A csi-bantam inhibitor and its use in the preparation of a medicament for treating liver fibrosis caused by clonorchis sinensis infection

Abstract

The present application relates to a kind of Csi-Bantam inhibitor and its application in the preparation of the drug for treating liver fibrosis caused by Clonorchis sinensis infection, belong to biological manufacturing technical field, the present application has found that Csi-Bantam analog can be combined with the 3'-UTR region of Trim35 gene, by inhibiting the function of Csi-Bantam further blocks its PI3K / Akt signal pathway, can significantly inhibit the activation of hepatic stellate cell (activated hepatic stellate cell is the core driving cell of liver fibrosis, is also the main source of extracellular matrix) and the excessive secretion of type I, type III collagen fiber and other extracellular matrix, to effectively improve the process of liver fibrosis.The present application first discloses the new use of Csi-Bantam inhibitor in the preparation of liver fibrosis treatment drug.This discovery provides a new idea for the targeted treatment of liver fibrosis, and has important clinical transformation value for the treatment of liver fibrosis caused by parasitic infection and other factors.

Description

Technical Field

[0001] This invention relates to the field of biomanufacturing technology, specifically to a Csi-Bantam inhibitor and its application in the preparation of a drug for treating liver fibrosis caused by Clonorchis sinensis infection. Background Technology

[0002] The central link in liver fibrosis is the activation and proliferation of hepatic stellate cells (HSCs), also known as lipid storage cells, which are the main effector cells in liver fibrosis. HSCs are in a quiescent, lipid-rich state. When the liver is stimulated by exogenous factors, HSCs are activated. Activated HSCs express large amounts of α-smooth muscle actin (α-SMA) and simultaneously transform into myofibroblasts, which are involved in liver wound healing. Activated HSCs can upregulate the expression of type I collagen α1 (Col1α1) and type III collagen α1 (Col3α1) genes, producing large amounts of extracellular matrix (ECM) to form scars. Simultaneously, pro-inflammatory and anti-inflammatory cytokines are produced, leading to fibrosis in the host liver tissue. Therefore, inhibiting the activation and proliferation of HSCs can provide an important technical approach for the treatment of liver fibrosis.

[0003] The PI3K / Akt signaling pathway plays a crucial role in the proliferation, survival, and metabolism of hepatic fibrosis cells (HSCs). PI3K activates Akt, regulating various cellular processes and promoting HSC activation and ECM deposition. Studies have shown that activation of the PI3K / Akt signaling pathway is closely related to the progression of liver fibrosis. Furthermore, the PI3K / Akt signaling pathway also promotes autophagy in HSCs and the maintenance of the fibrotic phenotype by regulating mTORC1 activity. In recent years, drugs targeting the PI3K / Akt signaling pathway have shown anti-fibrotic effects in preclinical studies. The PI3K / Akt signaling pathway promotes ECM deposition and fibrosis development by regulating HSC proliferation, survival, and metabolism. In addition, gene therapy strategies have also shown anti-fibrotic effects in preclinical studies; siRNA, by inhibiting PI3K or Akt expression, significantly improved liver pathological features in mouse models of liver fibrosis and reduced the expression of liver fibrosis-related genes. Previous studies have shown that PI3K / Akt promotes the inflammatory response and maintenance of the fibrotic phenotype in HSCs by regulating the NF-κB and STAT3 signaling pathways, while the Trim35 gene inhibits the classical PI3K / Akt liver fibrosis signaling pathway by inactivating Akt signaling.

[0004] Clonorchis sinensis is an important foodborne parasite that infects the bile ducts and gallbladder of its hosts, causing clonorchiasis. This disease is mainly distributed in East and Southeast Asia, with 35 million people infected globally and approximately 200 million at risk of infection. my country is the country most severely affected by this disease, with about 13 million infected. Clonorchis sinensis can parasitize the bile ducts of various mammals, including humans. Infection is often chronic, with no obvious clinical symptoms in the early stages, but later causing pathological changes primarily characterized by liver fibrosis, and even inducing liver cancer.

[0005] Extracellular vesicles (EVs) are a collective term for tiny membrane-bound vesicles actively secreted by cells. EVs carry bioactive molecules (such as proteins, non-coding RNAs, lipids, and carbohydrates) and play an important role in intercellular signaling. EVs secreted by parasites can mediate communication between parasites and their hosts by delivering parasite-derived miRNAs to host cells, which then bind to host target genes and inhibit gene expression in the host cells, thus playing a crucial regulatory role in the parasite-host interaction.

[0006] MicroRNAs (miRNAs) are a group of endogenous, non-coding RNAs widely distributed in eukaryotes, regulating gene expression at the posttranscriptional level. These small, evolutionarily highly conserved miRNAs are 21–23 bases in length. They typically cause target gene breakage by directly binding to the 3'-UTR site and completely binding to the mRNA, or by binding to the 2nd–8th base of the mRNA, leading to translational repression or degradation of the target gene. Most miRNAs originate from introns and cleave and degrade target mRNA or inhibit protein translation; the mechanisms depend on miRNA-mRNA binding complementarity. Perfect complementarity leads to transcript degradation, while incomplete complementarity leads to translational repression, the mechanisms of which depend on miRNA-mRNA binding complementarity.

[0007] Studies have shown that miRNAs carried by clonorchis sinensis EVs can regulate the progression of liver fibrosis in the host by modulating the activation of hepatic choriocarcinoma cells (HSCs). Therefore, identifying key parasite-derived miRNAs that regulate liver fibrosis and synthesizing corresponding miRNA mimics could potentially benefit the treatment and alleviation of liver fibrosis caused by Clonorchis sinensis, providing new insights and references for the treatment of liver fibrosis. Summary of the Invention

[0008] To achieve the objectives stated in the background art, the present invention provides an inhibitor of Csi-Bantam and its application in the preparation of a drug for treating liver fibrosis caused by Clonorchis sinensis infection.

[0009] The technical solution adopted in this invention is: the application of a Csi-Bantam inhibitor in the preparation of a drug for treating liver fibrosis caused by Clonorchis sinensis infection. The inhibitor, by binding to Csi-Bantam, inhibits the expression and function of Csi-Bantam, blocks the binding of Csi-Bantam to the 3'-UTR region of the Trim35 gene, relieves the inhibition of Trim35 gene expression by Csi-Bantam, restores normal protein expression of the Trim35 gene, and thereby inhibits the PI3K / Akt signaling pathway and suppresses hepatic stellate cell activation.

[0010] The miRNA inhibitors for treating liver fibrosis are Csi-Bantam-inhibitor and LV3-Csi-Bantam-inhibitor, containing the following nucleic acid sequence: 5'-ACCAGCTTTAGTCGCGATCTCA-3'.

[0011] The Csi-Bantam-inhibitor and LV3-Csi-Bantam-inhibitor contain the nucleotide sequence of Csi-Bantam.

[0012] The Csi-Bantam-inhibitor is a chemically synthesized mature miRNA double strand, the nucleotide sequence of which is SEQ ID NO. 1 or the nucleotide sequence of SEQ ID NO. 1 obtained by adding, deleting, modifying and / or conserving substitution of at least one nucleotide.

[0013] The LV3-Csi-Bantam inhibitor described above is a Csi-Bantam inhibitor packaged in a Lentivirus vector, which ensures its stability.

[0014] The miRNA molecule of Clonorchis sinensis used to treat liver fibrosis is Csi-Bantam, and its in vitro synthetic inhibitors are Csi-Bantam-inhibitor and LV3-Csi-Bantam-inhibitor.

[0015] The miRNA inhibitor of Clonorchis sinensis is an inhibitor of Csi-Bantam in the extracellular vesicles of Clonorchis sinensis. Csi-Bantam is a single-stranded structure, and Csi-Bantam-inhibitor is a Csi-Bantam inhibitor, a chemically synthesized mature double-stranded miRNA.

