Application of LSR in the preparation of drugs for preventing and treating IBD

Abstract

The present invention discloses the application of LSR (lipid metabolism activated lipoprotein receptor) in the preparation of drugs for preventing and treating IBD (inflammatory bowel disease). By designing and constructing a mouse model of knocking out the Lsr gene and a mouse model of overexpressing LSR, the relationship between LSR and the occurrence and development of IBD inflammation in the intestine of the body is studied. The results show that LSR itself has a protective and improving effect on the occurrence and development of IBD. LSR has a new use for preventing and treating IBD, and has no side effects, a significant therapeutic effect, and no complications. It can fundamentally cure IBD and prevent the occurrence of IBD.

Description

Technical Field

[0001] The invention relates to a new application of LSR, in particular to an application of LSR in preparing medicines for preventing and treating IBD, and belongs to the technical field of medicines. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, recurrent, nonspecific inflammatory disease that occurs in the gastrointestinal tract and is caused by the interaction of multiple factors. It mainly includes ulcerative colitis (UC) and Crohn's disease (CD), as well as indeterminate colitis (IC) that cannot be determined as UC or CD by pathology, and neonatal necrotizing enterocolitis (NEC). Among them, UC mainly affects the colorectum, while CD can affect any part of the digestive tract. In addition, IBD can also have some extraintestinal clinical manifestations, but the clinical features are relatively hidden. Depending on the specific site of the lesion, it may manifest as fever, mucosal inflammation, abdominal pain, diarrhea, and bloody stools.

[0003] The current strategies for the treatment of IBD are mainly to induce remission (eliminate symptoms) and maintain remission (prevent relapse). Traditional treatment drugs for IBD include aminosalicylic acid preparations, glucocorticoids, and immunosuppressants, among which:

[0004] (1) Aminosalicylic acid preparations: 5-aminosalicylic acid works by inhibiting NF-kB and scavenging free radicals. It is clinically used to treat tuberculosis, mild and moderate ulcerative colitis, and maintenance treatment of ulcerative colitis.

[0005] (2) Glucocorticoids: Corticosteroids have the function of inhibiting inflammatory response and relieving clinical symptoms. They can be used in the acute phase of IBD and are not used as maintenance treatment. Commonly used drugs include prednisone, methylprednisone, hydrocortisone, dexamethasone, budesonide, etc.

[0006] (3) Immunosuppressants: They are mainly used for maintenance treatment after hormone ineffectiveness or dependence and hormone-induced remission. Commonly used immunosuppressants in clinical practice include azathioprine, thiopurine, cyclosporine and tacrolimus.

[0007] Except for aminosalicylic acid preparations, glucocorticoids and immunosuppressants have many side effects, and the therapeutic effects vary greatly from person to person. They are often accompanied by a variety of intestinal and extraintestinal complications. More importantly, the therapeutic effects are limited.

[0008] According to incomplete statistics, more than 5 million people suffer from IBD worldwide, and the incidence rate varies depending on the region and race. Epidemiological surveys show that the incidence and prevalence of IBD worldwide are increasing year by year, and the incidence rate of IBD in my country is also showing an increasing trend year by year, and has become a common digestive system disease in my country. As a chronic, idiopathic disease of the intestine, IBD has not yet been clarified in terms of etiology and pathology, and the clinical efficacy is not ideal enough. At present, IBD has been listed as a refractory disease in the world. Due to its long course of disease, difficulty in curing, high cost, difficulty in control, easy deterioration, and even disability, IBD brings great mental stress and economic burden to patients. Therefore, early detection and early treatment are the key to improving the prognosis of IBD at present. In-depth exploration of the pathogenesis of IBD and finding effective intervention targets not only have important theoretical value, but also have very urgent practical significance.

[0009] Lipolysis stimulated receptor (LSR) is a tricellular tight junction (tTJ) molecule localized in most epithelial tissues. LSR is a member of the angulin family, also known as angulin-1, and is a newly discovered tTJ molecule. LSR contains an extracellular N-terminal Ig-like domain, a transmembrane region, and a cytoplasmic C-terminal tail.

[0010] So far, the functions of LSR that have been discovered include: acting as a barrier and fence between cells; participating in liver lipid metabolism; participating in cell migration, infiltration and proliferation; and being a component of the blood-brain barrier. However, the role of LSR in the intestine has not been reported, and whether it is involved in the occurrence and development of IBD is also unclear. Summary of the invention

[0011] The purpose of the present invention is to explore new uses of LSR in preventing and treating IBD by studying the relationship between LSR and the occurrence and development of IBD inflammation in the intestine of the body.

[0012] In order to achieve the above object, the present invention adopts the following technical solution:

[0013] By designing and constructing a mouse model with Lsr gene knockout and a mouse model with LSR overexpression, the relationship between LSR and the occurrence and development of IBD inflammation in the intestine was studied. The results showed that:

[0014] (1) Knockout of the Lsr gene: It can significantly increase the level of inflammatory factors in IBD, significantly enhance the level of inflammatory cell infiltration in IBD, increase the damage to intestinal tissue morphology in IBD, destroy the function and distribution of Paneth cells in IBD, and increase the incidence of IBD;

[0015] (2) Overexpression of LSR: It can significantly reverse the above phenomenon. Specifically, it can significantly reduce the level of inflammatory factors in IBD, significantly weaken the level of inflammatory cell infiltration in IBD, and at the same time improve the damage to intestinal tissue morphology in IBD, improve the function and distribution of Paneth cells in IBD, and significantly reduce the incidence of IBD.

