skin lesions and non-skin lesions in atopic dermatitis patients are mainly characterized by T cell infiltration of CD4 expression. Alarmins release, triggered by disruption of the epidermal barrier, activates inflammatory epidermal dendritic cells and type 2 immune-mediated responses, activated Th2 cells release IL-4 and IL-13, IL-31, promoting IgE production, and cytokines further activate B cells and plasma cells to promote inflammation, itch and antigen specific IgE production. In addition, macrophages are also involved in the pathogenesis of AD, however their role in AD is less recognized. LPS stimulates macrophages to pro-inflammatory M1 type macrophages, which secrete a variety of pro-inflammatory factors, IL4 stimulates macrophages to M2 type, and studies have shown that in AD Th2 cell activation predominates, macrophages may be prone to M2 subtype in the presence of IL4, IL13, etc.

Treatment of AD currently involves the use of corticosteroids, calcineurin inhibitors, uv light, immunosuppressants, but still has many adverse side effects. Targeted therapies such as ANTI-IL-4 receptor antibodies, ANTI-IL-31 receptor antibodies, ANTI-IGE antibodies, ANTI-IL-5 antibodies, janus kinase inhibitors, phosphodiesterase 4 inhibitors, ANTI-IL-12/-23P 40 antibodies, and the like have also attracted attention, however targeted therapeutic drugs have also been reported to have some side effects. Therefore, the search for new drugs for the treatment of AD is of paramount importance.

Oxidative Stress (ROS) refers to the excessive production of reactive oxygen species (reactive oxygen species, ROS) and reactive nitrogen species (reactive nitrogen species, RNS) in the body, which is insufficient to remove excessive ROS and RNS, resulting in damage to biological macromolecules such as tissue cells, proteins and nucleic acids. Oxidative stress is an important pathogenesis of AD, and AD patients may exhibit significant Reactive Oxygen Species (ROS) -related damage compared to normal controls, and ROS levels and severity of AD are positively correlated, with increased oxidative stress as AD exacerbates, and insufficient antioxidant capacity exists in AD.

Nano cerium oxide is an inexpensive and environment-friendly rare earth oxide, and cerium oxide has the capacity of resisting oxidative stress due to the self-regeneration cycle of Ce3+/Ce4+ and oxygen vacancies of the surface of cerium oxide, and is considered as a candidate medicament with huge therapeutic potential in nano medicine due to redox regulation and activities of various enzyme samples (superoxide dismutase, catalase, peroxidase and the like).

Disclosure of Invention

The invention aims to provide a novel medicament for treating atopic dermatitis, in particular to the PN-CeO which is researched and found by the inventor ₂ Atopic dermatitis can be treated by modulating oxidative stress.

To achieve the above object, the present invention provides a porous nanorod-shaped cerium oxide (PN-CeO) ₂ ) Application of PN-CeO in preparation of medicine for treating atopic dermatitis ₂ Treatment of atopic dermatitis by blocking oxidative stress of atopic dermatitis, in particular, PN-CeO ₂ Can reduce Reactive Oxygen Species (ROS) levels, down regulate inflammatory factors, up regulate skin barrier repair molecule levels. With a proper concentration of PN-CeO ₂ Can be used for treating mice model of DNFB-induced atopic dermatitis, and can improve skin injury, epidermis thickening, mast cell infiltration, spleen enlargement, reduce scratching times, etc., and has PN-CeO with certain concentration ₂ Can reduce the level of Reactive Oxygen Species (ROS) of keratinocyte HaCaT induced by tumor necrosis factor (TNF alpha) and the level of macrophage ROS induced by bacterial Lipopolysaccharide (LPS) -induced THP-1 monocyte, reduce the release of inflammatory factors, promote the expression of related molecules of skin barrier, and has better cell compatibility and tissue compatibility.

Further, the PN-CeO ₂ Synthesis by a simple two-step hydrothermal method, specifically, first of all, ce (OH) ₃ The precursor forms Ce (OH) under hydrothermal conditions at 100deg.C ₃ /CeO ₂ Is then dehydrated and oxidized under a second hydrothermal treatment at 160 c to form cerium oxide porous nanorods.