[0016] Csi-Bantam-inhibitor was synthesized by Beijing Qingke Biotechnology Co., Ltd., and LV3-Csi-Bantam-inhibitor was synthesized by Suzhou Jima Gene Co., Ltd.

[0017] The present invention also discloses the application of the above-described inhibitors of Clonorchis sinensis miRNA in the preparation of drugs for treating Clonorchis sinensis disease.

[0018] In this article, "Csi-Bantam-inhibitor" refers to an in vitro inhibitor of Csi-Bantam; "LV3-Csi-Bantam-inhibitor" refers to an in vivo inhibitor of Csi-Bantam; "Csi-Bantam-mimics" refers to in vitro mimics of Csi-Bantam; "LV3-Csi-Bantam-mimics" refers to in vivo mimics of Csi-Bantam; "siRNA NC", "mimics NC", and "NC mimics" refer to in vitro mimics that are obtained through bioinformatics screening and have almost no interaction with the coding RNA; "LV3-NC" refers to in vivo mimics that are obtained through bioinformatics screening and have almost no interaction with the coding RNA; "inhibitor NC" or "NC inhibitor" refers to an inhibitor that is obtained through bioinformatics screening and has almost no interaction with the coding RNA.

[0019] This application has the following beneficial technical effects: This application has identified a new miRNA, namely Csi-Bantam, from the extracellular vesicles of Clonorchis sinensis, and synthesized corresponding in vitro inhibitors, in vivo inhibitors (LV3-inhibitor), in vitro mimics, and in vivo mimics (LV3-mimics) based on it. In vivo and in vitro experiments have demonstrated that this new miRNA inhibitor can treat liver fibrosis. Attached Figure Description

[0020] Figure 1 Image: Diagram showing the isolation and identification of extracellular vesicles from Clonorchis sinensis.

[0021] Figure 2 Figure: Clonorchis sinensis can induce activation of hepatic stellate cells.

[0022] Figure 3 The results show that Csi-Bantam is highly expressed in CsEVs and that Csi-Bantam inhibitors can suppress HSC activation.

[0023] Figure 4 The result is: Csi-Bantam-inhibitor can inhibit liver fibrosis caused by Clonorchis sinensis infection.

[0024] Figure 5 To: Verify that the Trim35 gene is a direct target of Csi-Bantam.

[0025] Figure 6 The results show that Csi-Bantam promotes liver fibrosis by inhibiting the Trim35 gene and regulating the PI3K / Akt signaling pathway.

[0026] Figure 7 Image of mouse primary hepatic stellate cells identified by Desmin fluorescent staining. Detailed Implementation

[0027] The present invention will be further illustrated below with reference to embodiments and accompanying drawings. All RNA sequences disclosed in this invention can be obtained by artificial synthesis or other biological methods.

[0028] Example 1

[0029] 1. Overview of the research on which this application is based:

[0030] This application investigates the effects of Clonorchis sinensis extracellular vesicles containing parasite miRNAs on host liver fibrosis at two levels: in vivo and in vitro (within HSC-T6 cells) in mice.

[0031] To screen miRNAs from CsEVs that play a key role in liver fibrosis, this experiment cultured adult Clonorchis sinensis worms in vitro, collected and purified EVs, and collected primary HSCs using a Clonorchiasis-infected mouse model. High-throughput sequencing and related analyses were performed on these samples to screen for Csi-Bantam, which can participate in host liver fibrosis. The regulatory role of Csi-Bantam in the host was verified through both in vitro and in vivo experiments. In in vitro experiments, we synthesized Csi-Bantam mimics / inhibitors and transfected them into the HSC-T6 cell line. Changes in HSC activation-related marker genes were detected using qRT-PCR and Western blotting. In in vivo experiments, we synthesized two plasmids that can overexpress and silence Csi-Bantam, packaged them into lentiviruses, and transfected them into a Clonorchiasis-infected mouse model. Related analyses were performed on liver tissue and primary HSCs in these mice.

[0032] 2.1 Experimental Apparatus

[0033] A summary of the main experimental instruments used in this application.

[0034] Table 1. Main Instrument Names and Brands

[0035]

[0036] 2.2 Reagents

[0037] Summary of the main reagents in this application.

[0038] Table 2. Main Reagent Names and Brands

[0039]

[0040] 2.3 Preparation of main reagents

[0041] (1) Artificial digestion solution: 20 g Pepsin + 20 mL hydrochloric acid, dissolved in 1 L distilled water.

[0042] (2) Physiological saline: 9 g of NaCl dissolved in 1 L of distilled water.

[0043] (3) 1× Locke's solution: 9 g NaCl, 0.4 g KCl, 0.2 g NaHCO3 and 0.2 g CaCl2 dissolved in 1 L of distilled water.

[0044] (4) 15% Optiprep separation solution: 2.5 mL of 60% Optiprep separation solution + 7.5 mL of PBS. Type IV collagenase digestion solution: 5 mg of type IV collagenase powder dissolved in 10 mL of DMEM medium.

[0045] (5) Magnetic bead sorting buffer: MACS BSA Stock Solution + autoMACS RinsingSolution.

[0046] 2.4 Primers

[0047] The primers used in this study were synthesized by Beijing Qingke Biotechnology Co., Ltd., and their specific sequences are shown in Table 3.

[0048] Table 3 Primer sequence information

[0049]

[0050] 2.5 Experimental Materials

[0051] Laboratory animals: Female BALB / c mice used in the experiments were purchased from the Department of Laboratory Animal Science, Harbin Medical University. Rabbits used in the experiments were also purchased from the Department of Laboratory Animal Science, Harbin Medical University. Minnows used in the experiments were collected from Zhaoyuan County, Daqing City, Heilongjiang Province. The HSC-T6 cell line used in the experiments was purchased from Hunan Fenghui Biotechnology Co., Ltd.

[0052] 2.6 Experimental Methods and Results

[0053] 2.6.1 Collection of Clonorchis sinensis metacercariae:

[0054] Previous studies in our laboratory have shown that minnows are the dominant fish species for Clonorchis sinensis infection. Therefore, this experiment collected minnows to isolate Clonorchis sinensis metacercariae.

[0055] The infection status of metacercariae was tested on minnows collected from epidemic areas. Muscle around the dorsal fin of minnows was cut off and pressed into slides for microscopic examination to observe whether there were metacercariae parasitizing.

[0056] Samples of topmouth gudgeon infected with metacercariae were chopped into rice-grain-sized pieces and placed in a 2500 mL Erlenmeyer flask. 10 mL of artificial digestion solution was added per gram of fish meat. The flask was placed in a constant temperature shaker at 37℃ and shaken at 200 r / min for 6 h. The digested suspension was filtered through a 200-mesh copper screen and allowed to stand for 20 min. The supernatant was discarded, and an appropriate amount of physiological saline was added. The mixture was allowed to stand for another 20 min, and the supernatant was discarded. The above operation was repeated until the solution was completely clear. The precipitate at the bottom of the flask was collected, and the metacercariae of Clonorchis sinensis were isolated under a stereomicroscope based on their morphological characteristics. The metacercariae were then stored in physiological saline for later use.

[0057] 2.6.2 Collection of Clonorchis sinensis:

[0058] Each rabbit was infected with 500 Clonorchis sinensis metacercariae. After 8 weeks of rearing, the feces were examined for Clonorchis sinensis eggs using a method of ether hydrochloride and water washing and precipitation. If eggs were detected, the rabbits were euthanized in accordance with animal welfare procedures.

