Application of Atf3-siRNA in the preparation of drugs for treating liver fibrosis caused by Schistosoma japonicum

Abstract

The present invention discloses the use of Atf3-siRNA in the preparation of a drug for treating liver fibrosis caused by schistosomiasis japonicum, and relates to the field of biomedicine technology. The present invention discloses the use of Atf3-siRNA in the preparation of a drug for treating liver fibrosis caused by schistosomiasis japonicum, and the base sequence of Atf3-siRNA is 5'-CTGAGATTCGCCATCCAGAAT-3'. The results of the embodiments of the present invention show that compared with the siNC group, the degree of liver fibrosis in mice in the siAtf3 group is relatively alleviated, and there are still many gray-white worm egg nodules on the surface of the liver, which are reddish-brown in color and relatively soft in texture. Compared with the siNC group, the range of granulomas in the liver of mice in the siAtf3 group was significantly reduced, and the blue collagen was significantly reduced. In summary, ATF3 knockdown significantly alleviated the liver fibrosis lesions caused by Schistosoma japonicum infection.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and more specifically, relates to the application of Atf3-siRNA in preparing a medicine for treating liver fibrosis caused by schistosomiasis japonicum. Background Art

[0002] Schistosomiasis is a serious zoonotic parasitic disease caused by trematodes of the genus Schistosoma. Currently, there are more than 140 million schistosomiasis-infected patients worldwide, making it the second largest parasitic disease after malaria. Its main pathological manifestation is liver fibrosis, which can significantly affect the physiological function of the liver and lead to death of patients in the late stage. A survey data on schistosomiasis showed that although the number of new cases of infection in my country has dropped significantly after years of active prevention and control, there are still more than 1.8 million people who screen positive for schistosomiasis, of which there are still nearly 30,000 cases of late-stage schistosomiasis. Therefore, it is of great clinical significance to explore the molecular mechanism of liver fibrosis caused by schistosomiasis and to find potential molecular targets for intervention in liver fibrosis.

[0003] Activation transcription factor 3 (ATF3) is a member of the ATF / CREB transcription factor family, which is closely related to the cell's response to stress, including endoplasmic reticulum stress and oxidative stress. Unlike other ATF / CREB proteins, ATF3 is expressed at a low level in quiescent cells, but can be rapidly induced by a variety of cellular stresses and cytokines (such as IL-1β, TNF-α and TGF-β), so it is considered to be an adaptive response gene. Studies have shown that ATF3 plays an important role in the fibrosis process. For example, in systemic sclerosis fibroblasts, overexpression of ATF3 enhances the profibrotic effect of TGFβ. In the mouse liver fibrosis model induced by the chemical reagent carbon tetrachloride, ATF3 expression in the liver is upregulated and participates in the occurrence of liver fibrosis by promoting the transcription of the profibrotic factor ACTA2 (protein named α-SMA) / COL1A1. However, the role of Atf3 in the process of liver fibrosis caused by Schistosoma japonicum infection has not been reported.

[0004] In recent years, RNAi technology has experienced rapid development, and has also brought new hope and revolutionary changes to disease treatment. RNAi technology is used to specifically introduce siRNA into mammalian and human cells to reduce the expression of target genes, thereby causing a decrease in the expression of target proteins, achieving efficient and specific gene therapy. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide the use of Atf3-siRNA in the preparation of a drug for treating liver fibrosis caused by Schistosoma japonicum.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] The invention relates to the use of Atf3-siRNA in the preparation of a drug for treating liver fibrosis caused by schistosomiasis japonicum, wherein the base sequence of the siRNA is 5'-CTGAGATTCGCCATCCAGAAT-3'.

[0008] The gene ID of the Atf3 gene is: NM_007498.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] 1) The present invention utilizes gene recombination technology to design a 21-nucleotide Atf3 small interfering fragment; constructs an RNAi interference system of the Atf3 gene, and the base sequence of the RNAi interference system is 5'-CTGAGATTCGCCATCCAGAAT-3'.