PN-CeO of the invention ₂ Atopic dermatitis can be treated by regulating oxidative stress, in the above scheme, a porous nanorod-like cerium oxide (porous nanorods of ceria, PN-CeO) is synthesized ₂ ) Compared with non-porous nano rod-shaped cerium oxide (NR-CeO) ₂ ) Cerium oxide nanoparticles (NP-CeO) ₂ ) Having a maximum surface area (141 m 2/g) and a maximum surface Ce ³⁺ (32.8%) by normalizing the catalytic activity to surface Ce ³⁺ Total amount, PN-CeO ₂ Not only has higher active site, but also provides higher average internal bionic activity for the catalytic center, and has optimal catalytic performance.

Drawings

Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:

FIG. 1 shows the application of 50ug/ml (500 ul/2 times/day) and the subcutaneous injection of 100ug/ml (500 ul/1 times/day) of PN-CeO ₂ Treatment of atopic dermatitis mice lesions, visual observation of negative control mice skin, atopic dermatitis model mice lesions and hormone-coated group (positive control) mice, HE staining, skin thickness quantification, mast cell staining, and counting; wherein negative control mice were untreated, mice of model and treatment groups were modeled using DNFB.

FIG. 2 shows PN-CeO ₂ Mice treated for atopic dermatitis were evaluated for changes in the number of scratching times, spleen index changes, and histocompatibility.

FIG. 3 shows PN-CeO with different concentration gradients and action times ₂ Toxicity assessment of keratinocytes HaCaT and macrophages;

in the figure: A. d, G the toxicity detection of cerium oxide on HaCaT cells for 24h; B. e, H the method comprises the steps of applying cerium oxide to HaCaT48h morphological change and CCK8 toxicity detection; C. f, I it is the morphological changes of cerium oxide on macrophages for 24h and the toxicity test of CCK 8.

FIG. 4 shows 50ug/ml PN-CeO ₂ Downregulation of ROS in TNFα -stimulated HaCaT cells and the effects of expression of the antioxidant stress-associated molecules Nrf2 and HO-1 mRNA.

FIG. 5 shows 50ug/ml PN-CeO ₂ Effects on expression of tnfα -stimulated HaCaT cell inflammatory factors and skin barrier related molecules.

FIG. 6 shows 50ug/ml PN-CeO ₂ Downregulation of LPS-stimulated M1-type macrophage ROS, effects on M1-type macrophage pro-inflammatory molecule mRNA expression, and effects on IL 4-stimulated M2-type macrophage molecule mRNA expression.

FIG. 7 shows 50ug/ml PN-CeO ₂ The M1 type macrophage which acts on LPS stimulation influences the effect of the secretion factor of the supernatant, the supernatant further stimulates HaCaT cells and can influence the expression of the inflammatory factors of the HaCaT cells and the skin barrier related molecules。

Fig. 8 is a schematic representation of the synthesis of porous and non-porous CeO2 nanorods.

FIG. 9 shows PN-CeO ₂ A dark field Transmission Electron Microscope (TEM) image of (a).

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.

The invention discovers that porous nano rod-shaped cerium oxide (PN-CeO) ₂ ) Can be used for treating atopic dermatitis by regulating oxidative stress, and adopting PN-CeO with proper concentration ₂ Can be used for treating mice model of DNFB-induced atopic dermatitis, and has PN-CeO with certain concentration ₂ Can reduce the level of Reactive Oxygen Species (ROS) of keratinocyte HaCaT induced by tumor necrosis factor (TNF alpha) and the level of macrophage ROS induced by bacterial Lipopolysaccharide (LPS) -induced THP-1 monocyte, reduce the release of inflammatory factors, promote the expression of related molecules of skin barrier, and has better cell compatibility and tissue compatibility. These all suggest PN-CeO ₂ Can be used for treating atopic dermatitis. Specifically:

PN-CeO ₂ is synthesized by (a)

The PN-CeO ₂ The Ce (OH) is synthesized by a simple two-step hydrothermal method ₃ The precursor forms Ce (OH) under hydrothermal conditions at 100deg.C ₃ /CeO ₂ Is then dehydrated and oxidized under a second hydrothermal treatment at 160 c to form cerium oxide porous nanorods.

