CN109568583B - Application of RIPK3 as target in preparation of medicine for preventing and treating psoriasis - Google Patents

Abstract

The inventor researches and discovers that RIPK3 is positively correlated with the development of psoriasis, and the scale and skin damage thickness can be improved by adopting an RIPK3 inhibitor preparation with proper concentration to treat an imiquimod mouse psoriasis model. Moreover, knocking out the expression of RIPK3 in HEKa cells can reduce the release of inflammatory factors in psoriasis models of HEKa cells. Therefore, RIPK3 can be used as a target point in the preparation of medicaments for preventing and treating psoriasis. Wherein the medicine takes RIPK3 as a medicine target at the gene level and/or the protein level. The medicine is an inhibitor preparation aiming at RIPK3 and a carrier for knocking out RIPK3 by siRNA, and the medicine is DNA or RNA for preventing the expression or transcription of RIPK 3.

Description

Application of RIPK3 as target in preparation of medicine for preventing and treating psoriasis

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of RIPK3 in preparation of medicines for preventing and treating psoriasis.

Background

Psoriasis is an inflammatory, immune-mediated chronic skin disease characterized by keratinocyte proliferation and abnormal differentiation, inflammatory cell infiltration. Psoriasis is clinically characterized by silvery-white scales overlying the basis of erythema, and long-term disease progression seriously affects the quality of life of patients. The typical histopathology of psoriasis is: epidermal parakeratosis, often accompanied by hyperkeratosis; the granular layer disappears, the spinous layer is thick, the epicutaneous process extends, the lower end is widened and can be inosculated with the adjacent epicutaneous process, the nipple of the dermis extends upwards, and the spinous layer above the dermal nipple is thinned. The ground cells may have mild spongiform, even vacuolization or edematous degeneration. Capillary vessels in the papilla dilate and are congested, and peripheral lymphocytes and neutrophils infiltrate. Areas of parakeratosis are often seen with neutrophil accumulation, known as Munro's abscess.

The psoriasis treatment methods are various, the medicines for treating the psoriasis comprise corticosteroids, tretinoin, vitamin D3 derivatives, calcineurin inhibitors and the like, but the curative effect of the existing treatment methods cannot satisfy patients due to unclear diseases, the diseases are easy to relapse, and the clinical application of the medicines is limited due to the adverse reaction of the medicines for treating the psoriasis or the price of the medicines.

Receptor-interacting protein kinase 3(Receptor-interacting protein kinase 3, RIPK3), also known as RIP 3. Originally, Yu PW et al (Yu P W, Huang B C B, Shen M, et al.identification of RIP3, a RIP-like kinase at activities for apoptosis and NF-. kappa.B [ J ]. Current Biology, 1999, 9 (10): 539-542.

) It was found in the yeast two-hybrid system in 1999. The product of RIPK3 is a protein, structurally similar to a member of the serine/threonine protein kinase receptor-interacting protein kinase family, and is therefore designated RIPK 3.

RIPK3 has a molecular weight of 53kDa and is located on chromosome 14q11 and encodes 518 amino acids, with a kinase domain in the N-segment and a rhem domain at the C-terminus that interacts with a variety of molecules. The protein encoded by RIPK3 is predominantly localized to the cytoplasm, but nucleoplasmic shuttle can occur. RIPK3 is highly expressed in lymphocytes, monocytes and NK cells. Increased expression in diseases such as inflammatory bowel disease, irritable bowel disease, chronic liver inflammation, and Steven-Johnson syndrome, and decreased expression in tumors such as pancreatic cancer, hepatocellular carcinoma, cervical cancer, ovarian tumor, esophageal squamous cell carcinoma, and melanoma.

Disclosure of Invention

The inventor researches and discovers that RIPK3 is positively correlated with the development of psoriasis, the scale and skin lesion thickness can be improved by adopting an RIPK3 inhibitor preparation with proper concentration to treat an imiquimod mouse psoriasis model, and the release of inflammatory factors in the psoriasis model of HEKa cells can be reduced by knocking out the expression of RIPK3 in the HEKa cells. Therefore, RIPK3 can be used as a target point in the preparation of medicaments for preventing and treating psoriasis. Wherein the medicine takes RIPK3 as a medicine target at the gene level and/or the protein level. The medicine is an inhibitor preparation aiming at RIPK3 and a carrier for knocking out RIPK3 by siRNA, and the medicine is DNA or RNA for preventing the expression or transcription of RIPK 3.

