Use of creld2 in the preparation of a medicament for treating acute pancreatitis

Abstract

The application belongs to the technical field of biotechnology, and discloses application of CRELD2 in preparation of a drug for treating acute pancreatitis. The application constructs Creld2 gene knockout mice and pancreas acinar cell specific Creld2 overexpression mice, and uses caerulein to induce an acute pancreatitis model, so as to clarify the functional role of the Creld2 gene in the caerulein-induced acute pancreatitis, and to prove that Creld2 gene knockout can aggravate pancreas injury, and pancreas acinar cell specific overexpression of Creld2 can reduce pancreas injury. Through the two-way evidence of gene knockout aggravating pancreas injury and gene overexpression reducing pancreas injury, it is firstly proved that CRELD2 has a protective effect on the caerulein-induced acute pancreatitis, and a new strategy for intervention of acute pancreatitis based on CRELD2 is provided.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of CRELD2 in the preparation of drugs for treating acute pancreatitis. Background Technology

[0002] Acute pancreatitis (AP) is an acute abdominal condition caused by the abnormal activation of pancreatic enzymes, which digest the pancreas and surrounding tissues and organs. It is primarily characterized by local inflammation of the pancreas, and in severe cases, can lead to organ dysfunction, most commonly acute lung injury. AP is a common digestive system disease requiring hospitalization, with a high incidence rate that has been significantly increasing in recent years, and it often results in numerous sequelae. Currently, apart from symptomatic and supportive treatment, there are no specific effective drug treatments, leading to a poor overall prognosis and making it a disease of great concern to clinicians. Developing new therapeutic drugs could provide more options for the clinical treatment of acute pancreatitis.

[0003] Cysteine-rich with EGF-like domains (Creld) is a novel protein family belonging to the EGF superfamily. Initially named cirrin due to its multiple cysteine-rich EGF-like repeat sequences, it was later designated Creld by the Human Genome Nomenclature Committee. CRELD2, short for Cysteine ​​Rich with EGFLike Domains 2, is the second member of the Creld protein family to be discovered. As a novel, functional secretory protein, CRELD2 has attracted considerable research attention since its discovery.

[0004] Given the current lack of effective targeted intervention strategies to alleviate pancreatic damage and halt disease progression in clinical practice, the crotonin-induced acute pancreatitis (AP) model is a classic model simulating human acute pancreatitis (especially the mild edematous type) and is widely used in early pathological event studies of AP. The functional role of CRELD2 in the pathogenesis of AP remains unclear, particularly regarding its protective effect against crotonin-induced pancreatic damage; no reports have been published on this aspect. Therefore, elucidating the function of CRELD2 in AP and developing novel intervention strategies based on this understanding is of significant scientific and clinical value. Summary of the Invention

[0005] This invention aims to provide the application of CRELD2 in the preparation of drugs for treating acute pancreatitis. By constructing Creld2 knockout mice and Creld2 overexpression mice, the effect of Creld2 on pancreatic damage is verified in a crotonin-induced pancreatitis model, with the aim of discovering the protective effect of Creld2 on acute pancreatitis and providing new targets and ideas for targeted intervention of acute pancreatitis.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides the application of a CRELD2 overexpression reagent in the preparation of a drug for treating acute pancreatitis, wherein the amino acid gene sequence of CRELD2 (NP_083996.1) is shown in SEQ ID NO.1.

[0008] SEQ ID NO.1: MHLLLAAAFGLLLLLPPPGAVASRKPTMCQRCRTLVDKFNQGMANTARKNFGGGNTAWEEKTLSKYEFSEIRLLEIMEGLCDSSDFECNQLLEQQEEQLEAWWQTLKKEHPNLFEWFCVHTLKACCLPGTYGPDCQECQGGSERPCSGNGYCSGDGSRQGDGSCQCHTGYKGP LCIDCTDGFFSLQRNETHSICSACDESCKTCSGPSNKDCIQCEVGWARVEDACVDVDECAAETSPCSDGQYCENVNGSYTCEDCDSTCVGCTGKGPANCKECIAGYTKESGQCTDIDECSLEEKACKRKNENCYNVPGSFVCVCPEGFEETEDACVQTAEGKVTEENPTQPPSREDL*.