PN-CeO ₂ Characterization: PN-CeO synthesized by High Resolution Transmission Electron Microscope (HRTEM) for two-step hydrothermal method ₂ The morphology, diameter, length, pore size, etc. of the nanomaterial to define the morphological features of the nanomaterial, as shown in fig. 8 and 9: synthesized PN-CeO ₂ In the shape of a bar, straightThe diameter is about 6.0nm, the length is about 60.0nm, the porous characteristic is provided, the pore diameter is 2.0-4.0nm, and the pore volume is 0.378cm ³ 。

【1】 Establishment of atopic dermatitis mouse model and PN-CeO ₂ Treatment process

25 6 week old Balb/c mice were randomly divided into 5 groups of 5 mice each. Group 1, blank control (negative control), group 2, model, group 3, PN-CeO ₂ Smearing group, group 4 PN-CeO ₂ A subcutaneous injection group; group 5: hydrocortisone application group. The flow chart (fig. 1A) shows the experimental procedure. After the mice are adapted for 4 days, the backs of the mice are dehaired by using an electric clipper and dehairing paste, the dehairing area is 2cm multiplied by 2cm, and an AD mouse model is built 3 days after dehairing.

Day 1 and day 2 of the first week, mice were sensitized by applying 100ul 1% high concentration DNFB to their backs in acetone/olive oil=3/1;V/V. Day 1 and day 4 of the second week mice were back-smeared with 100ul of 0.4% low concentration DNFB to maintain sensitization. On

days

1 and 4 of the third week,

groups

2, 3, 4, and 5 continued to be sensitized with 100ul of 0.4% low concentration DNFB, while treatments were performed daily for

groups

3, 4, and 5 for 5 consecutive days on days 1 to 5 of the third week. Wherein, group 3 is given 50ug/ml PN-CeO ₂ 500 ul/time, 2 times/day, group 4: 100. Mu.g/ml PN-CeO ₂ PN-CeO of group 3 and group 4, dissolved in PBS, 500 ul/1/day ₂ The doses were 50 ug/day; group 5 was applied 1 day with 100ul 1% hydrocortisone.

After depilation, the skin of the back of the mouse is photographed before and after the treatment, and the cervical dislocation method is adopted to kill the mouse after the treatment is finished. After back skin was taken, skin tissue and all organs were washed with cold PBS and fixed in 4% paraformaldehyde.

As can be seen from fig. 1 and 2: external application of conventional medicine for treating atopic dermatitis and PN-CeO ₂ After either the smear or subcutaneous injection treatment, the mice had significantly improved skin lesions symptoms (fig. 1B), and the number of scrapings was down-regulated (fig. 2A).

【2】PN-CeO ₂ HE staining and hypertrophy for treating atopic dermatitis mice skin lesionsEvaluation of cell staining and spleen body Mass index and evaluation of histocompatibility

The detection method comprises the following steps:

1. HE staining and skin thickness measurement

(1) Soaking the tissue for 24 hours by using 4% paraformaldehyde for tissue fixation;

(2) After trimming the tissue, carrying out gradual dehydration by using ethanol with low concentration to high concentration;

(3) Immersing the tissue in xylene to make the tissue transparent, and immersing the tissue in paraffin for wax immersion and embedding;

(4) Slicing the wax block into slices by using a slicing machine, flattening in hot water, sticking the slices onto a glass slide, and drying the slices;

(5) Dewaxing with xylene, adding slices into ethanol with high concentration to low concentration, and adding into distilled water;

(6) Hematoxylin staining, tap water washing, 1% hydrochloric acid alcohol solution color separation for a few seconds, and tap water washing;

(7) Dyeing with eosin dye liquor, and washing with tap water;

(8) Slicing, dehydrating with ethanol, and transparentizing with xylene;

(9) Sealing with neutral resin.

Skin thickness measurement: the vertical distance from basal layer to stratum corneum was measured at 5 positions randomly selected on the skin pathology digital section using ndp.view 2 software, and the average value was taken as the epidermis thickness of the mouse. The results show that the conventional medicament for treating atopic dermatitis is externally applied and PN-CeO is adopted ₂ After either the smear or subcutaneous injection treatment, the mouse epidermis was thinned (fig. 1C).