The RIPK3 inhibitor preparation is prepared from the following raw materials:

GSK' 872 powder 5 mg; 2.6076ml of DMSO; 7.3924ml of physiological saline with the mass concentration of 0.9 percent.

The RIPK3 inhibitor formulation was prepared by the following steps:

weighing 5mg GSK' 872 powder, centrifuging at 2000rpm for 1min, dissolving in 2.6076ml DMSO, shaking, and dissolving in 7.3924ml 0.9% NaCl solution to obtain suspension.

The siRNA knockdown RIPK3 vector sequence is as follows:

h-RIPK3 Forward：5’-GCUGAAAGAAGUGGUGCUUTT，

Reverse：5’-AAGCACCACUUCUUUCAGCTT

compared with the prior art, the invention has the following beneficial technical effects:

the RIPK3 inhibitor preparation has good safety, the RIPK3 inhibitor preparation specifically inhibits RIPK3, and the auxiliary materials have no toxic or side effect on the body, thereby having good safety.

Drawings

FIG. 1 is a Western blot of skin tissue proteins of mice treated with 0.5mg/ml RIPK3 inhibitor formulations for imiquimod mice skin lesions, and control mice, where veh is 0.1% sodium azide for control and RIPK3 is 0.5mg/ml RIPK3 inhibitor formulations.

FIG. 2 is a PASI score for 0.5mg/ml RIPK3 treated imiquimod mice. Wherein A is the PASI score for erythema, B is the PASI score for scaling, C is the PASI score for skin thickness, and D is the total PASI score.

FIG. 3 is a graph showing the immunohistochemistry and semi-quantitative comparison of RIPK3 in normal skin tissues and psoriatic skin lesion tissues in humans.

FIG. 4 shows RIPK3 western blot and its relative expression in normal skin tissues and psoriatic skin lesion tissues in humans.

FIG. 5 shows that total mRNA was extracted from HEKa cells and HEKn cells 24 hours after stimulation with M5, and relative expression change of RIPK3 mRNA was measured by real-timePCR.

FIG. 6 shows the relative expression changes of RIPK3 protein measured by western blot at 24 hours after HEKa cells and HEKn cells were stimulated with M5 to extract total cell protein.

FIG. 7 is a western blot of the relevant molecules detected by extracting total cell protein 48 hours after applying si-negative and si-RIPK3 knockout RIPK3 to HEKn cells.

FIG. 8 shows that 48 hours after applying si-negative and si-RIPK3 to knock out RIPK3 on HEKn cells, total mRNA of the cells is extracted, and real-timePCR is used for detecting the relative expression change of the psoriasis-related inflammatory factors.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The invention discovers that the expression of RIPK3 is increased in skin lesions of patients with psoriasis and skin lesions of imiquimod mice, and the expression of RIPK3 is increased after the expression of M5(IL-17A, IL-22, IL-1a, OSM and TNF-a mixture) is used for stimulating HEKa cells to simulate the level of psoriasis cells; these suggest that RIPK3 is positively correlated with the development of psoriasis, and the invention is described in further detail below.

1) Establishment of imiquimod mouse model

BALB/c mice were purchased for 6-8 weeks, after anaesthesia with 2% chloral hydrate, the back hairs were shaved off to a size of about 2 x 2cm, 62.5mg of imiquimod cream was applied topically, once a day, 100ul of RIPK3 inhibitor formulation (cam number: S8465) was applied topically at different concentrations (0.1mg/ml, 0.25mg/ml and 0.5mg/ml), once a day, photographed and PASI scored daily for 7 days, and on the eighth day after cervical spine sacrifice, skin lesions were removed, a portion was immunohistochemically made, and a portion of the protein was extracted as WB.