[0059] Remove the liver and gallbladder, cut the liver along the bile duct with ophthalmic scissors, and remove the Clonorchis sinensis parasites from the bile duct with a brush.

[0060] The worms were washed three times with 1×PBS preheated to 37°C. Following previous research, *Clonorchis sinensis* was cultured in 1× Locke's solution at pH 7.3. One hundred adult *Clonorchis sinensis* worms were placed in a 25 cm³ container with 5 mL of Locke's solution added. 2 The cell culture flasks were filled with 100 mg / mL streptomycin and 100 U / mL penicillin. The incubator environment was set at 37°C, 5% CO2, and a certain level of humidity.

[0061] Replace the culture medium after 3 hours, and then collect and replace the culture medium every 3 hours thereafter, for a total of 24 hours. Observe the culture medium condition each time the culture medium is collected, and remove any dead insects.

[0062] 2.6.3 Isolation and Identification of CsEVs:

[0063] To obtain relatively pure CsEVs, density gradient centrifugation, the "gold standard" for EV separation, was used to collect the samples. A schematic diagram of CsEV extraction using density gradient centrifugation is shown below. Figure 1 As shown in a.

[0064] Place the culture medium collected in section 2.6.2 in a horizontal centrifuge and centrifuge at 4°C and 500×g for 10 min, discarding the precipitate, which at this stage mainly consists of parasite eggs. Centrifuge at 4°C and 2,000×g for 10 min, discarding the precipitate, which at this stage mainly consists of cells and dead cells. Centrifuge at 4°C and 12,000×g for 30 min, discarding the precipitate, which at this stage mainly consists of cell debris.

[0065] The supernatant was filtered through a 0.22 μm filter membrane. The filtered liquid was centrifuged at 100,000 × g for 90 min at 4 °C. The supernatant was gently aspirated and discarded. Then, 1 × PBS solution was added to the liquid, and the mixture was centrifuged at 100,000 × g for 90 min at 4 °C. The supernatant was gently aspirated and discarded. The liquid was resuspended in 200 µL of PBS solution to obtain EVs. The obtained EVs solution was stored at 4 °C for later use.

[0066] To verify whether the separated particles were EVs, they were detected using transmission electron microscopy and particle size analysis.

[0067] Transmission electron microscopy (TEM) examination: EVs were added to a 200-mesh coated copper grid, and excess liquid was removed with filter paper. The grid was negatively stained with 0.5% lead acetate solution for 10 min at room temperature. After being placed under an incandescent lamp for 10 min, the grid was then loaded onto the sample holder of the TEM and exposed to an 80 kV electron beam for image capture. Images are shown below. Figure 1 As shown in b. Characterization of CsEVs by transmission electron microscopy revealed that these vesicles are spherical, approximately 80 nm in diameter, and possess a lipid bilayer membrane structure (e.g., ...). Figure 1 (as shown in b).

[0068] Particle size analysis: The NanoFCM N30E particle size analyzer was used to analyze the particle size and concentration of EVs. Results are as follows: Figure 1 As shown in c. Particle size analysis indicates that the separated CsEVs are enriched between 50 and 150 nm, reaching a peak at 120 nm. The particle size conforms to the standard size for EVs, and the concentration reaches 4.33 × 10⁻⁶. 10 Particles / mL. The above experiments show that CsEVs can be successfully separated and identified (e.g., Figure 1 (as shown in c).

[0069] 2.6.4 Isolation, extraction, and identification of primary mouse HSCs:

[0070] Six-week-old female BALB / c mice were orally infected with Clonorchis sinensis metacercariae (50 metacercariae per mouse). After culturing for 35 days, fecal samples were examined daily. Clonorchis sinensis eggs were detected in the feces using a method of ether hydrochloride and water washing and precipitation. After the eggs were detected, the mice were cultured for another week to ensure that the parasites matured.

[0071] Simultaneously, uninfected mice were cultured for the same number of days as a negative control group. Then, referring to the method for isolating primary mouse HSCs by Weiskirchen et al., and with appropriate modifications, primary mouse HSCs were extracted as follows:

[0072] 1) Anesthetize mice with ether, and then immerse the part below their neck in 75% alcohol for disinfection.

[0073] 2) Liver irrigation: Fix the mouse, cut open the abdominal cavity, and move the intestinal tissue to the right side to fully expose the portal vein. Insert an indwelling intravenous catheter 5-10 mm from the porta hepatis and secure it with surgical sutures. Slowly infuse 5 mL of preheated PBS (37°C) into the indwelling catheter using a syringe. Once the liver has expanded, make a small incision near the liver border in the posterior vena cava to release the PBS and blood, and continue infusing 10 mL of preheated PBS (37°C) until the liver turns a light yellow color. Be careful not to inject air.

[0074] 3) Digesting the liver: After ligating the vena cava, perfuse the liver with 5 mL of preheated type IV collagenase digestion solution until the liver swells. Let it stand for 3 min, separate the liver, place it in a clean petri dish, rinse the liver with an appropriate amount of PBS to remove residual blood around the liver, separate the gallbladder, transfer it to a new petri dish, add 5 mL of preheated type IV collagenase digestion solution, crush the liver, transfer it to a 15 mL centrifuge tube, shake it on a shaker at 37°C at 180 r / min for 10 min, and add 1 mL of FBS to stop digestion.

[0075] 4) Incubate the cell suspension at 4℃ for 5 min to allow undigested large tissue fragments to settle. Transfer the supernatant to a new 50 mL centrifuge tube, add 40 mL of pre-cooled DMEM medium, and mix thoroughly by pipetting. Centrifuge at 4℃, 800×g, for 5 min.

[0076] 5) Discard the supernatant, add 40 mL of pre-cooled DMEM medium, pipette the precipitate into a single-cell suspension, and centrifuge at 4°C, 50×g for 5 min.

[0077] 6) Carefully aspirate the supernatant and transfer it to a new 50 mL centrifuge tube. Centrifuge at 4°C, 800×g for 7 min. Discard the supernatant and collect the precipitated cells.

[0078] 7) Resuspend the precipitate with 2 mL of 15% Optiprep separation buffer, then add 15% Optiprep separation buffer to a final volume of 10 mL. Transfer the solution to a 15 mL centrifuge tube. Tilt the centrifuge tube 45° and carefully add 2 mL of DMEM culture medium along the tube wall using a syringe, being careful not to disrupt the interface between the two liquids and ensuring a clear boundary. Centrifuge at 4°C, 1400×g, without acceleration or deceleration for 20 min. After centrifugation, a distinct white film layer will be visible at the interface between the DMEM and 15% Optiprep separation buffers.

[0079] 8) Use a 1 mL pipette to transfer 2 mL of the cell layer solution into a 15 mL centrifuge tube, add pre-cooled PBS or DMEM to 10 mL, centrifuge at 4°C, 800×g for 7 min, discard the supernatant, and resuspend the cells in 2 mL of DMEM medium containing 20% ​​FBS for later use.

[0080] 9) Centrifuge at 300×g for 10 min at 4℃, discard the supernatant and collect the cell pellet. Add 90 µL of buffer, resuspend, add 10 µL of CD11b magnetic beads, mix well, and incubate at 4℃ for 15 min. Add 1 mL of buffer, pipette, centrifuge at 300×g for 10 min at 4℃, discard the supernatant and collect the cell pellet. Add 500 µL of buffer and resuspend. Place the MS adsorption column in a miniMACS magnetic rack and wash the adsorption column with 500 µL of buffer.

[0081] 10) Add cell suspension and wash three times with 500 µL buffer (wait until the adsorption column is clear before the next wash). Collect the eluent as HSCs. Count the freshly isolated primary HSCs using a cell counting chamber. Mix 10 µL of 0.4% trypan blue with 90 µL of cell suspension, count the cells using a cell counting chamber, and calculate the viability.