2. Mast cell toluidine blue staining

(1) Paraffin sections dewaxed to water: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethanol I5 min-absolute ethanol II 5min-75% ethanol 5min, and washing with tap water.

(2) Toluidine blue staining:

the tissue slice is put into dye liquor for 2-5min, washed with water, slightly differentiated by 0.1% glacial acetic acid, the reaction is stopped by running water, the differentiation degree is controlled under a microscope, and the slice is baked in an oven after running water.

(3) Transparent sealing piece: slicing into clean xylene, transparent for 10min, and sealing with neutral resin.

(4) Microscopic examination, image acquisition and analysis.

Mast cell count: mast cell counts were performed by randomly selecting 5 locations on the pathodigital section of skin using ndp.view 2 software. The results show that the conventional medicament for treating atopic dermatitis is externally applied and PN-CeO is adopted ₂ Following either the smear or subcutaneous injection treatment, mice had reduced mast cell infiltration (fig. 1D).

3. Spleen body mass index: after the mice were sacrificed, the spleen weight and body weight of the mice were weighed, and the ratio of the two was the spleen body mass index. The results show that the conventional medicament for treating atopic dermatitis is externally applied and PN-CeO is adopted ₂ After either the smear or subcutaneous injection treatment, the spleen index of the mice decreased (fig. 1B).

4. Evaluation of histocompatibility: mice were sacrificed by cervical dislocation after treatment. Heart, liver, spleen, kidney tissue was harvested, after which skin tissue and all organs were washed with cold PBS and fixed in 4% paraformaldehyde. HE staining of heart, liver, spleen and kidney showed PN-CeO ₂ Has better tissue compatibility.

【3】 Treatment of HaCaT cells and macrophages

The HaCaT cells are stimulated by 10ng/mL TNF alpha to act on the HaCaT cells for 48 hours, and an AD cell model is established; the phorbol ester PMA is adopted to induce THP-1 cells for 48 hours to obtain macrophages, then 200ng/ml LPS and 20ng/ml IL4 are respectively used for stimulating the macrophages for 24 hours, M1 and M2 type macrophages are generated, and the macrophages in the AD immune environment are simulated.

The specific operation is as follows:

(1) Cell resuscitation

a) Taking out the cell freezing tube from the liquid nitrogen tank, and immediately placing the cell freezing tube in a 37 ℃ water bath box to enable the cell freezing tube to be rapidly melted within 1 min;

b) Adding into prepared DMEM medium containing 5ml 10% FBS, centrifuging at 800rpm for 5min, and discarding supernatant;

c) Cells were resuspended by adding 5ml of DMEM medium containing 10% FBSInoculating to 25cm ² Placing in culture flask at 37deg.C and 5% CO ₂ Culturing in an incubator, and replacing the culture medium the next day;

(2) Cell culture

Cells were routinely cultured at 37℃constant temperature, 5% CO ₂ In the incubator, the medium was changed every 2 d.

(3) Cell passage

a) When the cell coverage reaches 80% or more, passaging can be performed. The old medium is firstly discarded and washed 2 times with PBS;

b) 2ml of 0.25% pancreatin is added for digestion, the mixture is placed at 37 ℃ for about 5min, the cells are seen to be round under an inverted microscope, the cell gap is enlarged, at the moment, 4ml of DMEM culture medium containing 10% FBS is added immediately to terminate the digestion, a pipetting gun is used for gently blowing the cell suspension, and the cell suspension is transferred into a centrifuge tube; THP-1 is suspension cell, and is directly centrifugated without PBS cleaning and pancreatin digestion;

c) The cell suspension was transferred to a centrifuge tube, centrifuged at 800rpm for 5min, the supernatant was discarded, fresh medium was added for resuspension, and the culture was continued in a new flask. The typical passaging ratio is 1:2-4.

