Pab-miR396a-5p as well as pharmaceutical composition and application thereof

Abstract

The invention discloses pab-miR396a-5p as well as a pharmaceutical composition and application thereof, and belongs to the technical field of biological medicines. After HaCaT cells are treated by virtue of pa-miR396a-5p or a pa-miR396a-5p simulant, 3 '-UTR of HSP90 can be targeted, and NF-kB and JAK-STAT signal channels can be inhibited, so that an IL-17 signal is down-regulated, and an anti-inflammatory effect is achieved. An animal experiment that the lipid nanoparticles deliver the pa-miR396a-5p simulant shows that the skin inflammation of a psoriasis mouse is remarkably reduced, and the PASI score is improved. Therefore, the pa-miR396a-5p can be used as an active component of an anti-inflammatory drug, is applied to treatment of inflammatory diseases, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to pab-miR396a-5p and a pharmaceutical composition and use thereof, and specifically relates to the use of pab-miR396a-5p in the preparation of anti-inflammatory disease drugs. Background Art

[0002] Psoriasis is a chronic inflammatory skin disease characterized by recurrent erythema with a high prevalence worldwide. Its occurrence is closely related to genetics, immune disorders and environmental factors. Abnormal activation of the immune system leads to excessive proliferation of keratinocytes and inflammatory responses in the surface layer of the skin, especially the activation of cytokines such as IL-17 and TNF-α. Traditional treatments, such as steroids, vitamin D analogs, and systemic immunosuppressants such as cyclosporine, can provide symptom relief, but are also accompanied by adverse side effects, including skin irritation, immunosuppression, and susceptibility to infection. The emergence of biologics targeting IL-17 has revolutionized the treatment of psoriasis, achieving better disease control by specifically regulating inflammatory pathways. However, these therapies are often costly, require long-term use, and have the risk of immune-related side effects, which highlights the importance of developing natural, effective and safe anti-inflammatory drugs.

[0003] In recent years, exosomes (EVs), as a natural biological nanovesicle, have become an emerging carrier for drug delivery and immune regulation due to their good biocompatibility, low toxicity and stability. EVs are mainly derived from cells and have a double-layer lipid membrane structure, which can encapsulate and deliver a variety of bioactive molecules, such as proteins, lipids, RNA, etc. In particular, the role of small RNA (such as miRNA) in EVs has received increasing attention. Studies have shown that miRNA in EVs can regulate cell-to-cell communication, regulate immune responses, and inhibit or activate the expression of specific genes, and has great application potential.

[0004] Plant-derived exosome-like nanovesicles (EVPs) have gradually emerged as a promising biocompatible nanocarrier for therapeutic applications. These nanovesicles, with a phospholipid bilayer structure, naturally encapsulate bioactive molecules such as proteins, lipids, and nucleic acids, including miRNAs. In particular, EVPs have been shown to regulate intercellular communication and immune responses through miRNA delivery, and their membrane structure can effectively protect miRNAs from enzymatic degradation, improving their stability and bioavailability. Currently, many studies have explored the anti-inflammatory effects of EVPs from plants such as ginger, turmeric, and ginseng in various models. However, the specific mechanisms of these effects, especially the role of endogenous miRNAs, are still unclear; in addition, whether miRNAs packaged in plant-derived exosome-like vesicles can exert cross-domain regulatory effects on mammalian cells to regulate inflammatory diseases (such as psoriasis) remains to be fully explored.

[0005] Perilla has a long history of medicinal use in my country and has antibacterial, antiviral, anti-inflammatory, and anti-allergic effects. In recent years, studies have found that Perilla has multiple pharmacological effects such as regulating immunity, anti-oxidation, and lipid-lowering. Summary of the invention

[0006] In order to solve the above-mentioned problems existing in the prior art, the present invention provides exosome-like nanovesicle functional miRNA derived from perilla leaves, which exerts anti-inflammatory effects by effectively delivering functional miRNA to target cells. It has broad application prospects, especially in the treatment of inflammation-related diseases, and has important clinical application value.

[0007] The present invention also provides a pharmaceutical composition containing the above-mentioned exosome-like nanovesicle functional miRNA derived from perilla leaves.

[0008] The present invention also provides the use of the above-mentioned exosome-like nanovesicle functional miRNA derived from perilla leaves.

[0009] The present invention adopts the following technical solutions to solve the above technical problems: A functional miRNA of exosome-like nanovesicles derived from perilla leaves, characterized in that the functional miRNA is pab-miR396a-5p, as shown in SEQ ID NO.1, and the specific sequence is: 5′- TTCCACAGCTTTCTTGAACT-3′.