[0082] Cell culture: Freshly isolated primary HSCs were counted and seeded into 6-well plates, 10 cells per well. 6 Cells were cultured in DMEM medium containing 20% ​​FBS and 1% penicillin-dextrose anhydride, with the medium changed every 24 hours. The culture environment was 37°C and 5% CO2. Morphological changes were observed after 6 days of culture. 10 cells were then harvested. 5 Cells were placed in 24-well cell culture plates containing cell spreaders and cultured in DMEM medium containing 20% ​​FBS and 1% penicillin antibiotics, with the medium changed every 24 hours. The culture environment was 37°C and 5% CO2. 2 After culturing for 3 days, the cells were washed gently with PBS for 3 min, repeated twice. Then, they were fixed with 4% paraformaldehyde for 20 min, washed gently with PBS for 3 min, repeated three times. 0.3% Triton X-100 was added and incubated at room temperature for 20 min, followed by a gentle wash with PBS. The cells were blocked with goat serum at 37°C for 30 min. After blocking, sufficient primary antibody for Desmin (diluted 1:100) was added directly, and the cells were incubated at 37°C for 60 min, washed gently with PBS for 3 min, repeated three times. Sufficient secondary antibody (diluted 1:300) was added, and the cells were incubated at 37°C in the dark for 30 min, washed gently with PBS for 3 min, repeated three times. The cell slides were then removed and mounted with an anti-fluorescence quencher. Desmin belongs to the third class of intermediate filament proteins and is one of the marker proteins for HSCs. Detecting the presence of Desmin is the gold standard for identifying HSCs. Desmin fluorescent staining is used to identify HSCs, such as... Figure 7 As shown, a large amount of red fluorescent Desmin can be observed inside the cells. The above experimental results indicate that primary mouse HSCs were successfully isolated.

[0083] 2.6.5 High-throughput sequencing:

[0084] Total RNA was extracted from primary HSCs of positive and uninfected mice infected with Clonorchis sinensis, as well as isolated EVs, obtained in the above experiments. High-throughput sequencing was performed by Nanjing Paisennong Gene Technology Co., Ltd. to obtain raw data for analysis. First, adapter removal and quality filtering were performed on the raw data. The filtered sequences were then deduplicated (i.e., identical sequences were merged, and their abundance was recorded). The deduplicated sequences were then aligned with the Rfam and miRBase databases to obtain annotation information for various sRNAs. The analysis then focused on miRNAs, including statistical analysis of miRNA expression levels, screening for target genes, prediction of target genes, and validation of these target genes.

[0085] Table 4 Csi-miRNA screening in host HSCs

[0086]

[0087] 2.6.6 Csi-miRNA Screening:

[0088] The miRNA sequencing results of three groups of samples—primary HSCs from Clonorchis sinensis-infected positive mice, primary HSCs from uninfected negative mice, and isolated EVs—were cross-referenced. Parasite-derived miRNAs coexisting in both Clonorchis sinensis-infected primary HSCs and isolated EVs, but not found in primary HSCs from negative mice, were selected and ranked from highest to lowest expression level in vivo. Functional queries were performed on these Csi-miRNAs, and Csi-Bantam was ultimately selected for further research.

[0089] To verify the accuracy of the high-throughput sequencing results, Csi-Bantam was selected, and its specific stem-loop primer RT-Csi-Bantam was designed. The sample miRNA was reverse transcribed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit under the following conditions:

[0090] Table 5 Reverse Transcription Conditions

[0091]

[0092] The reverse transcription products were validated by qRT-PCR, with U6 as an internal control. Three technical replicates were performed for each sample. -ΔΔCt The experimental data were analyzed using a relative quantitative method. The qRT-PCR reaction conditions were as follows:

[0093] Table 6 qRT-PCR Reaction Conditions

[0094]

[0095] The validated Csi-miRNAs were compared with the mirBase database to screen for parasite-specific miRNAs for further research.

[0096] 2.6.7 HSC-T6 cell line culture

[0097] HSC-T6 cell lines were cultured in DMEM medium containing 10% FBS and 1% penicillin antibiotics, with the medium changed every 24 h. The culture environment was 37°C, 5% CO2, and the cells were passaged when they reached 70-80% confluence.

[0098] 2.6.8 Experiments on CsEVs internalizing host HSCs

[0099] To verify whether CsEVs can be internalized by host HSCs and thus play a regulatory role, CsEVs were stained with PKH67 dye. However, directly adding PKH67 dye to CsEVs would lead to heterogeneous staining and reduced cell viability. Therefore, 5 µg of CsEVs were first added to 1 mL of Diluent C, followed by 2 µL of PKH67 dye. The two mixtures were combined, allowed to stand for 4 min, and then 4 mL of FBS was added to stop staining. The mixture was then incubated for 1 min. 9 mL of DMEM was added and mixed thoroughly. The mixture was then transferred to a 100 kDa ultrafiltration centrifuge tube and centrifuged at 4°C and 6000×g for 40 min. The waste liquid was discarded, and the cells were resuspended in 100 µL of DMEM.

[0100] Poly-L-lysine cell spread sheets were placed in 12-well plates. HSC-T6 cells were cultured for 24 h (2 × 10⁶ cells per well) in DMEM containing 10% FBS, 1% 100 U penicillin, and 100 mg / mL streptomycin. 5 (Number of cells). Add 5 µg of PKH67-labeled CsEVs or 100 µL of PBS (control group), and then culture the cells at 37°C and 5% CO2 for 4 h. Remove the culture medium and wash the cells three times with PBS. Fix the cells with 4% paraformaldehyde solution for 15 min, and wash three times with PBS. Stain the cell nuclei with DAPI for 5 min. Remove the cell slides and invert them onto a slide coated with an anti-fluorescence quencher. Observe the cells using a confocal microscope. Figure 2As shown in Figure a, DAPI-labeled cell nuclei emit blue fluorescence, Desmin-labeled cytoplasm emits red fluorescence, and PKH67-labeled CsEVs emit green fluorescence. In the fluorescence micrograph after image fusion processing, PKH67-labeled green fluorescence signals can be detected in the blue nucleus region labeled with DAPI and its surrounding area. These green fluorescently labeled EVs are mainly located in the cytoplasm of the recipient cells (red fluorescent region), and this spatial distribution characteristic confirms that CsEVs can be effectively taken up and internalized by the host HSCs. After Merge processing, green fluorescent CsEVs can be seen both inside and around the blue nucleus, and CsEVs are mainly distributed in the cytoplasm of the recipient cells, proving that CsEVs can be internalized into the host HSCs.