(4) Cell cryopreservation

a) When the cell coverage rate reaches more than 80% and the state is better, the cell freezing can be carried out. The old medium is firstly discarded and washed 2 times with PBS;

b) Digestion with 0.25% pancreatin, stop digestion with DMEM medium containing 10% FBS, transfer into centrifuge tube, centrifuge at 800rpm for 5min; THP-1 is suspension cell, and is directly centrifugated without PBS cleaning and pancreatin digestion;

c) Discarding the supernatant, adding 1ml of cell cryopreservation liquid, blowing uniformly, and transferring into a cryopreservation tube;

d) And (3) placing the freezing tube into a freezing box, placing the freezing box into a refrigerator at the temperature of minus 80 ℃ for overnight, and finally transferring the freezing tube into a liquid nitrogen tank for long-term storage.

In addition, THP-1 cells are recovered, cultured, passaged and the like by using a special culture medium for THP-1;

【4】PN-CeO ₂ cell compatibility assays

(1) Selecting HaCaT cells in logarithmic growth phase, digesting the cells by pancreatin, and preparing single cell suspension by using a cell culture medium;

(2) Cell plating: after HaCaT cell counting 100 μl of cell culture medium/well and 3×10 were added to 96 well plates ³ Cells/well, 6 duplicate wells per group. A blank group to which only an equal amount of medium was added was also set. And PBS is added into the hole of the outermost circle of the reserved 96-well plate, and the PBS is not used as an index detection hole, so that experimental errors caused by excessive evaporation of liquid are prevented. After the plating is completed, placing the 96-well plate in a cell incubator, and conventionally culturing cells; the THP-1 cells were centrifuged and resuspended to give cells, and 100. Mu.L/well of PMA-containing cell culture medium and 3X 10 were added to 96-well plates ³ Cells/well, 6 duplicate wells per group. And simultaneously setting a blank control group with only an equal amount of culture medium, and forming the adherent macrophages after PMA is stimulated for 48 hours.

(3) Cell treatment: using PN-CeO of different concentrations ₂ Stimulating the cells for 24 hours or 48 hours;

(4) Cell viability detection: the original culture medium in a 96-well plate is sucked and removed, the cells are washed for 2 times by PBS, 10 mu L of CCK-8 reagent is added to each well during detection, the concentration of the CCK-8 reagent is diluted to be 10% by serum-free DMEM culture medium, the CCK-8 reagent is added to each to-be-detected hole in the 96-well plate, and after the CCK8 reagent is added to the to-be-detected cells, the cells are incubated in a cell incubator for about 2 hours to react so as to generate yellow formazan products. Before detection, ensuring that no bubble exists in each hole to be detected so as not to interfere with a detection result, and measuring an Optical Density (OD) value at a wavelength of 450nm by using an enzyme-labeled instrument;

(5) Data were analyzed and plotted against a cell-free blank control as background control.

The cytotoxicity detection was carried out by CCK8 method by photographing the morphological changes of the cells before and after the cerium oxide treatment by an optical microscope, as shown in FIG. 3.

The results show that: PN-CeO ₂ After treatment, neither HaCaT cells nor macrophages had changed morphology, and CCK8 showed no toxicity, indicating PN-CeO ₂ Has good cell compatibility.

【5】 Detection of cellular ROS

Intracellular ROS detection using DCFH-DA single-stain method

(1) Selecting cells in logarithmic growth phase, conventionally digesting the cells with pancreatin, and preparing single-cell suspension with a cell culture medium;

(2) Cell plating: after cell counting 2mL of cell culture medium/well and 4×10 were added to 6-well plates ⁵ Individual cells/wells. After the plating is completed, placing the 6-hole plate in a cell incubator, and conventionally culturing cells;

(3) Cell treatment: the cells were cultured in complete medium diluted with DMEM cell culture medium containing 10% FBS, and 50ug/ml PN-CeO was used ₂ Pretreatment of HaCaT cells for 24 hours, and stimulation of 10ng/mL of TNF alpha for 48 hours; 200ng/ml LPS and 50ug/ml PN-CeO were used ₂ Stimulating macrophages for 24 hours;

(4) Cell collection: conventionally digesting the cells with trypsin, centrifuging at 1000rpm for 5min, and discarding the supernatant;

(5) Cell washing: washing the collected cell pellet with pre-chilled PBS for 2 times, centrifuging at 1000rpm at 4deg.C for 5min, and discarding the supernatant;