[0010] A pharmaceutical composition, comprising: pab-miR396a-5p as described above.

[0011] Furthermore, the pharmaceutical composition also includes a delivery vector, and the delivery vector contains the pab-miR396a-5p.

[0012] Furthermore, the delivery carrier is one of exosomes, lipid nanoparticles, polymer nanocarriers, inorganic nanocarriers or protein carriers.

[0013] Furthermore, the delivery vehicle is a lipid nanoparticle.

[0014] Furthermore, the pharmaceutical composition can be applied externally in the form of ointment, gel or cream.

[0015] A use of the above-mentioned perilla leaf-derived exosome-like nanovesicle functional miRNA as an active ingredient or the above-mentioned pharmaceutical composition is used to prepare a drug for preventing and / or treating anti-inflammatory diseases.

[0016] Further, it is used to treat skin inflammation, immune-mediated skin diseases, allergic diseases or chronic inflammatory diseases.

[0017] Furthermore, it is used to treat psoriasis.

[0018] Furthermore, the pab-miR396a-5p can indirectly inhibit the activation of NF-κB and JAK-STAT pathways by targeting and inhibiting the 3′-UTR of HSP90, thereby inhibiting inflammatory factors in the IL-17 signaling pathway and alleviating immune-mediated skin inflammation.

[0019] Furthermore, the inflammatory factor is one or more of IL-6, IL-17a, IL-1β or CCL2.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The pab-miR396a-5p in the present invention is non-cytotoxic and exerts a significant anti-inflammatory effect by effectively inhibiting the IL-17 signaling pathway. Through mimic and inhibitor transfection experiments and animal experiments of lipid nanoparticle delivery, the role of pab-miR396a-5p in significantly reducing skin inflammation in psoriasis mice and improving PASI scores was further verified (lipid nanoparticles LNPs encapsulating pab-miR396a-5p mimics can significantly reduce skin inflammation in psoriasis mice and improve PASI scores. Lipid nanoparticles LNPs encapsulating pab-miR396a-5p mimics can significantly reduce the levels of related IL-6, IL-17a, and CCL2 in the serum of psoriasis mice. Lipid nanoparticles LNPs encapsulating pab-miR396a-5p mimics can significantly downregulate p-p65 and p-STAT3 in psoriasis mice and inhibit the activity of the IL-17 signaling pathway).

[0021] The present invention provides a regulator (pab-miR396a-5p) based on natural plant sources as a new choice of anti-inflammatory drugs, which has the characteristics of high safety and significant effect.

[0022] In addition, the current cost of extracting exosome-like nanovesicles is high, while pab-miR396a-5p can be artificially synthesized at low cost and can be mass-produced, providing a feasible solution for the development of anti-inflammatory drugs and related products.

[0023] (2) Different from the existing anti-inflammatory mechanism, the present invention found that pab-miR396a-5p reduces its regulatory effect on the signaling pathway by targeting and inhibiting the 3′-UTR of plant HSP90 (which is unique to the present invention), thereby indirectly inhibiting the activation of the NF-κB and JAK-STAT signaling pathways to inhibit the IL-17 signaling pathway and exert an anti-inflammatory effect. This provides a theoretical basis for pab-miR396a-5p to regulate the inflammatory pathway and thus relieve inflammation.

[0024] (3) The anti-inflammatory effect of the miRNA of the present invention is significantly better than that of PLEVPs as a whole: In Example 3, the present invention compared the anti-inflammatory effects of PLEVPs and the miRNA of the present invention through in vivo experiments, and the results of related inflammation in serum showed (Appendix Fig.15 ), although the anti-inflammatory effect of the PLEVPs group was significantly different from that of the other groups, the anti-inflammatory effect was not as good as that of the miRNA of the present invention.

[0025] (4) The delivery vector of the present invention has been optimized to a certain extent to improve the therapeutic effect of pab-miR396a-5p: LNPs (lipid nanoparticles) prepared by microfluidic chip technology can efficiently encapsulate miRNA, and dynamic light scattering (DLS) and transmission electron microscopy (TEM) characterization prove that the nanoparticles are uniform and stable. This shows that LNPs can effectively encapsulate and deliver miRNA, thereby improving its stability and biological activity in vivo.

[0026] In addition, the results of in vivo experiments showed (Example 3 Fig.15 ), even LNPs without pab-miR396a-5p encapsulation can exert significant anti-inflammatory effects. This indicates that LNPs themselves can not only provide stable encapsulation function during delivery, but also have certain anti-inflammatory activity.