[0101] 2.6.9 Csi-Bantam mimics and inhibitors transfected into the HSC-T6 cell line

[0102] First, the optimal concentration of CsEVs for transfection was screened, with concentration gradients of 0, 1, 2, 5, 10, and 20 μg / mL. CCK8 assays were used to evaluate the cytotoxic effects of different concentrations of CsEVs on HSC-T6 cells, and qRT-PCR was used to detect the expression levels of related genes to determine the optimal stimulation concentration of 5 μg / mL. After determining the optimal stimulation concentration, a time-gradient experiment was further conducted, transfecting HSC-T6 cells with CsEVs at 2, 6, 12, 24, and 48 h. Cell viability and gene expression changes were detected using CCK8 and qRT-PCR to determine the optimal stimulation time for HSC-T6 cells with CsEVs as 12 h. These experimental results demonstrate that Clonorchis sinensis releases CsEVs after infecting the host, and Csi-miRNA can enter the host body along with CsEVs; however, the regulatory role of Csi-Bantam in the host remains unclear. Therefore, in order to verify the role of Csi-Bantam in the host, we designed and synthesized Csi-Bantam-mimics that can overexpress Csi-Bantam and Csi-Bantam-inhibitor that can silence Csi-Bantam. HSC-T6 cell lines were stimulated with 5 μg / mL CsEVs for 12 h, followed by transfection with Csi-Bantam-inhibitor for 12 h, and total RNA was extracted from the cells. Following the RNAiso Plus instructions, add 1 mL of RNAiso Plus to HSCs and homogenize. Incubate at room temperature for 5 min, then centrifuge at 12,000 × g for 5 min at 4 °C. Transfer the supernatant to a new 1.5 mL centrifuge tube. Add 200 µL of chloroform to the tube, vortex until the solution emulsifies and turns milky white. Incubate at room temperature for 5 min, then centrifuge at 12,000 × g for 15 min at 4 °C. Remove the centrifuge tube; at this point, the liquid in the tube has separated into three layers: a colorless supernatant, a middle white protein layer, and a colored lower organic phase. Transfer the supernatant to a new centrifuge tube, add 700 µL of isopropanol, incubate at room temperature for 10 min, then centrifuge at 12,000 × g for 10 min at 4 °C. Discard the supernatant. Add 1 mL of 75% ethanol, invert the tube to wash the walls, centrifuge at 7,500 × g for 5 min at 4 °C, aspirate and discard the upper layer, retaining the precipitate. Open the cap on the centrifuge tube, allow the precipitate to dry at room temperature for 5 min, then add 50 µL of RNase-free water to dissolve the precipitate and store at -80℃ for later use.

[0103] Reverse transcription: Total RNA extracted from cells was reverse transcribed into cDNA according to the PrimeScript™ II 1st Strand cDNA Synthesis Kit instructions. Reverse transcription was performed under the following conditions:

[0104] Table 7 Reverse transcription conditions

[0105]

[0106] qRT-PCR: The reaction system was based on the Mir-X miRNA qRT-PCR TB Green Kit instructions. GAPDH was used as an internal control. Three replicates were performed for each sample. Two [reaction parameters were specified]. -ΔΔCt Relative quantitative methods were used to analyze experimental data.

[0107] Table 8 qRT-PCR reaction conditions

[0108]

[0109] qRT-PCR was used to detect HSC activation-related marker genes (such as... Figure 2 (As shown in b). The three marker genes in the HSC-T6 cell line were significantly elevated after CsEV internalization, with α-SMA, Col1α1, and Col3α1 showing extremely significant increases, indicating that HSCs are activated by CsEVs. The above experiments demonstrate that CsEVs can be internalized by host HSCs and can activate host HSCs, leading to liver fibrosis. Comparison with the mirBase database revealed that among these Csi-miRNAs, Bantam is a parasite-specific miRNA and an interspecies conserved miRNA. Functionally, Bantam is closely related to liver fibrosis and matched multiple target genes associated with liver fibrosis. Therefore, Csi-Bantam was selected as the research subject for subsequent studies.

[0110] Csi-Bantam mimics and inhibitors were synthesized by Beijing Qingke Biotechnology Co., Ltd. Transfection reagents for Csi-Bantam mimics and inhibitors were prepared according to the Lipofectamine RNAiMAX manufacturer's instructions. Concentration gradients of Csi-Bantam mimics and inhibitors were established at 12.5 pmol, 25 pmol, 50 pmol, 100 pmol, and 150 pmol. Different concentrations of Csi-Bantam mimics or inhibitors were transfected into cells, and the cells were cultured at 37°C in a 5% CO2 incubator. Cells were cultured for 24 h post-transfection, with stimulation treatment performed during the first 12 h. After transfection, total RNA was extracted from the cells, and the expression level of Csi-Bantam was detected by qRT-PCR to screen for the optimal transfection concentration of 25 pmol.

[0111] When the cultured HSC-T6 cell line reaches a density of 70%-80%, it is transferred to a cell plate for further culture. The cells are evenly seeded into 6-well plates, approximately 2 × 10⁶ cells per well. 5 Cells were cultured per well for 12 h. HSC-T6 cell lines were stimulated with the optimal Csi-Bantam-mimics / inhibitor transfection concentration of 25 pmol obtained in the above experiments. The corresponding groups were as follows: HSC-T6 cell lines transfected with PBS were negative; transfected with the corresponding mimics and inhibitors were experimental groups (Negative + Csi-Bantam-mimics, Negative + Csi-Bantam-inhibitor); transfected with 5 μg / mL CsEVs were positive; and transfected with both CsEVs, mimics, and inhibitors were experimental groups (Positive + Csi-Bantam-mimics, Positive + Csi-Bantam-inhibitor). Total RNA and total protein were extracted from the cells, and marker genes related to HSC activation were detected by qRT-PCR and Western blotting. Protein sample preparation: Discard the culture medium in the cell plate. Prepare preheated PBS (37°C). Add 1 mL of PBS to each well, keeping the plate level and gently agitating the washed cells. Discard the waste solution. Add 1 mL of PBS to the wells. Use a clean cell scraper to collect cells and transfer them to 1.5 mL centrifuge tubes. Centrifuge at 4°C, 6,000×g for 5 min. Discard the PBS. Add the prepared lysis buffer. After adding the lysis buffer, briefly centrifuge using a pipette. Lyse at 4°C for 30 min. Centrifuge at 6,000×g for 5 min and collect the supernatant as the extracted total protein. Quantify the successfully extracted protein using BCA according to the Pierce™ BCA Protein Assay Kit instructions. Add 5× buffer and dilute the final concentration to 1 µg / µL. Boil in water for 5 min, then rapidly cool in an ice box and store at -80°C for later use.

[0112] Prepare a 12% SDS-PAGE gel according to the SDS-PAGE gel preparation kit instructions. Add 10 µL of protein to each well for electrophoresis. Electrophoresis conditions: first, maintain a constant voltage of 90 V for 30 min, then increase the voltage to 140 V and maintain for 75 min. After electrophoresis, remove the gel, cut off excess gel, rinse with distilled water, and soak for later use.

[0113] Cut the filter paper and PVDF membrane slightly larger than the colloid. Wet the filter paper with transfer buffer. Activate the PVDF membrane with methanol for 30 seconds and soak it in transfer buffer until there is no oil. Place the sponge, filter paper, colloid, PVDF membrane, filter paper, and sponge on the transfer apparatus, respectively. Use a scraper to remove air bubbles from each layer. Perform the transfer at a constant current of 250 mA for 90 minutes. Remove the transferred membrane, wash it with PBST for 5 minutes, and block it in 5% (m / v) skim milk at room temperature for 2 hours. Wash the membrane with PBST for 8 minutes each time, repeating 3 times.

[0114] Place the washed membrane in primary antibody and incubate at 4°C for 12 h. Wash the membrane with PBST for 8 min per wash, repeating 3 times. Transfer to secondary antibody and incubate at room temperature for 1 h. Wash the membrane with PBST for 8 min per wash, repeating 3 times.

[0115] Prepare the working solution by mixing BeyoECL Star solutions A and B in a 1:1 ratio and storing at room temperature away from light. Prepare the solution immediately before use. Place the membrane in an infrared fluorescence scanning imaging system, remove excess liquid, and add 1 mL of BeyoECL Star working solution per 10 cm² of membrane to ensure even coverage. Place the membrane in the instrument chamber for fluorescence detection, screen and adjust the captured images, and analyze the experimental results.