The control group is 0.1% sodium azide, and the treatment groups are respectively 0.1, 0.25 and 0.5mg/ml RIPK3 inhibitor preparation;

the results of PASI scores for imiquimod mice treated with the 0.5mg/ml RIPK3 inhibitor formulation are shown in figure 2, where a is the PASI score for erythema, B is the PASI score for scaling, C is the PASI score for skin thickness, and D is the total PASI score.

The results of the imiquimod mouse skin damage control experiment show that the symptoms of the mouse skin damage are obviously improved after the conventional external application of the medicament for treating psoriasis and the treatment of the RIPK3 inhibitor preparation.

2) Western blot detection of skin lesions of imiquimod mice treated by RIPK3 inhibitor preparation

The detection method comprises the following steps:

(1) extraction of Total cellular protein

a) Mixing RIPA lysate and PMSF uniformly in advance according to the proportion of 100: 1, and placing on ice;

b) discarding the culture medium in a six-hole plate or a 1.5cm culture dish, and washing twice with PBS;

c) adding 150 μ l of lysis solution into each well, and performing lysis on ice for 20 min;

d) cells were scraped thoroughly on ice and transferred to EP tubes;

e) repeatedly freezing and thawing with liquid nitrogen for 3 times;

f) centrifuging at 12000rpm at 4 deg.C for 15min, carefully aspirating the supernatant, and transferring to another EP tube;

g) protein concentration by BCA method: the solution A and the solution B of the kit are uniformly mixed according to the concentration of 50: 1, 98 mu l of the solution A and the solution B are added into each well of a 96-well plate, 2 mu l of the extracted protein or a standard protein product (0 mu g/mu l, 0.25 mu g/mu l, 0.5 mu g/mu l, 1 mu g/mu l and 2 mu g/mu l) is added, the mixture is uniformly stirred and mixed on a vibrator, then the mixture is placed into a 37 ℃ incubator for 30min, then OD562nm is measured on a spectrophotometer, and the protein concentration is calculated according to a standard curve.

h) Adding 1/4 volume of 5 Xloading buffer solution into protein, decocting at 95 deg.C for 5min, and storing at-20 deg.C.

(2) SDS-PAGE electrophoresis

a) Preparing lower layer gel of polyacrylamide gel electrophoresis, pouring the upper layer gel after the gel is solidified, inserting a clean and dry comb to ensure that no bubbles exist, and standing for 20-30min to polymerize the concentrated gel;

b) fixing the glue in a protein electrophoresis tank, adjusting the sample loading amount according to the protein concentration to ensure that the same content of protein (about 20-25 mu g) is in each hole, and simultaneously adding 5 mu l of a pre-dyed protein molecular weight marker to determine the molecular weight of the protein to be detected;

c) electrophoresis: after sample application, electrophoresis is carried out for 30min at 15mA, electrophoresis is carried out for about 60min at 25mA, and the electrophoresis is stopped until bromophenol blue reaches the bottom of the separation gel;

d) the gel was removed and placed in the transmembrane buffer.

(3) Rotary film

a) Preparing 2 pieces of filter paper and 1 piece of PVDF membrane, wherein the size of the filter paper is similar to that of SDS-PAGE gel;

b) the PVDF membrane needs to be soaked in methanol for 1min and then soaked in a transfer buffer solution for later use;

c) placing filter paper-gel-PVDF membrane-filter paper on the membrane rotating device from the cathode to the anode in sequence, and paying attention to no bubble between layers;

d) the film is rotated for 1.5-2h under the constant current of 230 mA.

(4) Sealing of

The PVDF membrane was blocked at room temperature for 1h with a blocking solution of 5% skimmed milk powder or BSA in TBST configuration.

(5) Inhibitor formulation incubation

a) The PVDF membrane is subjected to a band with a proper width according to the molecular weight of the inhibitor preparation;

b) rinsing with TBST, adding primary antibody prepared according to required concentration, and incubating overnight at 4 ℃;

c) recovering primary antibody, and rinsing the membrane with TBST for 10min 3 times;

d) adding a secondary antibody prepared according to the required concentration, and incubating for 1 h;

e) the membrane was rinsed 3 times with TBST for 10min each, and then the liquid was poured off.