[0009] Preferably, the CRELD2 overexpression reagent includes a CRELD2 overexpression virus or a CRELD2 overexpression recombinant plasmid.

[0010] Preferably, the CRELD2 overexpression recombinant plasmid is obtained by inserting a gene fragment expressing Creld2 into a vector.

[0011] Preferably, the system vector is pAAV8-Y447F+Y733F-Elastase promoter-3×FLAG-P2A-EGFP vector.

[0012] Preferably, the CRELD2 overexpressing virus is obtained by co-transfecting host cells with a CRELD2 overexpressing recombinant plasmid, collecting the viral supernatant, lysing the cell pellet, and purifying it by gradient centrifugation.

[0013] Preferably, the drug is administered via intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, or intraperitoneal injection.

[0014] Preferably, the injection dose of the drug is 2 × 10⁻⁶. 11 Transfected unit / mouse.

[0015] Preferably, the drug is used to reduce the activity of amylase and lipase in serum, alleviate acinar cell necrosis, inflammatory cell infiltration and interstitial edema, downregulate the levels of inflammatory and chemokine mRNA in pancreatic tissue, and reduce the number of apoptotic cells in pancreatic tissue.

[0016] Secondly, this invention provides the application of CRELD2 gene knockout animals in drug screening studies for the prevention, relief and / or treatment of pancreatitis.

[0017] Thirdly, this invention provides the application of CRELD2 gene overexpressing animals in drug screening studies for the prevention, relief and / or treatment of pancreatitis.

[0018] Compared with existing technologies, the beneficial effects of this solution are:

[0019] This invention, through the construction of Creld2 gene knockout mice and pancreatic acinar cell-specific Creld2 overexpression mice, and using a cefuroxime-induced acute pancreatitis model, demonstrates that Creld2 gene knockout exacerbates pancreatic damage, while pancreatic acinar cell-specific overexpression of Creld2 alleviates pancreatic damage. This dual evidence of gene knockout aggravating pancreatic damage and gene overexpression alleviating pancreatic damage clearly demonstrates for the first time that CRELD2 has a protective effect against cefuroxime-induced acute pancreatitis. Therefore, it is possible to develop CRELD2-targeting overexpression formulations to develop therapeutic drugs for acute pancreatitis. Attached Figure Description

[0020] Figure 1 The effect of constructing an AP animal model on CRELD2 expression levels in mice (A shows the results of qPCR detection of CRELD2 mRNA levels in pancreatic tissues of each group of mice; B shows the results of Western Blot detection of CRELD2 protein levels in pancreatic tissues of each group of mice; Vehicle refers to WT mice injected intraperitoneally with physiological saline; AP refers to the AP animal model constructed based on WT mice).

[0021] Figure 2To construct the effect of Creld2 knockout mice on CRELD2 expression levels (A is a schematic diagram of the Creld2 KO mouse knockout strategy, B is the result of qPCR detection of Creld2 mRNA levels in pancreatic tissues of each group of mice, and C is the result of Western Blot detection of CRELD2 protein levels in pancreatic tissues of each group of mice; **** represents p<0.0001).

[0022] Figure 3 To verify the effect of Creld2 on pancreatic injury in a crotonin-induced pancreatitis model using Creld2 knockout mice (A shows the results of serum amylase and lipase levels in mice, B shows the H&E staining results of pancreatic tissue from mice; * represents p<0.05; ** represents p<0.01; **** represents p<0.0001);

[0023] Figure 4 To verify the effects of Creld2 on inflammation, immune cell activation, and apoptosis in a crotonin-induced pancreatitis model based on Creld2 knockout mice (A: qPCR results of mRNA expression levels of inflammation and chemokines in pancreatic tissue; B: MPO immunofluorescence staining results; C: TUNEL staining results).