[0116] In qRT-PCR experiments, significantly increased Csi-Bantam levels were detected in HSC-T6 cell lines stimulated by CsEVs and those stimulated by CsEVs+Csi-Bantam-mimics, while Csi-Bantam levels decreased and tended to be negative in HSC-T6 cell lines stimulated by CsEVs+Csi-Bantam-inhibitor, demonstrating successful model establishment (e.g., Figure 3 (as shown in a). Regarding the marker genes related to HSC activation, the expression levels of the three marker genes in the CsEVs+Csi-Bantam-mimics stimulation group were significantly higher than those in the CsEVs stimulation group, while the expression levels of the three related marker genes α-SMA, Col1α1, and Col3α1 in the CsEVs+Csi-Bantam-inhibitor stimulation group were also lower than those in the CsEVs stimulation group (e.g., ...). Figure 3 As shown in b). Western blotting and qRT-PCR results were consistent: the expression levels of the three marker proteins in the CsEVs+Csi-Bantam-mimics stimulation group were significantly higher than in other groups, while the expression levels of the three proteins α-SMA, Col1α1, and Col3α1 in the CsEVs+Csi-Bantam-inhibitor stimulation group were lower than in the CsEVs stimulation group (e.g., ...). Figure 3 (as shown in c) Figure 3The first graph in Figure c is a band plot of the protein results, and the other three graphs are bar charts analyzing the results of α-SMA, Col1α1, and Col3α1, respectively. These experimental results indicate that Csi-Bantam-inhibitor can inhibit the activation of host HSCs.

[0117] 2.6.10 Construction of plasmids and viral vectors

[0118] The plasmids that can silence Csi-Bantam and the plasmids that can overexpress Csi-Bantam were synthesized by Suzhou Genegenes Co., Ltd.

[0119] DNA oligos were designed using Designer 3.0 software. Primers were provided by Suzhou Jima Gene Co., Ltd. The loop structure in the LV3-shRNA template was TTCAAGAGA to avoid termination signals. GATCC was added to the 5' end of the sense strand template to complement the sticky ends formed after BamHI digestion; AATTC was added to the 5' end of the antisense strand template to complement the sticky ends formed after EcoRI digestion. The DNA oligos were dissolved in TE buffer to a concentration of 100 μM. Annealing reaction systems were prepared according to the proportions in Table 9, using the corresponding sense and antisense oligo solutions.

[0120] Table 9 Annealing Reaction System

[0121]

[0122] Annealing was performed on a PCR instrument according to the following procedure: 95℃ for 5 min; 85℃ for 5 min; 75℃ for 5 min; 70℃ for 5 min; and storage at 4℃. After annealing, a 10 μM shRNA template was obtained. The template solution was diluted 50-fold to a final concentration of 200 nM for the ligation reaction. 10 μg of LV3 vector was digested according to the enzyme digestion system in Table 10.

[0123] Table 10 Enzyme digestion system

[0124]

[0125] Enzyme digestion was performed at 37℃ for 1 h, followed by agarose gel electrophoresis. The DNA was recovered using an Agarose Gel DNA Purification Kit Ver 2.0, and the concentration was determined using a UV spectrophotometer. The concentration was then diluted to 50 ng / µL for use. Vector ligation was performed according to the system in Table 11, at 22℃ for 1 h.

[0126] Table 11 Connection System

[0127]

[0128] Remove DH5α competent cells from -80℃ and place the centrifuge tubes containing the competent cells on ice for 1 min. After the competent cells thaw, add 10 µL of ligation product, gently mix the contents, and place on ice for 30 min. Place the centrifuge tubes on a test tube rack in a water bath preheated to 42℃ and incubate for 90 s without shaking. Quickly place the centrifuge tubes in an ice bath for 2 min to cool the cells. Add 800 µL of LB liquid medium (antibiotic-free) to each centrifuge tube, then transfer the centrifuge tubes to a 37℃ shaker and incubate at 180 rpm for 45 min to allow the bacteria to recover. Remove the bacterial culture from the shaker and centrifuge at 6,000 rpm for 1 min, discard the supernatant, and reserve 200 µL for plating. After the liquid on the plate has been absorbed, invert the plate and incubate at 37℃ for 16 h. Take 400 µL of LB solution containing AMP (50 μg / mL), place it in a 1.5 mL centrifuge tube, pick up a colony and place it in the solution, seal the tube and shake on a shaker for 12 h at 220 r / min.

[0129] Plasmids were extracted from bacterial culture using a plasmid miniprep kit; detailed instructions are provided in the kit's manual. Sequencing and identification were performed by Suzhou Gemma Gene Co., Ltd., and the plasmids were packaged into lentiviruses for titer and purity testing. The final results yielded lentiviruses capable of silencing Csi-Bantam (LV3-Csi-Bantam-inhibitor), overexpressing Csi-Bantam (LV3-Csi-Bantam-mimics), and an empty plasmid lentivirus (LV3-NC) as a control.

[0130] 2.6.11 Lentiviral vector transfection

[0131] Six-week-old female BALB / c mice were orally orally inoculated with Clonorchis sinensis metacercariae, with 50 metacercariae per mouse, to establish a Clonorchis sinensis infection mouse model. Lentiviral vector (1×10⁻⁶) 8 Primary HSCs and livers were extracted 42 days later after 100 µL of TU / ml, control vector, or PBS was injected via tail vein.

[0132] Mice were randomly divided into eight groups of 10 each. Group 1 was injected with 100 µL PBS as the negative control group. Group 2 was injected with LV3-NC as the negative control group (Negative + LV3-NC). Group 3 was infected with Clonorchis sinensis metacercariae for 14 days and then injected with 100 µL PBS as the positive control group. Group 4 was infected with Clonorchis sinensis metacercariae for 14 days and then injected with LV3-NC as the positive control group (Positive + LV3-NC). Groups 5 and 6 were injected with lentiviruses LV3-Csi-Bantam-mimics and LV3-Csi-Bantam-inhibitor, respectively, which overexpress and silence Csi-Bantam, as the experimental groups (Negative + LV3-Csi-Bantam-mimics and Negative + LV3-Csi-Bantam-inhibitor). Groups 7 and 8 were infected with Clonorchis sinensis metacercariae for 14 days and were injected with lentiviruses LV3-Csi-Bantam-mimics and LV3-Csi-Bantam-inhibitor that overexpress and silence Csi-Bantam, respectively, as experimental groups (Positive+LV3-Csi-Bantam-mimics, Positive+LV3-Csi-Bantam-inhibitor).

[0133] 2.6.12 Csi-Bantam inhibitor can inhibit liver fibrosis caused by Clonorchis sinensis infection.

[0134] To further verify that Csi-Bantam promotes HSC activation in the host after infection with Clonorchis sinensis, we established a mouse model of Clonorchis sinensis disease and injected LV3-Csi-Bantam lentivirus via tail vein. The mice were then euthanized at 42 days.

[0135] First, the established mouse model of Clonorchis sinensis infection was examined. In normal mice, the liver is red with a smooth, glossy surface. In mice infected with Clonorchis sinensis, the liver is purplish-red or dark red, enlarged, with a dull surface and white spots, and the gallbladder is enlarged (e.g., ...). Figure 4 (As shown in a). Since ALT and AST can enter the bloodstream from hepatocytes when the liver is damaged, leading to elevated ALT and AST levels in the blood, this is a signal indicating liver disease. Therefore, mouse serum was collected and ALT and AST levels were measured using both ALT and AST assay kits. Mice infected with Clonorchis sinensis showed significantly higher ALT and AST activities than uninfected mice (e.g., as shown in a). Figure 4 (As shown in b), the above structure indicates that the mouse model of Clonorchis sinensis infection was successfully established.