[0016] It can be seen that LSR itself has a protective and improving effect on the occurrence and development of IBD, and LSR has a new use in the prevention and treatment of IBD.

[0017] The benefit of the present invention lies in that: through research, it is found that LSR can significantly improve the pathological characteristics of IBD, has no side effects, has a significant therapeutic effect, and does not cause complications. It can fundamentally cure IBD and prevent the occurrence of IBD. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG1( a ) is a graph showing changes in body weight of three groups of mice;

[0019] FIG1( b ) is a graph showing the disease activity index scores of the three groups of mice;

[0020] FIG1( c ) is a graph showing the measurement results of the colon lengths of three groups of mice;

[0021] Figure 2 is the relative expression of Lsr gene in colon tissues of DSS WT group mice and control group mice;

[0022] Figure 3 It is a graph showing the levels of TNF-α, IL-1β and IL-6 in the serum of the three groups of mice;

[0023] Figure 4 This is the HE staining result of colon tissues of three groups of mice;

[0024] FIG5( a ) is a graph showing the relative expression of Lsr genes in colon tissues of three groups of mice;

[0025] FIG5( b ) is a graph showing changes in body weight of three groups of mice;

[0026] FIG5( c ) is a graph showing the disease activity index scores of the three groups of mice;

[0027] FIG5( d ) is a graph showing the measurement results of the colon lengths of the three groups of mice;

[0028] Figure 6 It is a graph showing the levels of TNF-α, IL-1β and IL-6 in the serum of the three groups of mice;

[0029] Figure 7 This is the HE staining result of colon tissues of three groups of mice;

[0030] Figure 8 is a graph of the relative expression of Lsr gene and IL-1α, TNF-α, IL-6, IL-2, and IFN-γ genes in the intestinal tissues of the three groups of mice;

[0031] Fig. 9 This is the HE staining result of small intestine tissue of three groups of mice;

[0032] Fig.10 This is the result of immunofluorescence staining of Paneth cells in the small intestine tissues of the three groups of mice;

[0033] Fig.11 is a graph of the relative expression of Lsr gene and IL-1α, TNF-α, IL-6, IL-2, and IFN-γ genes in the intestinal tissues of the three groups of mice;

[0034] Fig.12 This is the HE staining result of small intestine tissue of three groups of mice;

[0035] Fig.13 These are the results of immunofluorescence staining of Paneth cells in the small intestinal tissues of the three groups of mice. DETAILED DESCRIPTION

[0036] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] 1. Experimental Animals

[0038] 1. C57BL / 6J mice

[0039] C57BL / 6J mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0040] 2. Lsr loxP / loxP Mouse

[0041] oeLh loxP / loxP The mouse was constructed by the inventors using CRISPR Cas9 technology, and the construction process is as follows:

[0042] In vitro, a gene sequence containing loxP sites was inserted on both sides of the first exon of the Lsr gene. The recombinant gene sequence was then transferred into embryonic stem cells of C57BL / 6J mice by electroporation. The embryonic stem cells were then reimplanted into the uterus of pseudo-pregnant mice to develop into a complete embryo and produce a transgenic mouse. The transgenic mouse was then hybridized with C57BL / 6J mice (the purpose of hybridization was to remove the Neo transgene) to obtain Lsr loxP / - Mice, then Lsr loxP / - The mice were self-fertilized and eventually produced Lsr loxP / loxP Mouse.

[0043] 3.villin- Cre Mouse

[0044] villin- Cre Mice were purchased from Jackson Laboratory.

[0045] 4.villin- Cre+ / - / Lsr loxP / loxP LSR conditional knockout mice (KO) and villin- Cre- / - / Lsr loxP / loxP Wild-type mice (WT)

[0046] villin- Cre+ / - / Lsr loxP / loxP LSR conditional knockout mice (KO) and villin- Cre- / - / Lsr loxP / loxP Wild-type mice (WT) were generated by the inventors by loxP / loxP Mice and villin- Cre The mice were obtained by hybridization, and the specific process of obtaining them is as follows:

[0047] Let villin- Cre Mouse and Lsr loxP / loxP Mice were crossed to obtain villin- Cre+ / - / Lsr loxP / - Mice, then let villin- Cre+ / - / Lsr loxP / - With Lsr loxP / loxP Mice were crossed to obtain villin- Cre+ / - / Lsr loxP / loxP LSR conditional knockout mice (KO);

[0048] Let villin- Cre+ / - / Lsr loxP / loxP LSR conditional knockout mice (KO) and Lsr loxP / loxP Mice were crossed to obtain villin- Cre- / - / Lsr loxP / loxP Wild-type mice (WT).

[0049] 2. Study the effect of knocking out the Lsr gene on ulcerative colitis (UC)

[0050] 1. Experimental animal grouping and drug administration method

[0051] (1) Control group

[0052] villin- Cre- / - / LsrloxP / loxP Eight wild-type mice (WT), male, 8 weeks old, were given free access to distilled water for 6 consecutive days.