[0024] Figure 5 A map of adeno-associated virus vectors;

[0025] Figure 6 To construct the effect of Creld2 overexpression mice on CRELD2 expression levels (A: qPCR results of Creld2 mRNA levels in pancreatic tissues of each group of mice; B: Western Blot results of CRELD2 protein levels in pancreatic tissues of each group of mice; C: qPCR results of Creld2 mRNA levels in other tissues of each group of mice; D: Western Blot results of CRELD2 protein levels in other tissues of each group of mice);

[0026] Figure 7 To verify the effect of Creld2 on pancreatic injury in a crotonin-induced pancreatitis model based on Creld2 overexpression mice (A shows the results of detection of amylase and lipase levels in mouse serum, and B shows the results of H&E staining of mouse pancreatic tissue);

[0027] Figure 8 To verify the effects of Creld2 on inflammation, immune cell activation, and apoptosis in a crotonin-induced pancreatitis model based on Creld2 overexpression mice (A: qPCR results of mRNA expression levels of inflammation and chemokines in pancreatic tissue; B: MPO immunofluorescence staining results; C: TUNEL staining results). Detailed Implementation

[0028] To facilitate understanding of the present invention by those skilled in the art, the technical solution of the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] Example 1: Constructing an AP animal model mouse

[0032] In this embodiment, an AP animal model was constructed by inducing taurine, and the expression of CRELD2 in mice of each group was investigated by qPCR and Western Blot.

[0033] First, male C57BL / 6 strain wild-type mice (WT mice, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) aged 8-12 weeks with uniform weight were selected and divided into two groups (n=5 in each group). One group served as the control group (denoted as Vehicle), and the other group was used to construct the AP animal model (denoted as AP group).

[0034] The specific steps for constructing an AP animal model are as follows:

[0035] Mice were fasted for 12 hours prior to modeling, but given free access to water. Before administration, the weight of each mouse was measured, the volume of medication administered was calculated, and experimental records were kept. Mice were intraperitoneally injected with Caerulein at a dose of 50 μg / kg. Seven consecutive intraperitoneal injections were given, each 1 hour apart, with the same injection volume each time, thus establishing the AP animal model (AP mouse). A control group was operated on concurrently, except that Caerulein was replaced with physiological saline.

[0036] Timing began 12 hours after the first injection. Mice from each group were harvested, with some pancreatic tissue placed in RNAlater preservation solution and stored at 4°C for RNA extraction; the remaining pancreatic tissue was placed in 1.5 mL EP tubes, flash-frozen in liquid nitrogen, and then stored at -80°C for later use. qPCR was used to detect the expression level of Creld2 mRNA in pancreatic tissue, and Western blotting was used to detect the expression level of CRELD2 protein in pancreatic tissue.

[0037] The results showed that the expression level of Creld2 mRNA in pancreatic tissue of AP mice was significantly reduced compared with that of pancreatic tissue of Vehicle mice. The results of Western Blot detection of Creld2 protein expression level were consistent with the results of qPCR detection, suggesting that Creld2 may be involved in the occurrence and development of AP disease.

[0038] Example 2: Construction of Creld2 knockout mice

[0039] In this embodiment, Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd. was commissioned to construct Creld2 gene knockout mice (denoted as KO mice) using CRISPR / Cas9 gene editing technology. The specific procedures are as follows:

[0040] The gene sequence of the Creld2-201 transcript from mice was selected as the gene locus for the Creld2 KO mouse construction strategy. sgRNAs were designed into the non-conserved regions downstream of the Intron 2 and 3' UTR, respectively, causing approximately 7kb of genomic deletion, thereby achieving gene knockout. See [link to knockout strategy] for details. Figure 2 Based on the sgRNA design principles, eight sgRNAs were designed for the 5' and 3' target sites, respectively, and the corresponding target sequences are shown in Table 1.