[0136] Total RNA was extracted from primary mouse HSCs and detected by qRT-PCR. Csi-Bantam was detected in both groups infected with Clonorchis sinensis and those injected with LV3-Csi-Bantam lentivirus. The Csi-Bantam expression level was significantly higher in the group injected with LV3-Csi-Bantam-mimics than in the Clonorchis sinensis-infected group, while the Csi-Bantam expression level was lower in the group injected with LV3-Csi-Bantam-inhibitor than in the Clonorchis sinensis-infected group. LV3-NC had no effect on the experimental results, confirming the successful establishment of the model (e.g., ...). Figure 4 (As shown in c). When detecting HSCs activation-related marker genes, the expression levels of all three marker genes in the Clonorchis sinensis-infected group were significantly higher than those in the negative control group, and after injection of LV3-Csi-Bantam-inhibitor lentivirus, the expression levels were essentially the same as in the negative control group. In the group infected with Clonorchis sinensis and subsequently injected with LV3-Csi-Bantam-mimics lentivirus, the expression levels of the three marker genes α-SMA, Col1α1, and Col3α1 were significantly higher than in other groups (e.g., ...). Figure 4 (As shown in d). Total protein was extracted from primary mouse HSCs and detected by Western blotting. The results were basically the same as those above. The expression levels of the three marker genes in the Clonorchis sinensis infection group were significantly higher than those in the negative control group. The expression levels of the three marker genes were significantly increased after injection of LV3-Csi-Bantam-mimics lentivirus, and significantly decreased after injection of LV3-Csi-Bantam-inhibitor lentivirus (e.g., as shown in d). Figure 4 (as shown in e) Figure 4 The first figure in e is a bar graph of the protein results, and the other three figures are bar graphs of the α-SMA, Col1α1, and Col3α1 results.

[0137] The above experimental results demonstrate that after the host is infected with Clonorchis sinensis, LV3-Csi-Bantam-inhibitor can inhibit the activation of HSCs in the host, thereby inhibiting the production of liver fibrosis in the host.

[0138] 2.6.13 Csi-Bantam target gene prediction and function query

[0139] Target gene prediction for Csi-Bantam was performed using the miRBase database (https: / / www.mirbase.org). Functional searches for Csi-Bantam target genes were conducted using NCBI (https: / / www.ncbi.nlm.nih.gov) and Bing (https: / / cn.bing.com). The miRBase database predicted 199 target genes for Csi-Bantam. The top 20 target genes were selected for further research. Functional analysis of these top 20 target genes revealed that the Trim35 gene inhibits the classic PI3K / Akt liver fibrosis signaling pathway by inactivating Akt signaling. Since the PI3K / Akt signaling pathway is a crucial pathway for liver fibrosis, Trim35 was chosen for further investigation.

[0140] 2.6.14 Csi-Bantam targeting Trim35 gene information acquisition

[0141] The 3'-UTR sequence of the Trim35 gene was queried using the UCSC Genome Browser (https: / / genome.ucsc.edu). The binding site was predicted between the obtained 3' UTR sequence of the Trim35 gene and the mature Csi-Bantam sequence using STarMir (https: / / sfold.wadsworth.org / ). STarMir predicted one potential binding site between the 3'-UTR sequence of the Trim35 gene and the mature Csi-Bantam sequence. Specifically, a completely complementary binding site exists between the mature Csi-Bantam sequence and positions 1893 to 1911 of the Trim35 gene, with a minimum free energy of -27 kcal / mol and 18 hydrogen bond donors. This binding site was selected for further validation (e.g., ...). Figure 5 (as shown in a).

[0142] 2.6.15 Construction of Dual-Luciferase Vector

[0143] The 3'-UTR sequence of the Trim35 gene corresponding to Csi-Bantam and its surrounding sequences were constructed into the pmirGlo vector as the wild type (pmirGlo-Trim35-3' UTR-WT), and the 3'-UTR sequence of the Trim35 gene corresponding to Csi-Bantam was mutated and constructed into the pmirGlo vector as the mutant type (pmirGlo-Trim35-3' UTR-Mut).

[0144] 2.6.16 293T cell culture

[0145] 293T cells were cultured in DMEM medium containing 10% FBS (containing 1.5 mg / L Glutamine, 100 U / ml Penicillin, and 100 μg / ml Streptomycin) at 37 ºC in a 5% CO2 saturated humidity incubator.

[0146] 2.6.17 Sample transfection into 293T cells

[0147] After digesting and resuspending healthy 293T cells, an appropriate amount of cells / well was seeded into a 12-well plate and incubated overnight at 37°C.

[0148] Add 4 μL of GP-transfect-Mate to 125 μL of DMEM medium and mix well. Then add 100 pmol Oligo and 1 μg of plasmid to 125 μL of DMEM medium and mix well. After incubating at room temperature for 5 min, mix the two mixtures and incubate at room temperature for another 20 min. Aspirate the original medium from the 12-well plate, add the transfection complex, and incubate at 37°C in a 5% CO2 incubator. After 6 h, replace with fresh medium and incubate at 37°C.

[0149] Table 12 Transfection Groups

[0150]

[0151] 2.6.18 Luciferase Activity Assay

[0152] The predicted binding sites were used to construct dual-luciferase plasmids. Csi-Bantam mimics and pmirGlo-Trim35-3'-UTR wild-type (WT) or mutant (Mut) recombinant reporter plasmids were co-transfected into 293T cells. Cells were cultured at 37°C for 24 h after transfection, and the culture medium was removed. Cells were washed with 1×PBS, and the washing buffer was discarded. 250 μL of 1× cell lysis buffer was added to each well. 50 μL of cell lysis buffer was added to an ELISA plate. 10 μL of firefly luciferase reaction solution was added, and the plate was shaken to mix. The activity of firefly luciferase was detected within 30 min. 30 μL of Renilla luciferase reaction solution was added, and the plate was immediately shaken to mix. The activity of Renilla luciferase was then detected. The results showed that luciferase activity in the Csi-Bantam mimics-treated group was significantly lower in the WT group compared with the negative control group (mimics NC) 24 h after transfection. However, when a mutation occurred in the Csi-Bantam binding site in the 3'-UTR sequence of the Trim35 gene, there was no statistically significant difference in luciferase expression between the Csi-Bantam mimics-treated group and the co-transfected mimics NC group (e.g., ...). Figure 5 (as shown in b), indicating that Csi-Bantam can target the Trim35 gene.

[0153] 2.6.19 Verification that the Trim35 gene is a direct target of Csi-Bantam

[0154] We transfected HSC-T6 cell lines with PBS, Csi-Bantam-NC, and Csi-Bantam-mimics, respectively. Total RNA was extracted from the cells, and the expression of Csi-Bantam and Trim35 genes was detected by qRT-PCR. Compared with the Negative group and the Negative+Csi-Bantam-NC group, the expression level of Csi-Bantam in the Negative+Csi-Bantam-mimics group was significantly increased, indicating that Csi-Bantam-mimics can effectively upregulate Csi-Bantam expression in the HSC-T6 cell line (e.g., PBS, Csi-Bantam-NC, and Csi-Bantam-mimics). Figure 5 (See the first figure). Meanwhile, compared to the Negative group and the Negative+Csi-Bantam-NC group, the expression level of the Trim35 gene was significantly decreased in the Negative+Csi-Bantam-mimic group, indicating that upregulating Csi-Bantam expression in the HSC-T6 cell line can significantly inhibit Trim35 gene expression (as shown in Figure c). Figure 5 (See the second figure).