(6) Probe loading: 2, diluting DCFH-DA by using a serum-free DMEM culture medium according to a ratio of 1:1000, then incubating HaCaT and macrophage cells, adding 1mL of working solution into each sample, incubating for 30min at 37 ℃ in a dark place, and reversing and uniformly mixing every 3-5min to ensure that the probes and the cells are fully contacted;

(7) Cell washing: centrifuging at 1000rpm for 5min, discarding supernatant, washing cells with PBS for 2 times, centrifuging at 1000rpm for 5min, and discarding supernatant;

(8) Cell resuspension: add 500. Mu.L PBS to each sample, resuspend cells;

(9) And (3) detecting: filtering with a screen, and detecting with a flow cytometer;

the results show that: ROS levels were significantly elevated, PN-CeO, upon stimulation of HaCaT cells by TNFa ₂ Can effectively reverse the increase of ROS generated by TNF alpha, see figure 4; ROS level is obviously increased after LPS stimulates macrophages, PN-CeO ₂ LPS-induced ROS within macrophages can be reduced, see FIG. 6.

【6】 Extraction of cellular RNA, reverse transcription and real-time fluorescent quantitative PCR (rt-qPCR)

1. Extraction of cellular RNA

(1) Discarding the cell culture medium in the 6-well plate, and washing the cells with PBS for 2 times;

(2) Adding 1mL TRIzol into each hole, standing at room temperature for 5min, blowing with a pipetting gun, transferring into an EP tube, reversing for 10 times, and standing at room temperature for 5min;

(3) 200. Mu.L of chloroform (1/5 volume of TRIzol) was added, vigorously shaken for 15s, left standing at room temperature for 5min, and centrifuged at 12000rpm for 15min;

(4) Transferring the upper colorless aqueous phase (about 400 mu L) into a new prepared EP tube, accurately recording the transfer amount of the aqueous phase, adding isopropanol with the volume equal to that of the aqueous phase into the new EP tube for precipitating RNA, reversing the solution for 10 times to fully mix the aqueous phase and the isopropanol, standing the solution for 10 minutes at room temperature, centrifuging the solution for 15 minutes at 12000rpm at4 ℃, wherein white precipitation is visible at the bottom of the EP tube, and carefully discarding the supernatant;

(5) Adding 1mL of precooled 75% ethanol for washing RNA, blowing up the precipitate, washing impurities and residual isopropanol, and centrifuging at 7500rmp at4 ℃ for 5min;

(6) Carefully discarding the supernatant, inverting the EP tube, and drying at room temperature for 5-10min until the white RNA precipitate at the bottom of the EP tube is observed to be converted into a transparent state, and the precipitate cannot be completely dried;

(7) Recording and adding proper amount (about 10-50 mu L) of DEPC treated water according to the precipitation amount, and fully dissolving RNA;

(8) Determination of RNA purity and concentration: on an enzyme-labeled instrument ultramicro detection plate, adding DEPC (DEPC) treated water as a blank control to the 1 st hole on a sample adding hole, adding an RNA sample to be detected to the rest corresponding sample adding holes, wherein the concentration of the RNA and the OD 260nm value and the OD 260/OD 280 value are detected by the enzyme-labeled instrument. When the OD 260/OD 280 is between 1.8 and 2.0, this suggests that the purity of the extracted RNA sample is high and can be used in a subsequent series of experiments. The RNA concentration can be adjusted using DEPC treated water.

(9) Placing the extracted RNA in a refrigerator at-80 ℃ and preserving for standby.

2. Reverse transcription

(1) On ice, the reverse transcription system was formulated in RNase-free 0.2mL PCR tubes as follows:

(2) After the sample addition is completed, the mixture is evenly mixed and centrifuged, and the mixture is put into a PCR instrument for reverse transcription under the following reaction conditions: 37 ℃ for 15min;85 ℃,5s;4 ℃,0 ℃.

(3) After the reaction, the complementary DNA (cDNA) of the reverse transcription product is placed in a refrigerator at-20 ℃ and stored for standby.