[0011] 2) The present invention constructs a mouse schistosoma japonicum infection model with knockdown of ATF3, and the results show that the liver of mice in the siNC group exhibits a typical liver fibrosis phenotype, with a dark brown color, a hard and dense texture, and a large number of gray-white egg nodules on the surface. Compared with the siNC group, the degree of liver fibrosis in the siAtf3 group of mice was relatively alleviated, and there were still many gray-white egg nodules on the surface of the liver, which were reddish-brown in color and relatively soft in texture. Compared with the siNC group, the range of granulomas in the liver of mice in the siAtf3 group was significantly reduced, and the blue collagen was significantly reduced. In summary, ATF3 knockdown significantly alleviated the liver fibrosis caused by Schistosoma japonicum infection.

[0012] 3) The present invention constructed a mouse schistosoma japonicum infection model with knockdown of ATF3, and the results showed that compared with the siNC group mice, the mRNA levels of Acta2 and Col1a1 in the liver tissue of the siAtf3 group mice were significantly downregulated (P<0.01, P<0.05); compared with the siNC group mice, the protein levels of α-SMA and COL1A1 in the liver tissue of the siAtf3 group mice were significantly downregulated. In summary, ATF3 knockdown significantly alleviated the liver fibrosis caused by Schistosoma japonicum infection.

[0013] 4) The RNAi interference system of the Atf3 gene constructed by the present invention is used to prepare drugs for resisting liver fibrosis caused by Schistosoma japonicum. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1The mouse model infected with Schistosoma japonicum and the expression of ATF3 (A: Normal is the liver tissue of normal mice, 9W is the liver tissue of mice infected with Schistosoma japonicum at the 9th week; B: RT-qPCR experiment to detect the expression of Atf3 mRNA in the liver of normal mice and Schistosoma japonicum mice at different infection weeks; C: HE staining of the liver of normal mice and Schistosoma japonicum mice at different infection weeks; D: Immunohistochemical staining results of ATF3 in the liver of normal mice and Schistosoma japonicum mice at different infection weeks; *** indicates P<0.001 compared with the Normal group);

[0015] Figure 2 This is a Western blot experiment to detect the results of Atf3 RNAi target knockdown in 293T cells overexpressing the Atf3 gene;

[0016] Figure 3 It is a diagram of the knockdown efficiency of Atf3 in mouse liver tissue detected by Western blot and RT-qPCR experiments (in the figure, A: Western blot experiment to detect the expression of ATF3 protein in mouse liver tissue, WT is wild-type mouse, KD is ATF3 knockdown mouse; B: Western blot experiment quantitative statistical analysis of the knockdown efficiency of ATF3 in mouse liver tissue; C: RT-qPCR experiment to detect the expression of Atf3 mRNA in mouse liver tissue; ** indicates P<0.01 compared with the siNC group; *** indicates P<0.001 compared with the siNC group);

[0017] Figure 4 This is a graph showing the expression of ATF3 protein in liver tissue sections of ATF3 knockdown mice after infection with Schistosoma japonicum by immunohistochemistry (scale bar: 100 μm);

[0018] Figure 5 Figures of liver fibrosis in ATF3 knockdown mice infected with Schistosoma japonicum (A: gross specimens of livers of mice in the siNC group and siAtf3 group at the 9th week of Schistosoma japonicum infection; B: HE staining of liver tissues of mice in the siNC group and siAtf3 group at the 9th week of Schistosoma japonicum infection; C: Masson's trichrome staining of liver tissues of mice in the siNC group and siAtf3 group at the 9th week of Schistosoma japonicum infection; blue represents collagen fibers; scale bar: 100 μm);

[0019] Figure 6Figure 1 is the expression of fibrosis markers in ATF3 knockdown mice after Schistosoma japonicum infection (A: RT-qPCR experiment to detect the expression of Acta2 mRNA in the liver tissue of mice infected with Schistosoma japonicum at the 9th week; B: RT-qPCR experiment to detect the expression of Col1a1 mRNA in the liver tissue of mice infected with Schistosoma japonicum at the 9th week; * is compared with the siNC group, P<0.05, ** is compared with the siNC group, P<0.01);

[0020] Figure 7 This is the expression diagram of α-SMA and COL1A1 proteins in liver tissue of ATF3 knockdown mice after Schistosoma japonicum infection (scale bar: 100 μm). DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0022] The 8-week-old C57BL / 6 male mice used in the following examples were from the Experimental Animal Center of Nantong University; the positive snails infected with Schistosoma japonicum cercariae were provided by the Shanghai Institute of Parasitic Diseases, Centers for Disease Control and Prevention; other materials or experimental methods not specifically stated are all conventional materials or methods in the art.