[0155] Next, we transfected CsEVs into the HSC-T6 cell line, and simultaneously transfected it with PBS, Csi-Bantam-NC, and Csi-Bantam-inhibitor, respectively. Total RNA was extracted from the cells, and the expression of Csi-Bantam and Trim35 genes was detected by qRT-PCR. Compared with the Positive group and the Positive+NC inhibitor group, the expression level of Csi-Bantam in the Positive+Csi-Bantam-inhibitor group was significantly decreased, indicating that Csi-Bantam-inhibitor can significantly inhibit the expression of Csi-Bantam in the HSC-T6 cell line (e.g., ...). Figure 5 (As shown in the first figure). Meanwhile, compared to the Positive group and the Positive+NC inhibitor group, the expression level of the Trim35 gene was significantly increased in the Positive+Csi-Bantam-inhibitor group, indicating that downregulating Csi-Bantam expression in the HSC-T6 cell line can significantly increase Trim35 gene expression (e.g., ...). Figure 5 (d. The second figure is shown).

[0156] We administered PBS, LV3-NC, and LV3-Csi-Bantam-mimics via tail vein injection into mouse models, respectively. Total RNA was extracted from primary HSCs in these mouse models, and the expression of Csi-Bantam and Trim35 genes was detected by qRT-PCR and Western blotting. Compared with the Negative and Negative+LV3-NC groups, the expression level of Csi-Bantam in the LV3-Csi-Bantam-mimics group was significantly increased, indicating that LV3-Csi-Bantam-mimics can significantly express Csi-Bantam in primary mouse HSCs. Simultaneously, compared with the Negative and Negative+LV3-NC groups, the expression level of Trim35 gene in the LV3-Csi-Bantam-mimics group was significantly decreased, indicating that upregulating Csi-Bantam expression in primary mouse HSCs can significantly inhibit Trim35 gene expression (e.g., ...). Figure 5 (as shown in e). Figure 5 The first and second figures in e are qRT-PCR results. Figure 5 The third and fourth figures in e are the results of Western blotting.

[0157] Next, we injected PBS, LV3-NC, and LV3-Csi-Bantam-inhibitor into the tail vein of a Clonorchis sinensis-infected mouse model. Total RNA was extracted from primary HSCs of the mouse model, and the expression of Csi-Bantam and Trim35 genes was detected by qRT-PCR and Western blotting. Compared with the Positive and Positive+LV3-NC groups, the expression level of Csi-Bantam was significantly reduced in the Positive+LV3-Csi-Bantam-inhibitor group, indicating that LV3-Csi-Bantam-inhibitor can significantly inhibit Csi-Bantam expression in primary mouse HSCs. Simultaneously, compared with the Positive and Positive+LV3-NC groups, the expression level of Trim35 gene was significantly increased in the Positive+LV3-Csi-Bantam-inhibitor group, indicating that downregulating Csi-Bantam expression in primary mouse HSCs can significantly increase Trim35 gene expression (e.g., ...). Figure 5 (as shown in f) Figure 5 The first and second images in f are qRT-PCR results. Figure 5 The third and fourth figures in f are the results of Western blotting.

[0158] 2.6.20 Csi-Bantam promotes liver fibrosis by inhibiting the Trim35 gene and regulating the PI3K / Akt signaling pathway.

[0159] The PI3K / Akt signaling pathway plays a crucial role in the progression of liver fibrosis by regulating the activation of hepatic cysts (HSCs) and the massive production of endogenous fibrotic tissue (ECM). Some studies have reported that the PI3K / Akt pathway promotes ECM protein synthesis by regulating multiple downstream target genes. The accumulation of these ECM proteins is a key characteristic of liver fibrosis. Phosphorylation and activation of anti-apoptotic proteins (such as Bcl-2) inhibits apoptosis, thereby promoting HSC survival. Other studies have found that fibronectin 1 is upregulated in biliary atresia-related liver fibrosis and promotes HSC activation through the PI3K / Akt pathway, thus exacerbating liver fibrosis. The Trim35 gene belongs to the TRIM family of proteins, which possess E3 ubiquitin ligase activity and participate in the regulation of various cellular processes, including cell proliferation, apoptosis, immune responses, and signal transduction. TRIM family proteins typically contain three conserved domains: a RING domain, a B-box domain, and a coil-coil domain. Previous studies have shown that the Trim35 gene can exert a regulatory role in liver fibrosis by inhibiting the Akt signaling pathway. To investigate whether Csi-Bantam regulates the PI3K / Akt signaling pathway through the Trim35 gene, we used tail vein injection of PBS, LV3-NC, and LV3-Csi-Bantam-mimics into mouse models, respectively. Total RNA was extracted from primary HSCs in these mouse models, and Western blotting was used to detect the expression of proteins related to the PI3K / Akt signaling pathway (PI3K, p-PI3K, Akt, p-Akt). The protein banding results are shown below. Figure 6 As shown in Figure a, the analysis results showed that after injection of LV3-Csi-Bantam-mimics, the protein expression of p-PI3K and p-Akt was significantly upregulated compared with the PBS and LV3-NC injection groups. This indicates that increased Csi-Bantam expression can promote the PI3K / Akt signaling pathway (e.g., ...) in primary mouse HSCs. Figure 6 b、 Figure 6 (as shown in c).

[0160] Based on these research findings, this invention discloses a miRNA inhibitor that can significantly inhibit liver fibrosis caused by Clonorchis sinensis. The miRNA inhibitors inhibiting liver fibrosis are Csi-Bantam-inhibitor and LV3-Csi-Bantam-inhibitor, and their potential applications in the preparation of drugs for treating liver fibrosis.

[0161] The experimental data from this invention highlights the following key points:

[0162] 1) Csi-Bantam is abundant in the liver of the host in the extracellular vesicles of Clonorchis sinensis;

[0163] 2) Csi-Bantam from extracellular vesicles of Clonorchis sinensis is involved in the activation of HSCs and the development of host liver fibrosis;

[0164] 3) Csi-Bantam in the extracellular vesicles of Clonorchis sinensis can upregulate the mRNA and protein levels of α-SMA, Col3α1, and Col1α1, and can regulate the fibrotic process of mouse HSCs, indicating that Csi-Bantam can promote the host's liver fibrosis process through a cross-species approach.

[0165] 5) After inhibiting Csi-Bantam with LV3-Csi-Bantam-inhibitor, the degree of liver fibrosis caused by Clonorchis sinensis can be reduced, which brings a new direction to the development of drugs for the treatment of Clonorchis sinensis, especially the development of drugs for the treatment of liver fibrosis caused by Clonorchis sinensis infection.

[0166] 6) LV3-Csi-Bantam-inhibitor from extracellular vesicles of Clonorchis sinensis inhibits liver fibrosis through the PI3K / Akt signaling pathway.

[0167] Csi-Bantam-inhibitor and LV3-Csi-Bantam-inhibitor have great potential to become effective components in drugs for treating Clonorchis sinensis.

Claims

1. The application of a Csi-Bantam inhibitor in the preparation of a drug for treating liver fibrosis caused by Clonorchis sinensis infection, wherein the inhibitor, by binding to Csi-Bantam, inhibits the expression and function of Csi-Bantam, inhibits the binding of Csi-Bantam to the 3'-UTR region of the Trim35 gene, relieves the inhibition of Trim35 gene expression by Csi-Bantam, restores normal protein expression of the Trim35 gene, and thereby inhibits the PI3K / Akt signaling pathway and inhibits hepatic stellate cell activation; The inhibitors are Csi-Bantam-inhibitor and LV3-Csi-Bantam-inhibitor. The specific sequence of Csi-Bantam-inhibitor is: 5'-ACCAGCTTTAGTCGCGATCTCA-3'; the specific sequence of LV3-Csi-Bantam-inhibitor is: 5'-ACCAGCTTTAATCGCGATCTCAACCAGCTTTAATCGCGATCTCAACCAGCTTTAATCGCGATCTCA-3'.

2. Use according to claim 1, characterized in that: The LV3-Csi-Bantam inhibitor described above is a Csi-Bantam inhibitor packaged in a lentiviral vector, which ensures its stability.

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