(6) Development (operation in darkroom)

a) Taking equal volumes of chemiluminescence test solution A and B, and temporarily and uniformly mixing before use;

b) uniformly spreading the prepared luminous liquid on a PVDF membrane, reacting for 1min, then sucking the luminous liquid by using filter paper, placing a film, and adjusting exposure time according to exposure effect;

c) and taking out the film, immersing the film into a developing solution, rinsing the film by using clear water, fixing the film, and scanning, acquiring and analyzing the image after drying the film.

(7) Strip method for membrane regeneration

a) Rinsing the PVDF film after the luminescence for 5min by using TBST;

b) adding appropriate amount of membrane regeneration solution, and incubating at room temperature for 30 min;

c) rinsing with TBST for 5min × 3 times;

d) and (4) re-blocking, incubating the inhibitor preparation, and developing, which are the same as steps (4), (5) and (6).

The Western blot detection results of the skin tissue protein of mice in the control group and the skin lesions of mice treated by the RIPK3 inhibitor preparation are shown in figure 1, and the treatment effect is negatively correlated with the RIPK3 content, and the RIPK3 content of the group with better treatment effect is lower.

3) RIPK3 detection in normal skin tissue and psoriatic lesion tissue, including immunohistochemical staining and western blot

1) Immunohistochemical staining

(1) Treatment of clinical specimens

The specimens obtained during the operation were thoroughly washed with physiological saline to remove blood and contaminants, and then the tissue blocks were fixed in 4% paraformaldehyde for 24 hours.

(2) Treatment of slides

a) Ultrasonically cleaning the glass slide detergent, and soaking the glass slide detergent in a mixed solution of potassium dichromate and concentrated sulfuric acid for 24 hours;

b) taking out the acid vat, washing the acid vat with self-distilled water until the acid vat is completely clean, and placing the acid vat in an oven to dry the acid vat overnight at 60 ℃;

c) to prevent tissue loss, slides were treated with 3-Aminopropyl-3-ethoxysilane (3-Aminopropyl-Triethoxysilane, APES). Diluting APES stock solution and acetone at a ratio of 1: 50 to obtain working solution, placing the cleaned slide into the newly-configured APES working solution, standing for 30s, and taking out;

d) taking out the slide, pausing for 10s, then putting in acetone solution for 30s, and rinsing out the unbound APES;

e) and then placing in an oven for drying for 2 hours, and boxing for later use.

(3) Paraffin embedding and tissue sectioning

a) Embedding tissues: firstly, adding some liquid paraffin into a mould, slightly cooling, placing tissues to be embedded into the paraffin, arranging the tissues in order, covering a plastic mould box, finally adding a little liquid paraffin, and cooling to enable the tissues to become solid;

b) slicing: taking the embedded tissue from the mold, placing on a paraffin slicer, adjusting the thickness of the slice to be 4 μm, and continuously slicing;

c) the slices were placed in an oven at 60 ℃ for 90min and then transferred to an oven at 37 ℃ overnight.

(4) Immunohistochemical staining procedure

a) Deparaffinizing the tissue slices in xylene for 10min × 2 times;

b) sequentially adding into anhydrous alcohol, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol for 10min respectively;

c) placing in PBS and hydrating for 5min × 3 times;

d) 3% H freshly prepared is added dropwise₂O₂Incubating at room temperature for 20min to block endogenous peroxidase, and washing with PBS for 5min × 3 times;

e) placing the slices in a pressure cooker containing 0.01M sodium citrate buffer solution (pH6.0), heating until the pressure valve starts to inject air for 2min, then leaving the heat source, cooling, taking down the air valve, taking out the slices, and steaming PBS for 5min × 3 times;

f) adding 5% goat serum dropwise, sealing at 37 deg.C for 20min, and removing without washing;

g) adding a primary antibody: dropwise adding diluted primary anti-working solution (RIPK3 antibody dilution ratio is 1: 100), and standing overnight at 4 deg.C;