[0023] Example 1 Establishment of a mouse model of liver fibrosis caused by Schistosoma japonicum infection and changes in the expression of ATF3 / fibrosis-related proteins

[0024] Eight-week-old C57BL / 6 male mice were selected for inoculation with Schistosoma japonicum cercariae. The specific steps were as follows:

[0025] Choose a sunny day, 2h to 3h before the experiment, place the positive snails in dechlorinated water, maintain the temperature at 24℃ to 25℃, and wait for the cercariae to escape; when inoculating, select 8-week-old mice, fix them on a mouse fixing stand, remove the abdominal fur to expose the skin and keep it moist; pick up the cercariae with an inoculation loop under a microscope, gently tap them on a coverslip of appropriate size and count them, and the cercariae are generally controlled at 13 to 15; turn the coverslip containing cercariae upside down and press it against the moist skin of the mouse's abdomen, keep contact for 15 minutes, so that the cercariae enter the mouse body and infect the mouse; normal mice from the same batch and 6 mice randomly selected at the 6th and 9th weeks of infection were killed, and their liver tissues were collected.

[0026] 1) HE staining

[0027] ① After dewaxing, transparency and hydration, take out the paraffin sections from xylene, drain them slightly, and remove the xylene in gradient ethanol.

[0028] ② After removing the slices from ethanol, soak them in distilled water twice, each time for 5 minutes.

[0029] ③ Take the slices out of the distillation, drain them slightly, and soak them in hematoxylin solution for 15 minutes.

[0030] ④ After the slices are taken out from the hematoxylin staining, they are rinsed with tap water and then differentiated. After staining under the microscope, they are rinsed with distilled water twice, each time for 3 minutes.

[0031] ⑤ Eosin staining: Use freshly prepared 1% eosin staining solution for 3 minutes, rinse the sections with 70% ethanol, and perform gradient ethanol dehydration. Then, soak the sections in equal volumes of xylene and anhydrous ethanol for 5 minutes, xylene I for 5 minutes, and xylene II for 15 minutes.

[0032] ⑥ Take out the slices, drain them slightly, and seal them with drops of neutral resin. After the resin solidifies, observe and photograph them under an upright microscope.

[0033] 2) Immunohistochemical staining

[0034] ① After dewaxing, transparency and hydration, remove the paraffin sections from xylene, drain slightly, and remove xylene in gradient ethanol. After removing the sections from ethanol, soak them in distilled water twice, 5 minutes each time.

[0035] ②Then dewax the sections for antigen retrieval. The specific steps are as follows: Place the slides in EDTA buffer at pH 9.0, maintain in boiling water in a pressure cooker for 20 minutes, and after returning to room temperature, rinse with PBS three times, each time for 5 minutes.

[0036] ③ Add 1 drop of peroxidase blocking reagent in the kit and incubate at room temperature for 15 minutes to block endogenous peroxidase, then continue washing with PBS for 3 times, 5 minutes each time.

[0037] ④ Incubate with ATF3 primary antibody (dilution ratio 1:500) at room temperature for 1 hour, 4°C overnight, and rinse with PBS for 5 minutes three times the next day.

[0038] ⑤ Incubate with secondary antibody at room temperature for 30 minutes, then wash with PBS three times, 5 minutes each time.

[0039] ⑥ Add DAB color developing solution to the slices, incubate for 5 minutes, rinse with tap water for 5 minutes, then counterstain with hematoxylin for 20 seconds, rinse with running water, differentiate with hydrochloric acid-ethanol for 5 seconds, continue to rinse with running water, and blue with warm water for 5 minutes.