[0053] (2)DSS WT group

[0054] The villin- Cre- / - / Lsr loxP / loxP Eight wild-type mice (WT), male, 8 weeks old, were allowed to drink freely 3% (w / v) dextran sulfate sodium (DSS) solution prepared with distilled water (used to induce acute colitis model in mice) for 6 consecutive days.

[0055] (3)DSS KO group

[0056] The villin- Cre+ / - / Lsr loxP / loxP Eight LSR conditional knockout mice (KO), male, 8 weeks old, were allowed to drink 3% (w / v) dextran sulfate sodium solution prepared with distilled water freely for 6 consecutive days.

[0057] 2. Disease Activity Index Score

[0058] The disease activity index (DAI) score is a comprehensive score based on weight change, stool consistency, and rectal bleeding, which can be used to assess the severity of enteritis.

[0059] The mice in each group were weighed and their body weights were recorded every day. The body weight on day 0 was used as the benchmark to calculate the body weight changes, where a weight loss of <1% was scored as 0 points, a weight loss of 1% (inclusive) to 5% was scored as 1 point, a weight loss of 5% (inclusive) to 10% was scored as 2 points, a weight loss of 10% (inclusive) to 20% was scored as 3 points, and a weight loss of ≥20% was scored as 4 points.

[0060] The fecal status of mice in each group was observed daily, where normal fecal morphology was scored as 0 points, soft but formed feces was scored as 1 point, soft and unformed feces was scored as 2 points, and diarrhea-like feces was scored as 3 points.

[0061] The rectal bleeding of each group of mice was counted daily, where a negative result was scored as 0 points, a positive result but no visible blood was scored as 1 point, a positive result with visible blood in the feces was scored as 2 points, and a positive result with obvious bleeding was scored as 3 points.

[0062] 3. Sampling and sample processing

[0063] The mice in each group were anesthetized with isoflurane ether, and then the eyeballs were removed for blood collection. The blood was centrifuged at 4500g / min for 15min, and the serum was collected and stored at -80℃ for use.

[0064] After removing the eyeballs for blood collection, the mice in each group were killed by cervical dislocation, and the abdominal cavity was immediately opened to expose the abdominal cavity. The colon tissue (the intestinal segment from the end of the cecum to the anus) was found, and the colon tissue was cut off and the length was measured.

[0065] The complete colon of each group of mice was made into a "Swiss roll" and fixed with a 4% (w / v) paraformaldehyde solution at 4°C for 24 h. The colon tissue of the mice was then paraffin-embedded and sliced ​​into 5 μm sections and stained with HE. The pathological changes of the colon tissue were observed under an optical microscope.

[0066] 4. Enzyme-linked immunosorbent assay

[0067] The serum of the mice in each group was collected, and the inflammatory factors TNF-α, IL-1β and IL-6 were detected according to the instructions in the kit.

[0068] 5. RNA extraction and real-time quantitative PCR

[0069] The small intestinal samples were homogenized in Trizol (Ambion, USA) to obtain total RNA. The RNA was reverse transcribed using a reverse transcription system kit according to the instructions provided by the manufacturer (Takara, Japan), and then fluorescent quantitative PCR was performed using a ThermoFisher QS3 PCR instrument according to the instructions in the SYBR Green PCR Master Mix (ABI, USA) kit. The internal reference gene was β-actin, based on ΔCT=Ct gene -Ct β-actin Calculate the relative expression level of each target gene mRNA and use 2 -ΔCt Value representation.

[0070] 6. Data Analysis

[0071] Prism 8 software was used to analyze the experimental data. Unpaired t-test was used for comparison between two groups, and one-way ANOVA was used for comparison between multiple groups. The data were expressed as mean ± standard error (Mean ± SEM), and P < 0.05 was interpreted as statistically significant.

[0072] 7. Experimental results

[0073] (1) Weight changes

[0074] The weight changes of the three groups of mice are shown in Figure 1(a). It can be seen from Figure 1(a):

[0075] As time went on, the body weight of mice in the control group showed a relatively stable upward trend, while the body weight of mice in the DSS WT group and the DSS KO group showed a downward trend, among which the body weight of mice in the DSS KO group decreased more significantly (P<0.001).

[0076] (2) DAI rating

[0077] Since DAI is a method for evaluating the severity of enteritis and a comprehensive score based on weight change, stool hardness and stool bleeding, we recorded and scored the weight, stool hardness and bleeding of each group of mice every day. The final DAI score results are shown in Figure 1(b). As shown in Figure 1(b):

[0078] From the third day, the DAI of both DSS WT and DSS KO mice began to increase, but the increasing speed of DSS WT mice was lower than that of DSS KO mice.

[0079] (3) Changes in colon length

[0080] Colon shortening is an indirect reflection of the course of colitis. The length of the colon cut off from each group of mice after being killed is shown in Figure 1(c). It can be seen from Figure 1(c) that:

[0081] Compared with the control group mice, the colons of both DSS WT and DSS KO group mice were significantly shortened, among which the colons of DSS KO group mice were particularly shortened.

[0082] The results shown in Figures 1(a), 1(b) and 1(c) preliminarily indicate that LSR plays an important role in maintaining intestinal homeostasis and that Lsr gene deletion can aggravate the development of inflammatory bowel disease.