[0027] Therefore, after encapsulating miRNA by LNPs, the present invention can further enhance the anti-inflammatory effect of pab-miR396a-5p and improve its therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Results of screening functional miRNAs of perilla leaf-derived exosome-like nanovesicles (PLEVPs) co-cultured with IL-6-stimulated HaCaT cells. (A) Schematic diagram of the experimental process; (B) Principal component analysis (PCA) plot; (C) Heat map analysis of differentially expressed small RNAs; (D) Small RNA volcano plot, with pab-miR396a-5p highlighted; (E) Gene (GO) enrichment analysis; (F) KEGG pathway enrichment analysis.

[0029] Figure 2 The expression verification and target identification results of pab-miR396a-5p. (A) qPCR verification of the expression level of pab-miR396a-5p in the PLEVPs treatment group and the control group; (B) Dual luciferase assay verification of the interaction between pab-miR396a-5p and Hsp90.

[0030] Figure 3 In vitro safety evaluation of pab-miR396a-5p mimics and inhibitors. (A) Schematic diagram of the experimental process; (B) Cell viability assay; (C) Cell live / dead staining.

[0031] Figure 4 The expression level of pab-miR396a-5p in HaCaT cells after transfection with pab-miR396a-5p mimics and inhibitors.

[0032] Figure 5 Analysis of gene levels related to the IL-17 signaling pathway in HaCaT cells after transfection with pab-miR396a-5p mimics and inhibitors.

[0033] Figure 6 Immunofluorescence staining of IL-17, HSP90, p-p65, and p-STAT3 in HaCaT cells after transfection with pab-miR396a-5p mimics and inhibitors.

[0034] Figure 7 Analysis of HSP90, IL-17, p-p65, and p-STAT3 protein levels in HaCaT cells after transfection with pab-miR396a-5p mimics and inhibitors.

[0035] Figure 8 The expression levels of IL-17a, IL-6, IL-1β and CXCL10 in the culture supernatant of HaCaT cells after transfection with pab-miR396a-5p mimics and inhibitors.

[0036] Fig. 9Particle size distribution of lipid nanoparticles (LNPs) encapsulating pab-miR396a-5p mimics and NC mimics.

[0037] Fig.10 Transmission electron microscopy (TEM) images of lipid nanoparticles (LNPs) encapsulating pab-miR396a-5p mimics and NC mimics.

[0038] Fig.11 Lipid nanoparticles (LNPs) encapsulating pab-miR396a-5p mimics and NC mimics were applied to the back skin of mice during different treatment periods.

[0039] Fig.12 Figure 2 Spleen size of mice after treatment with lipid nanoparticles (LNPs) encapsulating pab-miR396a-5p mimics and NC mimics.

[0040] Fig.13 Comparison of body weight, PASI score, total skin thickness profile, and spleen index of mice after treatment with lipid nanoparticles (LNPs) encapsulating pab-miR396a-5p mimics and NC mimics.

[0041] Fig.14 HE staining results of mouse skin after treatment with lipid nanoparticles (LNPs) encapsulating pab-miR396a-5p mimics and NC mimics.

[0042] Fig.15 Results of IL-6, IL-17a, and CCL2 levels in mouse serum after treatment with lipid nanoparticles (LNPs) encapsulating pab-miR396a-5p mimics and NC mimics.

[0043] Fig.16 (A) IF staining of p-p65 and p-STAT3, and (B) IHC analysis of IL-17 and HSP90 in mouse skin after treatment with lipid nanoparticles (LNPs) encapsulating pab-miR396a-5p mimics and NC mimics. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the scope of protection claimed by the present invention is not limited to the scope described in the examples.

[0045] Example 1: Screening and verification of functional miRNAs in exosome-like nanovesicles (PLEVPs) derived from Perilla leaves

[0046] 1 Experimental methods

[0047] 1.1 Cell culture Human epidermal keratinocytes HaCaT cells and 293T cells were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (China). HaCaT cells were maintained at 37°C and 5% CO2 and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin.

[0048] 1.2 Co-culture of PLEVPs with IL-6-stimulated HaCaT cells HaCaT cells were seeded in 12-well culture plates and cultured to 70-80% confluency. 25 ng / mL IL-6 was added for 24 hours to activate the inflammatory response. During the IL-6 stimulation, the culture plates were returned to 37°C and 5% CO. 2 After 24 hours, add PLEVPs and place the culture plate back to 37°C and 5% CO 2 The cells were incubated in an incubator for 24 hours to ensure that PLEVPs were effectively internalized by HaCaT cells. After treatment, the cell culture medium was removed and the cells were gently washed twice with RNase-free PBS buffer to remove residual culture medium and exosome-like nanovesicles. Then, total RNA of the cells was extracted using Trizol reagent. The extracted total RNA was stored at -80°C for subsequent analysis.