[0040] ⑦ After dehydration, the sections were sealed with neutral resin after being treated with 70% ethanol, 80% ethanol, 95% alcohol, and anhydrous ethanol for 1 min each, and xylene for 8 min twice. The sections were observed and photographed under an upright microscope after staining. The dark brown staining area was the positive area.

[0041] The results are as follows Figure 1 As shown, the livers of mice infected with Schistosoma japonicum at the 9th week and mice not infected during the same period were observed with naked eyes; the surface of the liver of normal mice was bright red and soft to the touch; the surface of the liver of mice infected with Schistosoma japonicum at 9 weeks was brown and covered with gray-white dot-shaped egg nodules, and the texture became hard, indicating that the mouse model of Schistosoma japonicum infection was successfully established ( Figure 1 A). The RT-qPCR experiment was used to detect the changes in the expression of Atf3 mRNA in the liver tissue of mice at different infection weeks. The results showed that after the mice were infected with Schistosoma japonicum, the expression of Atf3 mRNA gradually increased in the 6th week after infection and reached a peak in the 9th week (P<0.001) ( Figure 1 B). HE staining of mouse liver tissue sections further confirmed that the mouse model of Schistosoma japonicum infection was successfully established; as the infection time prolonged, the number of granulomas in the mouse liver tissue gradually increased, and the liver fibrosis gradually worsened ( Figure 1 C). The immunohistochemical staining results of ATF3 showed that the expression of ATF3 protein increased with the extension of infection time, and the expression was most significant in the 9th week of infection, which was consistent with the trend of RT-qPCR results ( Figure 1 D).

[0042] Example 2

[0043] 1. Construction of AAV-siAtf3 adeno-associated virus expression system

[0044] For the Atf3 gene sequence (gene ID: NM_007498), an RNAi interference system specifically targeting the Atf3 gene was constructed to construct an AAV-siAtf3 adeno-associated virus expression system. The target sequence of the RNAi interference system of the Atf3 gene is shown in Table 1:

[0045] Table 1 Target sequences of RNAi interference system of Atf3 gene

[0046]

[0047] The linearized GV478 vector was obtained by digestion with restriction endonuclease BsmBI, the primers were annealed to form double-stranded DNA, and the linearized vector was ligated with the double-stranded DNA and transformed.

[0048] The final synthesized shRNA sequence and complementary sequence are shown in Table 2.

[0049] Table 2 Viral vector construction framework

[0050]

[0051] 2. RNAi target screening in 293T tool cells overexpressing Atf3 gene

[0052] The above-mentioned Atf3 siRNA adeno-associated virus vector plasmid was transferred into LB medium containing ampicillin antibiotics, and the Atf3 siRNA adeno-associated virus vector plasmid was amplified and extracted.

[0053] 293T cells in logarithmic growth phase were cultured at 4×10 5 / mL density was inoculated into 24-well plates and cultured at 37°C until the cell density reached 70% to 80%. Atf3 siRNA adeno-associated virus vector plasmid was transfected into 293T cells according to the experimental groups.

[0054] The groups are as follows:

[0055] Lane 1: 293T cell group without transfection of any plasmid (empty cell group)

[0056] Lane 2: Overexpression plasmid

[0057] Lane 3 / 7: Co-transfection of overexpression plasmid and negative control adeno-associated virus vector plasmid cell group (negative control group)

[0058] Lanes 4-6 / 8-10: Cell groups co-transfected with overexpression plasmid and RNAi adeno-associated viral vector plasmid, containing RNAi adeno-associated viral vector plasmids with different interference sequences targeting the target gene, namely Atf3-RNAi (106605), Atf3-RNAi (106606), and Atf3-RNAi (106607).

[0059] After 48 hours of transfection, the cell proteins of each group were extracted for Western blot experiment verification to determine the Atf3 siRNA with the best interference effect.

[0060] From the Western Blot results ( Figure 2 ) It can be seen that the Atf3-RNAi (106606-1) target has a significant knockdown effect on the expression of the target gene, and is therefore an effective target. Therefore, in the subsequent animal in vivo model, the Atf3-RNAi (106606-1) target was used to perform a knockdown experiment in mice. The Atf3-RNAi (106606-1) target was named AAV-siAtf3 in subsequent experiments. The Atf3 siRNA (106606-1) target sequence is shown in SEQ ID NO.1.