3、qRT-PCR

(1) To 20. Mu.L of cDNA of the reverse transcription product, 40. Mu.L of DEPC-treated water was added (i.e., diluted 3-fold);

(2) On ice, 20. Mu.L of the reaction system was prepared in RNase-free 0.1mL PCR 8-mix as follows:

(3) After sample addition, centrifuging, and putting into a real-time quantitative PCR instrument, wherein the reaction conditions are as follows:

4. analysis of results: processing and analyzing the result by using a 2-delta CT method, and calculating and comparing the relative expression quantity of the target gene;

5. the primer sequences used during the experiment are shown in Table 1.

The detection results of the relative expression change of mRNA of Rt-qPCR detection gene are shown in FIG. 4, FIG. 5 and FIG. 6, and the results show that PN-CeO ₂ mRNA expression of Nrf2 (FIG. 4C) and HO-1 (FIG. 4D) in AD inflammatory cell model, PN-CeO can be increased ₂ Can reduce ROS, resistAntioxidant stress, during which Nrf2 is functionally activated.

TNFa stimulation can raise the levels of TNFa, IL6, MIF, RANTES, IL4 in HaCaT cells, while PN-CeO ₂ Can down-regulate the expression of these inflammatory factors; TNF alpha stimulation can reduce the level of Filaggrin in HaCaT cells, while PN-CeO ₂ Treatment may increase levels of Filaggrin. Indicating PN-CeO ₂ May be anti-inflammatory and promote repair of the skin barrier, see fig. 5.

The level of the proinflammatory factor RANTES and IL6 is obviously increased after LPS stimulates macrophages, PN-CeO ₂ Can reduce the elevated levels of LPS-induced RANTES (FIG. 6C) and IL6 (FIG. 6D) in macrophages. In addition, PN-CeO ₂ Can reduce the expression of CCL22 and IL-10 in M2 type macrophages, and all the factors are involved in the pathogenesis of AD. Indicating PN-CeO ₂ Can be involved in the pathogenesis of AD by regulating the secretion of macrophage cytokines.

Finally, due to PN-CeO ₂ Can change the expression of inflammatory factors of M1 type macrophages stimulated by LPS, change the inflammatory factors into serum-free DMEM after the stimulation is finished, and collect PN-CeO ₂ Supernatant of pretreated LPS-stimulated macrophages stimulated HaCaT keratinocytes, and found down-regulated inflammatory factors TNFα, IL1 β, IL6, TSLP, MIF, IL13, and up-regulated epidermal differentiation-related molecule Filaggrin (FIG. 7). Indicating PN-CeO ₂ Can influence the interaction of macrophages and keratinocytes to participate in the pathogenesis of AD.

Experiments on the whole show that the porous nano rod-shaped cerium oxide (PN-CeO) ₂ ) Can be used for treating atopic dermatitis by regulating oxidative stress, and adopting PN-CeO with proper concentration ₂ Can be used for treating mice model of DNFB-induced atopic dermatitis, and can improve skin injury, epidermis thickness, mast cell infiltration, splenomegaly, and reduce scratching times. And PN-CeO ₂ Can reduce the level of keratinocyte HaCaT (ROS) induced by tumor necrosis factor (TNF alpha) and macrophage ROS induced by bacterial Lipopolysaccharide (LPS) -induced THP-1 monocyte, reduce the release of inflammatory factor, and promote the expression of skin barrier related molecules. In vivo and in vitro experiments prove thatIt has better cell compatibility and tissue compatibility.

In view of this, PN-CeO ₂ Can be used as medicine for treating atopic dermatitis.

The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.

Claims

1. The application of porous nano rod-shaped cerium oxide in preparing medicament for treating atopic dermatitis is characterized in that PN-CeO ₂ Treatment of atopic dermatitis is achieved by blocking oxidative stress of atopic dermatitis.

2. The use according to claim 1, wherein the PN-CeO is ₂ Can reduce Reactive Oxygen Species (ROS) levels, down regulate inflammatory factors, up regulate skin barrier repair molecule levels.

3. The use according to claim 1, wherein said PN-CeO ₂ Synthesis by a simple two-step hydrothermal method, specifically, first of all, ce (OH) ₃ The precursor forms Ce (OH) under hydrothermal conditions at 100deg.C ₃ /CeO ₂ Is then dehydrated and oxidized under a second hydrothermal treatment at 160 c to form cerium oxide porous nanorods.