[0041] Table 1. sgRNA sequence information

[0042]

[0043] Oligos were synthesized according to the designed sgRNA sequence and ligated into the pCS-4G vector via Gibson. After sequencing verification, the Cas9 / sgRNA was microinjected into mouse zygotes and transplanted into surrogate mother mice to obtain F0 generation mice, which are Creld2 KO mice.

[0044] Pancreatic tissues were collected from 8-12 week old KO and WT mice. The expression level of Creld2 mRNA in the tissues was detected by qPCR, and the expression level of CRELD2 protein in the tissues was detected by Western Blot.

[0045] The results showed that CRELD2 was almost not expressed in the pancreatic tissue of KO mice compared with WT mice, indicating that KO mice were successfully constructed.

[0046] Example 3: Investigating the effect of Creld2 on pancreatic injury using Creld2 knockout mice

[0047] This embodiment uses Creld2 knockout mice constructed in Example 2 to verify the effect of Creld2 on pancreatic damage in a pancreatitis model induced by taurine.

[0048] First, following the AP mouse construction method in Example 1, AP animal models were constructed from Creld2 knockout mice and WT mice in Example 2, resulting in an AP animal model constructed from WT mice (denoted as WT+AP group) and an AP animal model constructed from KO mice (denoted as KO+AP group). Creld2 knockout mice and WT mice in Example 2 were treated with physiological saline, and the control groups were denoted as WT+Vehicle group and KO+Vehicle group, respectively.

[0049] Serum samples were collected from mice, and the levels of amylase and lipase in the serum were detected using an α-amylase assay kit and a lipase assay kit (purchased from Nanjing Jiancheng Biotechnology Institute). Pancreatic tissues were collected from the mice, and the degree of pancreatic tissue damage was assessed by H&E staining; the state of inflammation and immune cell activation was revealed by MPO immunofluorescence staining; and apoptotic cells were specifically identified by TUNEL staining. The expression levels of Il-6, Il-1β, Tnf-α, Ccl2, Cxcl1, Ly6g, and F4 / 80 mRNA in pancreatic tissues were detected by qPCR.

[0050] Serum test results showed that there were no significant differences in serum amylase and lipase levels between WT+Vehicle mice and KO+Vehicle mice; however, serum amylase and lipase levels were significantly increased in both WT and KO mice after AP animal model establishment treatment; compared with WT+AP mice, serum amylase and lipase levels were significantly increased in KO+AP mice, suggesting that Creld2 gene knockout can lead to increased serum amylase and lipase levels in AP animal models (see...). Figure 3 (A in the middle).

[0051] H&E staining results showed that the pancreatic tissues of WT+Vehicle and KO+Vehicle mice were basically normal and structurally regular; after AP animal model construction in WT and KO mice, the pancreatic tissue structure was blurred and disordered. Compared with WT+AP mice, the area and degree of pancreatic tissue necrosis in KO+AP mice were significantly more severe (see...). Figure 3 (B in the middle).

[0052] Compared with WT+AP mice, the levels of Il-6, Il-1β, Tnf-α, Ccl2, Cxcl1, Ly6g, and F4 / 80 mRNA in the pancreatic tissue of KO+AP mice were significantly upregulated; therefore, Creld2 gene knockout can significantly increase inflammatory cell infiltration and chemokine expression in the pancreas of AP animal models (see...). Figure 4 (A in the middle).

[0053] MPO immunofluorescence staining results showed no significant MPO positivity in the pancreatic tissues of WT+Vehicle and KO+Vehicle mice; compared with WT+AP mice, the number of MPO-positive cells in the pancreatic tissues of KO+AP mice was significantly increased (see...). Figure 4 (B in the text). This indicates that Creld2 knockout significantly increases neutrophil infiltration in pancreatic tissue, suggesting an exacerbated inflammatory response.