[0083] (4) Relative expression of Lsr genes

[0084] The results of the relative expression of Lsr gene in colon tissues of DSS WT group mice and control group mice are shown in Figure 2 As shown by Figure 2 It can be seen that:

[0085] The relative expression of Lsr gene in colon tissue of mice in the DSS WT group was significantly decreased.

[0086] Figure 2 The results shown further illustrate that LSR plays an important role in maintaining intestinal homeostasis and that Lsr gene deletion can aggravate the development of inflammatory bowel disease.

[0087] (5) Contents of TNF-α, IL-1β and IL-6

[0088] The test results of TNF-α, IL-1β and IL-6 levels in the serum of each group of mice are as follows Figure 3 As shown by Figure 3 It can be seen that:

[0089] Compared with the control group mice, the levels of TNF-α, IL-1β and IL-6 in the serum of DSS WT group mice and DSS KO group mice were significantly upregulated, among which the levels of TNF-α, IL-1β and IL-6 in the serum of DSS KO group mice were more significantly upregulated.

[0090] (6) HE staining

[0091] The results of HE staining of colon tissues of mice in each group are shown in Figure 4 As shown by Figure 4 It can be seen that:

[0092] (i) The colon tissue structure of mice in the control group was intact, the intestinal mucosa was intact, the morphology and number of goblet cells and crypts were normal, and there were few inflammatory cells;

[0093] (ii) The colon tissue structure of DSS WT and DSS KO mice was damaged, the mucosa was severely damaged, the number of goblet cells and crypts was reduced, the crypt morphology was damaged, and there was a large amount of inflammatory cell infiltration. The symptoms of DSS KO mice were more obvious.

[0094] 3. Study the effect of LSR overexpression on ulcerative colitis (UC)

[0095] 1. Experimental animal grouping and drug administration method

[0096] Twenty-four C57BL / 6 mice, male, 7 weeks old, were randomly divided into three groups: control group, DSS+AAV-CTL group, and DSS+AAV-Lsr group, with 8 mice in each group.

[0097] (1) Control group

[0098] C57BL / 6 mice were allowed to drink distilled water freely for 6 consecutive days.

[0099] (2)DSS+AAV-Lsr group

[0100] 7-week-old C57BL / 6J mice were injected intraperitoneally with 1×10 9 MOI LSR overexpression virus particles AAV-Lsr, after feeding for one week, the mice were allowed to freely drink 3% (w / v) dextran sodium sulfate solution prepared with distilled water for 6 consecutive days.

[0101] LSR overexpression virus particles AAV-Lsr were constructed using the following method:

[0102] (I) Construction of plasmid

[0103] (1) Full-length amplification of the Lsr target gene

[0104] The full-length coding sequence of the mouse Lsr gene was obtained from the NCBI database. The gene number is: NM_001164184.1. The primer sequence of the target gene was designed, where:

[0105] AAV-m-Lsr-B / KF:

[0106] ttgacctccatagaagacaccgggatccAAGCTTGCCCACCATGGCGCC;

[0107] AAV-m-Lsr-B / KR:

[0108] tgtagtcgttaattaaggtacCGAATTCGACGACTAAACTTTCCCGACT.

[0109] The design of the primers includes exchange pairing bases, a BamHI restriction site, a KpnI restriction site, an expression enhancement sequence and a 5' end partial sequence of the target gene.

[0110] Total RNA was extracted from mouse tissues and reverse transcribed into cDNA. The cDNA was used as a template for PCR amplification (PCR cycle parameters: 95°C, 5 min, 1 cycle; then 95°C, 30 s, 55°C, 30 s, 72°C, 60 s, 30 cycles, 72°C, 10 min, one cycle again; and finally 4°C, ∞, one cycle). Then, the mouse Lsr gene fragment was recovered by electrophoresis and gel cutting.

[0111] (2) Construction and extraction of recombinant plasmid

[0112] First, the vector pHBAAV-CMV-MCS-3flag-T2A-ZsGreen and the recovered mouse Lsr gene fragment were double-digested with restriction endonucleases BamHI and KpnI. The final enzyme digestion system was 50 μl, including: 10×CutSmart Buffer 5 μl, vector 10 μl, BamHI 1 μl, KpnI 1 μl, and the remaining system was made up with double distilled water.

[0113] Next, the linearized vector DNA was mixed with the purified PCR product at a molar ratio of 1:2 and HB infusion was performed. TM One-step cloning and ligation system, in HB infusionTM The reaction was carried out at 50°C for 30 minutes under the action of the master mix, followed by cooling in an ice-water bath for 5 minutes before transformation. 10 μl of the above reaction product was added to 100 μl of DH5α competent cells, mixed and placed on ice for 30 minutes, then heat-shocked at 42°C for 90 seconds, incubated on ice for 3 minutes, and then 200 μl of LB medium was added, and shaken and cultured in a shaker at 37°C for 1 hour.

[0114] Finally, all the above bacterial liquid was evenly spread on a plate containing ampicillin antibiotics and incubated in an incubator for 12 hours.