[0061] Example 3 Establishment and functional verification of ATF3 knockdown mouse schistosomiasis japonicum model

[0062] 1. Establishment of ATF3 knockdown mouse model of Schistosoma japonicum infection

[0063] 1) Prepare 30 C57BL / 6 male mice and divide them into two groups, namely AAV-siAtf3 group and AAV-siNC group (control insertion sequence: 5'-CGCTGAGTACTTCGAAATGTC-3'), with 15 mice in each group. Choose a sunny day, and place the positive snails in dechlorinated water 2h to 3h before the experiment, keep the temperature at 24℃ to 25℃, and wait for the cercariae to escape; when inoculating, select 8-week-old mice, fix them on a mouse fixing stand, remove the abdominal fur to expose the skin and keep it moist; pick up the cercariae with an inoculation loop under a microscope, gently tap them on a coverslip of appropriate size and count them, and the cercariae are generally controlled to be 13 to 15; turn the coverslip containing cercariae upside down and stick it tightly to the moist skin of the mouse abdomen, and keep contact for 15 minutes to allow the cercariae to enter the mouse body and infect the mouse.

[0064] 2) Two weeks after infection with Schistosoma japonicum, the two groups of mice were injected with about 100 μL of adeno-associated virus AAV-shAtf3 or AAV-shNC via the tail vein, and dynamic observation was performed for 7 weeks.

[0065] 3) At the 9th week of infection, the mice were killed and the liver tissue of each mouse was collected.

[0066] 4) The mouse liver tissue was divided into three parts: mRNA extraction, protein extraction, and tissue section preparation.

[0067] 2. Western blot and RT-qPCR detection

[0068] Western blot and RT-qPCR experiments were used to detect the knockdown efficiency of ATF3 in mouse liver tissue. All primer sequences involved in the RT-qPCR reaction are as follows:

[0069] mGapdh-F: 5'-TGGAAAGCTGTGGCGTGAT-3',

[0070] mGapdh-R: 5'-TGCTTCACCACCTTCTTGAT-3';

[0071] mAtf3-F: 5'-GAGGATTTTGCTAACCTGACACC-3',

[0072] mAtf3-R: 5'-TTGACGGTAACTGACTCCAGC-3';

[0073] mCol1a1-F: 5'-CACTCCTATCTCTCTCCTGTTGCCT-3',

[0074] mCol1a1-R: 5'-CACATCTTGTTCCTGTGTGCTCTCT-3';

[0075] mActa2-F: 5'-CACTCCTATCTCTCTCCTGTTGCCT-3',

[0076] mActa2-R: 5'-CACATCTTGTTCCTGTGTGCTCTCT-3'.

[0077] The RT-qPCR reaction system is: Forward Primer 0.4μL, Reverse Primer 0.4μL, ROX 0.4μL, SYBR Ex Taq II 10μL, cDNA2μL, ddH2O 6.8μL, Total 20μL.

[0078] The RT-qPCR reaction conditions were as follows: pre-denaturation at 95°C for 30 s; 95°C for 5 s, 63°C for 30 s, 72°C for 30 s, 40 cycles; 95°C for 15 s; 60°C for 30 s; 95°C for 15 s.

[0079] The results are as follows Figure 3 As shown, the expression of ATF3 in liver tissue of mice in the siAtf3 group was significantly lower than that in the siNC group (P<0.001, P<0.01).

[0080] 3. Immunohistochemistry

[0081] The knockdown of ATF3 in mouse liver was further detected by performing ATF3 immunohistochemical staining on mouse liver tissue sections (the steps were the same as those in Example 1).

[0082] The results are as follows Figure 4 As shown, the expression of ATF3 protein in the liver tissue of the mice in the siAtf3 group was significantly decreased compared with that in the mice in the siNC group.