[0054] TUNEL staining showed that the number of TUNEL-positive cells in the pancreatic tissues of WT+Vehicle and KO+Vehicle mice was very low, with no significant difference between the two groups; compared with WT+AP mice, the number of TUNEL-positive cells in the pancreatic tissues of KO+AP mice was significantly increased (see...). Figure 4 (C in the text). Therefore, it can be seen that the knockout of the Creld2 gene significantly increases the number of apoptotic cells in the pancreatic tissue of mice.

[0055] Example 4: Construction of CRELD2 gene-high expression mice

[0056] This embodiment uses the adeno-associated virus vector (AAV-GFP) purchased from Fubai Ao (Suzhou) Biomedical Technology Co., Ltd. (vector map shown in [link]). Figure 5 Using a high-expressing virus (AAV-Creld2), pancreatic tissue-specific CRELD2 gene-high-expressing mice (denoted as OE mice) were constructed. The specific procedures are as follows:

[0057] The gene fragment expressing CRELD2 was inserted into the AAV8(Y447F+Y733F)-Elastase promoter-3×FLAG-P2A-EGFP vector to construct a stable CRELD2-expressing recombinant plasmid, namely the CRELD2 overexpression recombinant plasmid. The CRELD2 overexpression recombinant plasmid was co-transfected into HEK293T cells, and the viral supernatant was collected. The cell pellet was lysed, centrifuged in gradients, and purified to obtain the CRELD2 overexpressing virus.

[0058] AAV-Creld2 was injected intraperitoneally into 8-week-old WT mice at a dose of 2 × 10⁻⁶. 11 Transfected mice were obtained by transfecting units (vg) per mouse, and OE mice were obtained after 4 weeks. AAV-Creld2 was replaced with AAV-GFP to obtain control mice (denoted as Ctrl mice).

[0059] Pancreas, heart, liver, spleen, lung, and kidney tissues were collected from Ctrl and OE mice. The expression level of Creld2 mRNA was detected by qPCR, and the expression level of Creld2 protein was detected by Western blot.

[0060] The results showed that, compared with the Ctrl group mice, the expression level of CRELD2 in the pancreatic tissue of OE mice was significantly increased, indicating that the OE mouse model was successfully constructed (see...). Figure 6 (A and B in the text). There was no significant difference in CRELD2 expression levels in the heart, liver, spleen, lung, and kidney tissues of mice in the Ctrl group and the OE group, suggesting that CRELD2 is specifically highly expressed in pancreatic tissue in OE mice (see A and B in the text). Figure 6 (C and D in the text).

[0061] Example 5:

[0062] This embodiment uses Creld2-overexpressing mice constructed in Example 4 to verify the effect of Creld2 on pancreatic damage in a pancreatitis model induced by taurine.

[0063] First, following the AP mouse construction method in Example 1, AP animal models were constructed using Creld2 overexpression and Ctrl mice from Example 4, resulting in an AP animal model constructed using OE mice (denoted as the OE+AP group) and an AP animal model constructed using Ctrl mice (denoted as the Ctrl+AP group). The Creld2 overexpression and Ctrl mice from Example 4 were treated with physiological saline, and the control groups were denoted as the OE+Vehicle group and the Ctrl+Vehicle group, respectively.

[0064] Serum samples were collected from mice, and the levels of amylase and lipase in the serum were detected using an α-amylase assay kit and a lipase assay kit (purchased from Nanjing Jiancheng Biotechnology Institute). Pancreatic tissues were collected from the mice, and the degree of pancreatic tissue damage was assessed by H&E staining; the state of inflammation and immune cell activation was revealed by MPO immunofluorescence staining; and apoptotic cells were specifically identified by TUNEL staining. The expression levels of Il-6, Il-1β, Tnf-α, Ccl2, Cxcl1, Ly6g, and F4 / 80 mRNA in pancreatic tissues were detected by qPCR.

[0065] Serum test results showed that there were no significant differences in serum amylase and lipase activities between OE and Ctrl+Vehicle mice and Ctrl+Vehicle mice. After establishing the AP animal model in both Ctrl and OE mice, serum amylase and lipase levels were significantly increased. Compared with Ctrl+AP mice, serum amylase and lipase levels in OE+AP mice were significantly decreased, suggesting that high expression of the Creld2 gene can reduce serum amylase and lipase levels in the AP animal model (see...). Figure 7 (A in the middle).