[0115] (3) Identification of recombinant plasmids

[0116] Pick 5 single colonies from the plate and place them in 5 LB culture media containing ampicillin respectively. Shake the culture medium at 37°C. After 12 hours, take 1 ml of the culture medium from each culture medium and send it to Sangon Biotech Co., Ltd. for sequencing to identify the accuracy of the recombinant plasmid. The remaining culture medium is preserved with 30% glycerol and stored in a -80°C refrigerator for later use.

[0117] (II) AAV-293 cell culture

[0118] (1) Recovery of AAV-293 cells:

[0119] ① Set the water bath temperature to 37°C;

[0120] ② Take out the frozen AAV-293 cells from the liquid nitrogen tank, quickly put them into a water bath and shake them quickly, trying to completely dissolve the cell solution within 1 minute;

[0121] ③ Transfer the cell solution to a 15 ml centrifuge tube, add 5 ml of fresh complete medium to the centrifuge tube, mix well and centrifuge at 1000 rpm for 5 min;

[0122] ④ Remove the supernatant, add 5 ml of fresh complete culture medium, mix the precipitate and transfer it to a T225 culture flask, add DMEM complete culture medium (DMEM + penicillin + streptomycin + 10% fetal bovine serum), and place the culture flask steadily in an incubator at 37°C and 5% CO2 for culture.

[0123] (2) The cells were digested with 0.25% trypsin for 5 min, then the trypsin was removed and fresh complete medium was added to mix well. The cells were then transferred to a 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and 5 ml of complete medium was added to the precipitate for resuspending.

[0124] (3) Take 50 μl of the mixed cells and place them in a 1.5 ml EP tube. Add 450 μl of DMEM complete medium and mix well. Take 10 μl of the cells and count them in a counting plate. Subculture the AAV-293 cells into a 100 mm culture dish (for transfection). The day of plating is recorded as the first day.

[0125] (4) Place the above culture dish in a 37°C, 5% CO2 incubator for culturing. After two days of culturing, observe the cell density. When the culture dish reaches 80% to 90% full, transfection can be performed.

[0126] (III) AAV packaging and concentration

[0127] (1) Plasmid amplification

[0128] The constructed AAV vector, packaging plasmid and auxiliary plasmid (vector plasmid carrying the target gene, pAAV-RC, pHelper, respectively) were extracted in large quantities using the Qiagen large-scale extraction kit. When the concentration was greater than 1 μg / μl and the A260 / 280 was between 1.7 and 1.8, they could be used to package the virus.

[0129] (2) The culture dish containing AAV-293 cells (the cells reach 80% to 90% monolayer confluence) is replaced with a culture medium, and then the cells are transfected using Lipofiter™ transfection reagent (purchased from Hanheng Biotechnology, for instructions, refer to the Lipofiter™ manual).

[0130] (3) LipofiterTM transfection reagent was mixed with DMEM medium without dual antibody and serum at a volume ratio of 1:25 to obtain mixed solution 1, the above-mentioned AAV vector, packaging plasmid and auxiliary plasmid were mixed at a mass ratio of 1:2:1 to obtain mixed solution 2, mixed solution 2 was mixed with DMEM medium without dual antibody and serum at a volume ratio of 1:25 to obtain mixed solution 3, mixed solution 1 and mixed solution 3 were mixed at a volume ratio of 1:1, and added dropwise to the pre-plated AAV-293 cells, cultured at 37°C and 5% CO2, replaced with DMEM complete medium after 6 hours, and the culture dish with the transfection plasmid was placed in an incubator and cultured for 72 hours.

[0131] (4) AAV virus collection: 72 h after transfection, cells were harvested from a 150 mm culture dish using a cell scraper and centrifuged at 150 g for 3 min to collect the cell pellet, which was then resuspended in 300 μl PBS. The centrifuge tube containing the cells was repeatedly frozen and thawed three times in liquid nitrogen and a 37°C water bath, followed by centrifugation at 2000 g for 5 min at 4°C to remove cell debris and collect the lysis supernatant containing AAV particles (crude virus extract).

[0132] (5) Purification of AAV:

[0133] ① Treatment with universal nuclease: add 0.1 μl Benonase enzyme to every 1 ml of crude virus extract, incubate in 37°C water bath for 1 hour to remove the cell genome and residual plasmid DNA in the crude virus extract, then centrifuge at 600 g for 10 minutes at 4°C and take the supernatant;

[0134] ② Column purification: AAV virus column purification was performed according to the Biomiga adeno-associated virus purification kit V1469-01 operating instructions;

[0135] ③ Centrifugation: Add the AAV virus sample liquid (4 ml) obtained by column purification into the ultrafiltration tube, centrifuge at 1400g for 30 minutes to obtain the AAV virus (about 1 ml), collect the purified AAV virus and store it at -80°C.

[0136] (6) AAV virus packaging titer determination (using qRT-PCR method):

[0137] Take 3μl AAV virus, add 10μl DNAse, 41μl high-pressure water and 6μl Dilution Buffer, mix well and place in a 37℃ water bath for 1h, then place in a metal bath and heat at 100℃ for 10min. After cooling naturally to room temperature, add 2μl proteinase K, place in a 55℃ water bath for 1h, then quickly place in a metal bath and heat at 100℃ for 10min. Cool naturally to room temperature again and centrifuge to allow the liquid on the tube wall to gather at the bottom of the tube. After treatment, dilute 10 times and use for qRT-PCR titer detection.