h) rewarming at room temperature for 30min, washing with PBS for 5min × 3 times;

i) adding a secondary antibody: adding biotin-labeled secondary antibody dropwise, incubating at 37 ℃ for 30min, washing with PBS for 5min × 3 times;

j) dripping horse radish peroxidase labeled streptavidin, incubating for 20min at 37 ℃, and washing for 5min × 3 times with PBS;

k) preparing DAB color developing solution according to the specification, dripping the DAB color developing solution on the slice, developing for 3-5min, and observing color developing reaction under a microscope;

l) washing with clear water, counterstaining with hematoxylin for 2min, and washing with clear water;

m) placing the slices in 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, and anhydrous alcohol for 3min respectively, and transparent xylene for 10min × 2 times;

n) neutral gum mounting, negative control PBS instead of primary antibody, the same procedure, observation under microscope, photography and analysis of the results.

(5) Determination of immunohistochemical staining results

Two doctors read the film in a double blind way respectively. Positive expression is the appearance of yellow, tan or brown granules in the cytoplasm and/or nucleus. 10 visual fields are randomly selected under a 400-fold microscope, the staining intensity of the cells is observed, and the percentage of positive cells is calculated. The positive rate is less than or equal to 5 percent and is 0min, 6 to 25 percent is 1min, 26 to 50 percent is 2min, 51 to 75 percent is 3min, and more than 75 percent is 4 min; the dyeing intensity is divided into 4 grades, the non-coloring is 0 grade, the light yellow is 1 grade, the brown is 2 grades, and the brown is 3 grades. The percentage of positive cells in each field was multiplied by the staining intensity to give the field score, which was finally scored as the average of the 5 field scores. 0 is negative (-), 1-4 are weak positive (+), 5-8 are moderate positive (+ +), and 9-12 are strong positive (+++).

The detection results are respectively shown in fig. 3 and fig. 4, and the results show that the content of RIPK3 in the skin injury tissues is obviously improved compared with that in the normal tissues. The above results indicate that RIPK3 is positively correlated with the development of psoriasis.

4) Detection of simulated psoriasis cells by using M5 to stimulate HEKa cells

Stimulating HEKa cells to simulate the level of psoriasis cells by using M5(IL-17A, IL-22, IL-1a, OSM and TNF-a mixture), and extracting RIPK3 mRNA for detection; the specific operation is as follows:

(1) cell resuscitation

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

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

c) 5ml of DMEM medium containing 10% FBS was added to resuspend the cells, and the cells were inoculated to 25cm²Placing in a culture flask at 37 deg.C and 5% CO₂Culturing in an incubator, and replacing the culture medium the next day.

(2) Cell culture

Conventional cell culture at 37 deg.C and 5% CO₂In the incubator, the medium was changed every 2 d.

(3) Cell passage

a) When the cell coverage rate reaches more than 80%, passage can be carried out. The old culture medium was discarded first and washed 2 times with PBS;

b) adding 2ml of 0.25% pancreatin for digestion, standing at 37 ℃ for about 5min, observing under an inverted microscope, until cells become round and cell gaps become large, immediately adding 4ml of DMEM medium containing 10% FBS to stop digestion, gently blowing cell suspension by using a pipette, and transferring into a centrifuge tube;

c) transferring the cell suspension to a centrifuge tube, centrifuging at 800rpm for 5min, discarding supernatant, adding fresh culture medium for resuspension, and inoculating to a new culture flask for continuous culture. The general passage ratio is 1: 2-4.