[0049] 1.3 miRNA sequencing HaCaT cells were exposed to IL-6 and then co-cultured with PLEVPs for 24 h. After culture, total RNA of cells and cells that internalized PLEVPs was extracted. RNA was extracted using Trizol reagent (Invitrogen, CA, USA). Small RNA library was constructed by NEBNextSmall RNA Library Prep Set for Illumina kit according to the instructions. RNA was reverse transcribed into cDNA and amplified by PCR, and then sequenced using Illumina Novaseq 6000 platform. Mature miRNAs were identified using the miRBase database and expression profiles were generated. Differentially expressed miRNAs were screened by q value < 0.05 and foldchange > 2 or < 0.5. GO and KEGG pathway enrichment analysis were performed for differentially expressed miRNAs. Sequencing and analysis were supported by OE BiotechCo., Ltd. (Shanghai, China).

[0050] 1.4 q-PCR detection HaCaT cells were seeded in 6-well culture plates and placed in a 37°C, 5% CO 2The cells were cultured in an incubator until the cell confluence was 70-80%. The cells were then randomly divided into two groups: control group (Control) and treatment group (PLEVPs). PLEVPs were added to the treatment group and incubated for 24 hours. After the experiment, the culture medium was discarded, and the cells were gently washed twice with RNase-free PBS. Then, miRNA was extracted from the cells using the BIOG Cell miRNA Extraction Kit (51094, Baidu, Changzhou, China), and the expression level of miR396a-5p was analyzed by qRT-PCR using the All-in-One™ miRNA qRT-PCR Detection Kit 2.0 (QP115, GeneCopoeia, Inc.) and All-in-One™ miRNA qPCR Primers.

[0051] 1000 ng of RNA was reverse transcribed using PrimeScript™ RT Master Mix (Takara). The reaction system was configured according to the kit instructions, with a total volume of 20 μL, and the reaction was performed at 42°C for 15 minutes and terminated at 85°C for 5 seconds to obtain the cDNA template. After reverse transcription, it was immediately used for qPCR or stored at -20°C for future use.

[0052] Real-time fluorescence quantitative PCR (qPCR) reactions were performed using the SYBR Green dye system (ChamQ Universal SYBRqPCR Master Mix, Vazyme) in a QuantStudio 3 real-time PCR instrument (Applied Biosystems). The total reaction system was 20 μL. The PCR reaction conditions were: 95°C pre-denaturation for 30 seconds, followed by 40 cycles of 95°C denaturation for 5 seconds and 60°C annealing / extension for 30 seconds. Primers should be designed for target genes (such as pab-miR396a-5p target genes). Finally, by comparing the changes in the expression levels of target genes in the Control group and the PLEVPs-treated group, the expression regulation of pab-miR396a-5p under PLEVPs treatment was evaluated.

[0053] 1.5 Dual luciferase reporter gene assay According to the results of small RNA sequencing, miR-pab-396a-5p may affect the IL-17 signaling pathway by targeting upstream regulatory genes. Bioinformatics analysis predicted that HSP90 was a potential target gene of pab-miR-396a-5p and showed high binding affinity. To verify this targeting relationship, the wild-type (WT) and mutant (MT) 3'-UTR sequences of HSP90 were cloned into the pmirGLO dual-luciferase reporter vector, respectively. Subsequently, the constructed reporter vector was co-transfected with pab-miR-396a-5p mimics or negative control (NC mimics) into 293T cells and divided into four groups: 1) NC mimics + hsp-WT, 2) pab-miR-396a-5pmimics + hsp-WT, 3) NC mimics + hsp-MT, and 4) pab-miR-396a-5pmimics + hsp-MT. After 48 hours of transfection, the luciferase activity was determined using a dual-luciferase reporter gene assay system to measure the luminescence values ​​of firefly luciferase and Renilla luciferase, respectively, and the ratio between the two was calculated. By analyzing the differences in luciferase activity between different groups, the direct targeted regulation of pab-miR-396a-5p on HSP90 was verified.

[0054] 2 Experimental Results The functional miRNA of the exosome-like nanovesicles derived from the perilla leaves of this example is pab-miR396a-5p, and its nucleotide sequence is shown in Table 1.

[0055] Table 1. Nucleotide sequence of pab-miR396a-5p

[0056] We co-cultured PLEVPs with IL-6-stimulated HaCaT cells and performed small RNA sequencing ( Figure 1 A) The results showed that the differentially expressed miRNAs were derived from PLEVPs and were successfully internalized. PCA analysis and heat map analysis showed ( Figure 1 BD), the miRNA expression in the PLEVPs-treated group was significantly different from that in the model group, and pab-miR396a-5p was significantly upregulated. GO and KEGG analysis showed that ( Figure 1 E and F), IL-17 signaling pathway is the main target of these miRNAs, demonstrating that pab-miR396a-5p can regulate this pathway.