[0138] (3) DSS+AAV-CTL group

[0139] 7-week-old C57BL / 6J mice were injected intraperitoneally with 1×10 9 After one week of feeding with MOI control virus particles AAV-CTL, mice were allowed to freely drink 3% (w / v) dextran sulfate sodium (DSS) solution prepared with distilled water (used to induce acute colitis model in mice) for 6 consecutive days.

[0140] The construction process of the control virus particle AAV-CTL is basically the same as the construction process of the LSR overexpression virus particle AAV-Lsr. The only difference is that when constructing the control virus particle AAV-CTL, the unmodified plasmid pHBAAV-CMV-MCS-3flag-T2A-ZsGreen is used instead of the recombinant plasmid. The unmodified plasmid is used as the AAV vector, and the AAV packaging, concentration and purification operations are performed in the same manner as the construction of the LSR overexpression virus particle AAV-Lsr.

[0141] 2. Disease Activity Index Score

[0142] Same as above.

[0143] 3. Sampling and sample processing

[0144] Same as above.

[0145] 4. Enzyme-linked immunosorbent assay

[0146] Same as above.

[0147] 5. RNA extraction and real-time quantitative PCR

[0148] Same as above.

[0149] 6. Data Analysis

[0150] Same as above.

[0151] 7. Experimental results

[0152] (1) Relative expression of Lsr genes

[0153] The detection results of the relative expression of Lsr gene in the colon tissue of each group of mice are shown in Figure 5(a). As shown in Figure 5(a):

[0154] Compared with those in the control group, the relative expression level of Lsr gene in the colon tissue of mice in the DSS+AAV-CTL group was significantly decreased, and the relative expression level of Lsr gene in the colon tissue of mice in the DSS+AAV-Lsr group was significantly increased (P<0.001).

[0155] (2) Weight changes

[0156] The weight changes of mice in each group are shown in Figure 5(b). It can be seen from Figure 5(b) that:

[0157] As time went on, the body weight of mice in the control group showed a relatively steady upward trend, while the body weight of mice in the DSS+AAV-CTL group and the DSS+AAV-Lsr group both showed a downward trend, among which the body weight of mice in the DSS+AAV-Lsr group decreased more slowly.

[0158] (3) DAI rating

[0159] The DAI score results of each group of mice are shown in Figure 5(c). It can be seen from Figure 5(c) that:

[0160] From the second day, the DAI of mice in the DSS+AAV-CTL group and the DSS+AAV-Lsr group began to increase, among which the DAI of mice in the DSS+AAV-Lsr group increased more slowly.

[0161] (4) Changes in colon length

[0162] The measurement results of the length of the colon cut off from each group of mice after being killed are shown in FIG5( d ). As shown in FIG5( d ),

[0163] Compared with the control group mice, the colons of the mice in the DSS+AAV-CTL group and the DSS+AAV-Lsr group were significantly shortened, among which the colon shortening phenomenon of the mice in the DSS+AAV-Lsr group was less severe.

[0164] The results shown in Figures 5(a), 5(b), 5(c) and 5(d) preliminarily indicate that overexpression of LSR can alleviate the development of IBD in mice.

[0165] (5) Contents of TNF-α, IL-1β and IL-6

[0166] The test results of TNF-α, IL-1β and IL-6 levels in the serum of each group of mice are as follows Figure 6 As shown by Figure 6 It can be seen that:

[0167] Compared with the control group mice, the levels of TNF-α, IL-1β and IL-6 in the serum of the DSS+AAV-CTL group mice and the DSS+AAV-Lsr group mice were significantly increased, among which the levels of TNF-α, IL-1β and IL-6 in the serum of the DSS+AAV-Lsr group mice increased more slowly.

[0168] It can be seen that overexpression of LSR inhibited the expression of various inflammatory factors in the serum of DSS-induced IBD mice.

[0169] (6) HE staining

[0170] The results of HE staining of colon tissues of mice in each group are shown in Figure 7 As shown by Figure 7 It can be seen that:

[0171] (i) The colon tissue structure of mice in the control group was intact, the intestinal mucosa was intact, the morphology and number of goblet cells and crypts were normal, and there were few inflammatory cells;

[0172] (ii) The colon tissue structure of mice in the DSS+AAV-CTL group was damaged, the mucosa was severely damaged, the number of goblet cells and crypts was reduced, the crypt morphology was damaged, and a large number of inflammatory cells infiltrated;

[0173] (iii) These symptoms were alleviated in the DSS+AAV-Lsr group compared with those in the DSS+AAV-CTL group.

[0174] It can be seen that after overexpression of LSR, the symptoms of DSS-induced IBD mice were alleviated.

[0175] 4. Study the effect of knocking out the Lsr gene on neonatal necrotizing enterocolitis (NEC)

[0176] 1. Experimental animal grouping and drug administration method

[0177] (1) Control group

[0178] villin- Cre- / - / Lsr loxP / loxP Wild-type mice (WT), male, 14 days old, 5 mice, no treatment.