(4) Cell cryopreservation

a) When the cell coverage rate is more than 80% and the state is good, the cells can be frozen. The old culture medium was discarded first and washed 2 times with PBS;

b) digesting with 0.25% pancreatin, stopping digestion with 10% FBS-containing DMEM medium, transferring into a centrifuge tube, and centrifuging at 800rpm for 5 min;

c) removing the supernatant, adding 1ml of cell cryopreservation solution, uniformly blowing and beating, and transferring to a cryopreservation tube;

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

Extraction and reverse transcription of total RNA from cells into cDNA

(1) Extraction of Total RNA from cells

a) Soaking a gun head, a gun head box, an EP tube and the like required by the experiment overnight by DEPC in advance to remove RNA enzyme;

b) discarding the culture medium in a six-hole plate or a 1.5cm culture dish, and washing twice with PBS;

c) adding 1ml Trizol, standing for 2min, blowing with sample gun, transferring into 1.5ml EP tube, gently inverting for 10 times, and standing on ice for 5min

d) Adding 200 μ l chloroform, reversing for 10 times to make it fully and uniformly mixed into milky white, standing on ice for 5min, and centrifuging at 12000rpm at 4 deg.C for 15 min;

e) the supernatant colorless and clear supernatant (about 300. mu.l) was carefully transferred to a new 1.5ml EP tube, an equal volume of isopropanol was added, gently inverted 10 times, and then left on ice for 20 min;

f) centrifuging at 12000rpm at 4 deg.C for 15min to obtain white precipitate at the bottom of the tube, and carefully discarding the supernatant;

g) adding 1ml of precooled 75% ethanol, washing the precipitate, and then centrifuging at 7500rpm at 4 ℃ for 5 min;

h) discarding supernatant, drying at room temperature for 5-10min to obtain transparent white precipitate at bottom, adding appropriate amount (30-50 μ l) of DEPC water, stirring, and standing at 55-60 deg.C for 10min to dissolve RNA;

i) determination of RNA purity and concentration: mu.l of the extracted RNA was pipetted into 99. mu.l of DEPC water (diluted 100 times) and measured on a spectrophotometer for OD260nm and OD280nm values, OD260/OD280 between 1.9 and 2.0 indicating a higher purity of the extracted RNA. The total RNA concentration (μ g/ml) was A260nm × 40 × dilution, which was required for the subsequent reverse transcription into cDNA, and the remaining RNA product was stored in a freezer at-80 ℃ until use.

(2) Reverse transcription reaction

a) Adding reagents into a PCR tube on ice according to a reverse transcription system, uniformly mixing, and then centrifuging for a short time;

5×RT super mix 4μl

RNA 2μg

DEPC water was added to a total volume of 20. mu.l

b) Putting the mixture into a PCR instrument, and carrying out reverse transcription under the conditions: at 25 ℃ for 10 min; 30min at 42 ℃; 5min at 85 ℃;

c) the reverse transcription product is stored at-20 ℃ for later use.

4) General PCR

(1) PCR amplification reaction

a) Add 80. mu.l DEPC water to the reverse transcribed 20. mu.l cDNA (i.e., dilute to 5-fold);

b) reagents were added to the PCR tubes on ice as follows, mixed well and centrifuged briefly. Primer sequences are shown in Table 1;

TABLE 1 PCR primer sequences

b) Placing into a PCR instrument, and reacting under the conditions shown in Table 2:

TABLE 2 PCR reaction conditions

(2) Agarose gel electrophoresis

a) Adding 2.5% agarose gel, cooling at room temperature for 20-30min to solidify into gel;

b) taking 10 mu l of each RNA sample, uniformly mixing 6 mu l of Marker, and adding into a sample adding hole;

c) electrophoresis: electrophoresis is carried out for 20-30min under the voltage of 110V, and the gel is observed by a gel ultraviolet analyzer and photographed.

5) Fluorescent real-time quantitative PCR (qRT-PCR)

(1) Adding reagents into a PCR tube on ice according to a system shown in the table 3, uniformly mixing, and then centrifuging for a short time;

TABLE 3 PCR reaction System

(2) The reaction was carried out in a real-time quantitative PCR apparatus under the conditions shown in Table 4:

TABLE 4 PCR reaction conditions

95℃	10min
			95℃	30s
58℃	30s	40 cycles
			72℃	30s
60℃	1min
			95℃	30s	Melting curve
61.5℃	15s

(3) And (4) judging a result: by 2^-ΔΔCtThe method of (1) analyzes the CT value and calculates the relative expression level of the gene.