[0066] H&E staining showed that the pancreatic tissue of Ctrl+Vehicle and OE+Vehicle mice was basically normal and structurally regular; after AP animal model construction in Ctrl and OE mice, the pancreatic tissue structure was blurred and disordered. Compared with Ctrl+AP mice, the area and degree of pancreatic tissue necrosis in OE+AP mice were significantly reduced (see...). Figure 7 (B in the middle).

[0067] The results showed that, compared with Ctrl+AP mice, the levels of Il-6, Il-1β, Tnf-α, Ccl2, Cxcl1, Ly6g, and F4 / 80 mRNA in pancreatic tissue of OE+AP mice were significantly downregulated (see [link to OE+AP mice]). Figure 8 (A in the text). Therefore, it can be seen that high expression of the Creld2 gene can significantly reduce inflammatory cell infiltration and chemokine expression in the pancreas of the AP animal model.

[0068] MPO immunofluorescence staining results showed no significant MPO positivity in the pancreatic tissues of Ctrl+Vehicle and OE+Vehicle mice; compared with Ctrl+AP mice, the number of MPO-positive cells in the pancreatic tissues of OE+AP mice was significantly reduced (see...). Figure 8 (B in the text). This indicates that high expression of Creld2 significantly reduces neutrophil infiltration in pancreatic tissue, suggesting a reduction in inflammatory response.

[0069] TUNEL staining results showed that the number of TUNEL-positive cells in the pancreatic tissues of Ctrl+Vehicle and OE+Vehicle mice was very low, with no significant difference between the two groups; compared with Ctrl+AP mice, the number of TUNEL-positive cells in the pancreatic tissues of OE+AP mice was significantly reduced (see...). Figure 8 (C in the text). Therefore, it can be concluded that high expression of the Creld2 gene significantly reduces the number of apoptotic cells in mouse pancreatic tissue.

[0070] In summary, this invention provides the first clear evidence that CRELD2 has a protective effect against taeniacin-induced acute pancreatitis, through dual evidence that gene knockout aggravates pancreatic damage and gene overexpression alleviates pancreatic damage.

[0071] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. The application of CRELD2 overexpression reagent in the preparation of drugs for treating acute pancreatitis, characterized in that, The amino acid sequence of CRELD2 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The CRELD2 overexpression reagent includes a CRELD2 overexpression virus or a CRELD2 overexpression recombinant plasmid.

3. The application according to claim 2, characterized in that, The CRELD2 overexpression recombination system is obtained by inserting a gene fragment expressing Creld2 into a vector.

4. The application according to claim 3, characterized in that, The vector is pAAV8-Y447F+Y733F-Elastasepromoter-3×FLAG-P2A-EGFP vector.

5. The application according to claim 1, characterized in that, The CRELD2 overexpressing virus was obtained by co-transfecting host cells with a CRELD2 overexpressing recombinant plasmid, collecting the viral supernatant, lysing the cell pellet, and purifying it by gradient centrifugation.

6. The application according to claim 1, characterized in that, The drug can be administered via intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, or intraperitoneal injection.

7. The application according to claim 1, characterized in that, The injection dose of the drug is 2×10 11 Transfected unit / mouse.

8. The application according to claim 1, characterized in that, The drug is used to reduce the activity of amylase and lipase in serum, alleviate acinar cell necrosis, inflammatory cell infiltration and interstitial edema, downregulate the mRNA levels of inflammatory and chemokine factors in pancreatic tissue, and reduce the number of apoptotic cells in pancreatic tissue.

9. Application of CRELD2 gene knockout animals in drug screening for the prevention, relief and / or treatment of pancreatitis.

10. Application of CRELD2 gene overexpression animals in drug screening for the prevention, relief and / or treatment of pancreatitis.

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