[0179] (2) TNBS WT group

[0180] The villin- Cre- / - / Lsr loxP / loxP Wild-type mice (WT), male, 14 days old, 5 mice, were administered 2,4,6-trinitrobenzenesulfonic acid (TNBS, for developing NEC-like lesions) at a dose of 50 mg / kg by both gavage and enema.

[0181] (3)TNBS KO group

[0182] The villin- Cre+ / - / Lsr loxP / loxP LSR conditional knockout mice (KO), male, 14 days old, 5 mice, were administered 50 mg / kg of 2,4,6-trinitrobenzenesulfonic acid (TNBS, for developing NEC-like lesions) by gavage and enema.

[0183] 2. Sampling and sample processing

[0184] The mice in the TNBS WT group and the TNBS KO group were killed by cervical dislocation 48 hours after drug administration. At the same time, the mice in the control group were killed by cervical dislocation. After the mice in each group were killed by cervical dislocation, the abdominal cavity was immediately opened to expose the abdominal cavity, and the small intestine tissue was found and removed. Then, it was divided into three parts according to different segments (duodenum, jejunum, and ileum) and made into "Swiss rolls". After that, it was fixed with a 4% (w / v) paraformaldehyde solution at 4°C for 24 hours, and then dehydrated with alcohol gradient. The small intestine tissue of a part of the mice was paraffin-embedded and sliced ​​5 times continuously. The intestinal tissues were divided into two groups and 4 groups, and the pathological changes of the small intestine were observed under an optical microscope. The intestinal damage was confirmed by histopathological grading and designated by a blinded reviewer: (1) no damage; (2) mild damage: rupture of the tips of the small intestinal villi or mild separation of the lamina propria; (3) moderate damage: rupture of the villi, obvious separation of the lamina propria and / or edema of the submucosal layer; (4) severe damage: transmural damage of the small intestine. The small intestinal tissues of another part of the mice were embedded in OCT and frozen sections were made for immunofluorescence staining.

[0185] 3. RNA extraction and real-time quantitative PCR

[0186] Same as above.

[0187] 4. Immunofluorescence staining

[0188] The specific process of immunofluorescence staining is as follows:

[0189] (1) Fixation: The tissue sections of each group were fixed with methanol at -20°C, taken out, and the fixative was washed away with PBS. The hydrophobic circle was drawn with a histological pen, and then washed twice with PBS, each time for 5 min;

[0190] (2) Serum blocking: 5% serum, blocking at room temperature for 1 h;

[0191] (3) Incubation with primary antibody: dilute rabbit anti-MPTX2 antibody 100-fold, add 50 μL of diluted primary antibody to each tissue, and incubate at 4°C for 12 h;

[0192] (4) Washing the primary antibody: Wash three times with blocking solution at room temperature, 10 min each time;

[0193] (5) Incubation with secondary antibody: dilute the rhodamine (TRITC)-conjugated goat anti-rabbit secondary antibody 300 times, add 50 μL of the diluted secondary antibody to each tissue, and incubate at room temperature for 90 min;

[0194] (6) Washing the secondary antibody: Wash three times with blocking solution at room temperature, 10 min each time;

[0195] (7) DAPI incubation: incubate DAPI at room temperature for 5 min, wash 4 times, 5 min each time;

[0196] (8) Sealing: Seal the film and take photos.

[0197] 5. Data Analysis

[0198] Same as above.

[0199] 6. Experimental results

[0200] (1) Relative expression of Lsr gene and IL-1α, IL-2, IL-6, IFN-γ and TNF-α genes

[0201] The results of the relative expression of Lsr gene and IL-1α, IL-2, IL-6, IFN-γ and TNF-α genes in the intestinal tissues of each group of mice are shown in Figure 2. Figure 8 As shown by Figure 8 It can be seen that:

[0202] (i) Compared with the control group, the relative expression levels of IL-1α, IL-2, IL-6, IFN-γ, and TNF-α genes in the intestinal tissues of the TNBS WT and TNBS KO mice were significantly upregulated (P<0.001), among which the relative expression levels of IL-1α, IL-2, IL-6, IFN-γ, and TNF-α genes in the TNBS KO group were more significantly upregulated (P<0.001).

[0203] (ii) Compared with the control group, the relative expression of Lsr gene in the intestinal tissue of TNBS WT group mice was significantly downregulated (P<0.001), and there was no Lsr gene expression in the intestinal tissue of TNBS KO group mice.

[0204] It can be seen that the loss of Lsr gene aggravates the development of NEC in mice.

[0205] (2) HE staining

[0206] The results of HE staining of small intestine tissue of mice in each group are shown in Fig. 9 As shown by Fig. 9 It can be seen that:

[0207] Compared with the control group mice, the intestinal segments of the TNBS WT group mice and the TNBS KO group mice showed varying degrees of damage, among which the intestinal damage of the TNBS KO group mice was more severe.

[0208] (3) Immunofluorescence staining

[0209] Paneth cells play an indispensable role in maintaining the stability of the infant's intestinal environment. NEC may be related to the loss of Paneth cells. To prove this phenomenon, we performed immunofluorescence staining on Paneth cells in the small intestinal tissues of each group. The staining results are shown in Figure 2. Fig.10 As shown by Fig.10 It can be seen that:

[0210] Compared with the control group mice, the number of Paneth cells in each segment of the intestinal tissue of the TNBS WT group mice and the TNBS KO group mice was significantly reduced. Among them, there were almost no Paneth cells in each segment of the intestinal tissue of the TNBS KO group mice.