The result of relative expression change detection of RIPK3 mRNA by real-timePCR is shown in figure 5, and the result shows that the expression of RIPK3 is increased after HEKa cells are stimulated to simulate the level of psoriasis cells

5) Detection of M5 stimulated HEKa cells after siRNA knockout of RIPK3

siRNAs aiming at RIPK3 are si-negative and si-RIPK3, and the sequences are respectively as follows:

h-RIPK3 Forward：5’-GCUGAAAGAAGUGGUGCUUTT，

Reverse：5’-AAGCACCACUUCUUUCAGCTT

Negative Forward：5’-UUCUCCGAACGUGUCACGUTT-3’

Control

Reverse：5’-ACGUGACACGUUCGGAGAATT-3’

siRNA transfection procedure

a) About 1.5-2X 10 h before transfection⁵The individual cells were seeded in 6-well plates in DMEM medium containing 10% FBS at 37 ℃ in 5% CO₂The cell is incubated in the incubator for 24 hours, and the cell fusion degree needs to reach 60-70% during transfection;

b) adding RNAimax at 5 μ l/well into 250 μ l/well Opti-MEM serum-reduced medium, mixing by gently turning upside down, and incubating at room temperature for 5 min;

c) respectively adding 5 μ l/well RIPK3-siRNA and negative control siRNA into 250 μ l/well Opti-MEM reduced serum culture medium, and mixing;

d) mixing the liquids in b) and c), gently inverting and mixing, and incubating for 20min at room temperature to form a transfection complex of siRNA and RNAimax;

e) dripping the corresponding mixture of siRNA and RNAimax into each hole, supplementing the total amount of liquid in each hole to be 2ml by using a pure DMEM culture medium, gently shaking the 6-hole plate to mix uniformly, and putting the 6-hole plate into a cell culture box after marking;

f) after the conventional culture for 6h, the medium was replaced by normal DMEM medium containing 10% FBS and 1% double antibody.

(3) Verification of transfection efficiency

a) RNA is extracted 24h after transfection, and qRT-PCR experiment is carried out to detect RIPK3 mRNA expression level;

b) protein is extracted 48h after transfection, and Western Blot experiment is carried out to detect the RIPK3 protein expression level.

FIG. 5 is a western blot 48 hours after applying si-negative and si-RIPK3 knockout RIPK3 to Heka cells to extract total cell protein and detect related molecules.

The results show a decrease in NLRP3 expression after RIPK3 is inhibited, suggesting that RIPK3 contributes to the progression of psoriasis by activating NLRP3 signaling.

The experimental detection shows that RIPK3 is positively correlated with the development of psoriasis, and when the RIPK3 inhibitor preparation with a proper concentration is used for treating an imiquimod mouse psoriasis model, scales and skin lesion thickness can be improved, and the effects of inhibiting the development of psoriasis and inhibiting the skin lesion from recovering to be normal are achieved by inhibiting NLRP3 signals.

Therefore, RIPK3 can be used as a target for preparing a medicament for preventing and treating psoriasis.

And RIPK3 can be used as a drug target at the gene level and/or protein level. Such as by DNA or RNA that blocks RIPK3 expression or transcription, or a drug that blocks or inhibits RIPK3 action NLRP3 signaling. Meanwhile, the corresponding inhibitor preparation aiming at RIPK3 and the carrier of siRNA knockout RIPK3 can be applied to the preparation of the medicament for preventing and treating psoriasis.

The embodiments given above are preferable examples for implementing the present invention, and the present invention is not limited to the above-described embodiments. Any non-essential addition and replacement made by the technical characteristics of the technical scheme of the invention by a person skilled in the art belong to the protection scope of the invention.

Claims (2)

1. The medicine for preventing and treating psoriasis is characterized by being prepared from the following raw materials:

GSK' 872 powder 5 mg; 2.6076ml of DMSO; 7.3924ml of physiological saline with the mass concentration of 0.9 percent.

2. The medicament of claim 1, prepared by the steps of:

weighing 5mg GSK' 872 powder, centrifuging at 2000rpm for 1min, dissolving in 2.6076ml DMSO, shaking, and dissolving in 7.3924ml 0.9% NaCl solution to obtain suspension.

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