[0083] 4. Detection of the effect of ATF3 knockdown on liver fibrosis in mice

[0084] In order to detect the effect of ATF3 knockdown on liver fibrosis in mice, the severity of liver fibrosis in mice in the siNC group and siAtf3 group was determined by naked eye observation, HE staining (same steps as in Example 1) and Masson's trichrome staining.

[0085] The steps of Masson's trichrome staining are as follows:

[0086] ① Dewax and hydrate the sections: first soak in xylene for 5 min, twice, then dehydrate with gradient ethanol, and finally rinse with tap water for 5 min.

[0087] ②Then the sections were stained with Weigert iron hematoxylin solution, rinsed gently with tap water after 5 minutes, differentiated with 1% hydrochloric acid alcohol for 5 seconds, and rinsed with tap water again.

[0088] ③ The sections were stained in the acid fuchsin stain solution for 5 minutes, rinsed with tap water and then treated in the phosphomolybdic acid solution for about 5 minutes. After removing the phosphomolybdic acid solution from the surface of the slides, they were counterstained in the aniline blue stain solution for 5 minutes, and then rinsed in a 1% glacial acetic acid aqueous solution until there was no obvious blue on the sections.

[0089] ④Finally, the sections were dehydrated with 95% alcohol and anhydrous ethanol, transparentized with xylene, and sealed with neutral gum.

[0090] ⑤Observe and photograph the stained sections under an upright microscope. Collagen fibers, cartilage, mucus, etc. are blue, cellulose, muscle fibers, red blood cells, etc. are red, and cell nuclei are purple-black.

[0091] The results are as follows Figure 5 As shown in A, the liver of mice in the siNC group showed a typical phenotype of liver fibrosis by naked eye observation. The color was dark brown, the texture was hard and dense, and a large number of gray-white worm egg nodules were visible on the surface ( Figure 5 A left figure). Compared with the siNC group, the degree of liver fibrosis in the siAtf3 group was relatively alleviated. There were still many gray-white egg nodules on the surface of the liver, which were reddish-brown in color and relatively soft in texture ( Figure 5 A (right).

[0092] The results are as follows Figure 5 B and Figure 5 As shown in C, compared with the siNC group, the range of granulomas in the liver of mice in the siAtf3 group was significantly smaller, and the blue collagen was significantly reduced.

[0093] In summary, ATF3 knockdown significantly alleviated liver fibrosis caused by Schistosoma japonicum infection.

[0094] 5. RT-qPCR detection of changes in the expression of liver fibrosis markers in mice infected with Schistosoma japonicum after ATF3 knockdown

[0095] RT-qPCR experiments were used to detect the expression changes of fibrosis genes Acta2 and Col1a1 mRNA, which are liver fibrosis markers, after ATF3 knockdown, to further verify the effect of ATF3 knockdown on liver fibrosis in mice.

[0096] The results are as follows Figure 6As shown, compared with those in the siNC group, the mRNA levels of Acta2 and Col1a1 in the liver tissue of the mice in the siAtf3 group were significantly downregulated (P<0.01, P<0.05).

[0097] 6. Immunohistochemical staining to detect the expression changes of α-SMA and COL1A1 proteins in mice infected with Schistosoma japonicum after ATF3 knockdown

[0098] The expression changes of fibrosis markers in liver tissue after ATF3 knockdown were detected by immunohistochemical staining experiment. The immunohistochemical staining steps were the same as those in Example 1. The dilution ratio of α-SMA and COL1A1 antibodies was 1:500.

[0099] The results are as follows Figure 7 As shown, compared with those in the siNC group, the levels of α-SMA and COL1A1 proteins in the liver tissue of the siAtf3 group mice were significantly downregulated.

[0100] In conclusion, knockdown of ATF3 significantly alleviated liver fibrosis caused by Schistosoma japonicum infection. Atf3-siRNA was used to prepare drugs against liver fibrosis caused by Schistosoma japonicum infection.

Claims

1. Atf3 - Use of siRNA in the preparation of a drug for treating liver fibrosis caused by Schistosoma japonicum, characterized in that: The base sequence of the siRNA is 5'-CTGAGATTCGCCATCCAGAAT-3'; Said Atf3 Gene ID of gene: NM_007498.

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