[0211] 5. Study the effect of LSR overexpression on neonatal necrotizing enterocolitis (NEC)

[0212] 1. Experimental animal grouping and drug administration method

[0213] C57BL / 6 mice, male, 7 days old, 24 mice, were randomly divided into three groups: control group, TNBS+AAV-CTL group, and TNBS+AAV-Lsr group, with 8 mice in each group.

[0214] (1) Control group

[0215] No action is taken.

[0216] (2)DSS+AAV-Lsr group

[0217] 7-day-old C57BL / 6J mice were intraperitoneally injected with 1×10 8 MOI LSR overexpression virus particles AAV-Lsr, after feeding for one week, 2,4,6-trinitrobenzenesulfonic acid was applied to this group of mice at a dose of 50 mg / kg by gavage and enema.

[0218] The method for constructing LSR overexpression virus particles AAV-Lsr is the same as above and will not be repeated here.

[0219] (3) TNBS+AAV-CTL group

[0220] 7-day-old C57BL / 6J mice were intraperitoneally injected with 1×10 8 MOI control virus particles AAV-CTL, after feeding for one week, 2,4,6-trinitrobenzenesulfonic acid was applied to the mice in the group at a dose of 50 mg / kg by gavage and enema.

[0221] The construction method of the control virus particle AAV-CTL is the same as above and will not be repeated here.

[0222] 2. Sampling and sample processing

[0223] The mice in the TNBS+AAV-CTL group and the TNBS+AAV-Lsr group were killed by cervical dislocation 48 hours after drug administration. At the same time, the mice in the control group were killed by cervical dislocation, and the subsequent sampling and sample processing methods were the same as above.

[0224] 3. RNA extraction and real-time quantitative PCR

[0225] Same as above.

[0226] 4. Immunofluorescence staining

[0227] Same as above.

[0228] 5. Data Analysis

[0229] Same as above.

[0230] 6. Experimental results

[0231] (1) Expression of LSR and various inflammatory factors

[0232] The results of the relative expression of Lsr gene and IL-1α, IL-2, IL-6, IFN-γ and TNF-α genes in the intestinal tissues of each group of mice are shown in Figure 2. Fig.11 As shown by Fig.11 It can be seen that:

[0233] (i) Compared with the control group, the relative expression level of Lsr gene in the intestinal tissue of mice in the TNBS+AAV-CTL group was significantly downregulated (P<0.001), while the relative expression level of Lsr gene in the intestinal tissue of mice in the TNBS+AAV-Lsr group was significantly upregulated (P<0.001), indicating that the mice in the TNBS+AAV-Lsr group overexpressed LSR;

[0234] (ii) Compared with the mice in the TNBS+AAV-CTL group, the relative expression levels of IL-1α, IL-2, IL-6, IFN-γ, and TNF-α genes in the intestinal tissues of the mice in the TNBS+AAV-Lsr group were significantly downregulated (P<0.001), indicating that LSR overexpression can alleviate the development of NEC in mice.

[0235] (2) HE staining

[0236] The results of HE staining of small intestine tissue of mice in each group are shown in Fig.12 As shown by Fig.12 It can be seen that:

[0237] Compared with the control group mice, the intestinal segments of the TNBS+AAV-CTL group mice and the TNBS+AAV-Lsr group mice showed varying degrees of damage, among which the damage to the intestinal segments of the TNBS+AAV-Lsr group mice was lighter.

[0238] It can be seen that overexpression of LSR can significantly alleviate the inflammatory damage in various segments of the intestine.

[0239] (3) Immunofluorescence staining

[0240] The results of immunofluorescence staining of Paneth cells in the small intestine tissue of each group of mice are shown in Fig.13 As shown by Fig.13 It can be seen that:

[0241] Compared with the control group mice, the number of Paneth cells in various sections of intestinal tissues of the TNBS+AAV-CTL group mice and the TNBS+AAV-Lsr group mice was significantly decreased. Among them, the number of Paneth cells in various sections of intestinal tissues of the TNBS+AAV-CTL group mice was particularly significantly reduced, which was almost invisible from the immunofluorescence staining images.

[0242] It can be seen that overexpression of LSR can significantly restore the number of Paneth cells in various intestinal tissues of mice.

[0243] In summary, overexpression of LSR can significantly reduce the level of inflammatory factors in IBD, significantly reduce the level of inflammatory cell infiltration in IBD, and improve the damage to intestinal tissue morphology in IBD, improve the function and distribution of Paneth cells in IBD, and significantly reduce the incidence of IBD. Therefore, making LSR into a drug (for example: LSR overexpression virus particles AAV-Lsr) and administering it in a suitable manner (for example: intravascular injection, muscle injection, tissue in situ injection and oral administration) can significantly improve the pathological characteristics of IBD, and has no side effects, significant therapeutic effects, no complications, and can fundamentally cure IBD and prevent the occurrence of IBD.

[0244] It should be noted that the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. Application of lipid metabolism activated lipoprotein receptor gene overexpression virus particles AAV-Lsr in the preparation of drugs for the treatment of neonatal necrotizing enterocolitis.

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