[0057] To verify the effect of pab-miR396a-5p, we performed qPCR analysis on cells treated with PLEVPs ( Figure 2 A), confirming that its expression was significantly increased. Dual luciferase reporter gene assay ( Figure 2 B), we confirmed that pab-miR396a-5p specifically binds to the 3'-UTR of HSP90, suggesting that it may have a regulatory role in animal cells.

[0058] Example 2: Pab-miR396a-5p targets mammalian HSP90 to regulate the IL-17 signaling pathway and inhibit psoriasis skin inflammation

[0059] 1 Experimental methods

[0060] 1.1 Cell viability and live / dead assays To evaluate the effects of mimics and inhibitors of pab-miR396a-5p and their negative controls (NC mimics and NC inhibitors) on HaCaT cell viability, HaCaT cells were first seeded into 96-well plates. Subsequently, mimics and inhibitors of pab-miR396a-5p and their negative controls (NC mimics and NC inhibitors) were transfected into the prepared cells using CALNP™ RNAi reagent (D-Nano Therapeutics, DN001) and then incubated for 24 hours. Next, 10 μL of CCK-8 reagent (Beyotime, C0037) was added to each well and incubated for another 2 hours. The absorbance (OD value) of each well was read at a wavelength of 450 nm using a microplate reader (Thermo Fischer Scientific Co., Ltd, China).

[0061] Note: mimics represent pab-miR396a-5p functional enhancement group, which simulates the function of natural miRNA, and is synthesized exogenously and transfected into cells to increase its expression level in cells. In the present invention, mimics refer to artificially synthesized pab-miR396a-5p miRNA, which is used to simulate the function of natural pab-miR396a-5p miRNA.

[0062] Inhibitors represent the pab-miR396a-5p functional loss group or silencing group, which inhibit the function of the miRNA by complementary binding to the endogenous miRNA and preventing it from binding to the target miRNA, so as to verify the specific role of the miRNA in regulating the target gene and signaling pathway.

[0063] To further evaluate cell death, the Calcein-AM / PI double-staining live / dead cell staining kit was used for detection. HaCaT cells were seeded in a 24-well plate with a cover glass, and treated with the corresponding pab-miR396a-5p mimics and inhibitors and their negative controls (NC mimics and NC inhibitors). The cells were treated with a mixed staining solution of Calcein-AM and PI prepared in serum-free medium and incubated at 37°C for 30 minutes in the dark. After staining, the cells were gently washed twice with RNase-free PBS, and immediately observed and photographed under a confocal laser scanning microscope after sealing. Calcein-AM stained live cells with green fluorescence, and PI stained dead cells with red fluorescence.

[0064] 1.2 q-PCR detection HaCaT cells treated with mimics and inhibitors of pab-miR396a-5p and their negative controls (NCmimics and NC inhibitors) were collected. Total RNA was extracted from cells using the YALEPIC® Human Cell and Tissue Total RNA Rapid Isolation Kit (YR23017, YALI Biotech Co., Ltd.), and reverse transcribed using the HiScript IV RTSuperMix for QPCR Kit (R423-01, Nanjing Vazyme Biotech Co., Ltd.). q-PCR detection was performed using ChamQ Universal SYBR qPCR Master Mix (Q711, Nanjing Vazyme Biotech Co., Ltd.) and PCR Array Plate (Shanghai WcGene Biotech Co., Ltd.).

[0065] 1.3 Immunofluorescence staining HaCaT cells were transfected with mimics and inhibitors of pab-miR396a-5p and their negative controls (NC mimics and NC inhibitors) respectively. After incubation for 24 hours, the cells were washed with PBS, fixed in 4% paraformaldehyde for 15 minutes, and then permeabilized with 0.2% TritonX-100 for 10 minutes. Then, the cells were blocked and the primary antibodies for IL-17, HSP90, p-p65 and p-STAT3 were added respectively, and incubated overnight at 4°C. The next day, after incubation with fluorescently labeled secondary antibodies for 1 hour, the cell nuclei were counterstained with DAPI, and after washing with PBS, the cells were observed and photographed under a fluorescence microscope to detect the regulatory effect of pab-miR396a-5p on the expression of related proteins.

[0066] 1.4 Western blot analysis HaCaT cells treated with mimics and inhibitors of pab-miR396a-5p and their negative controls (NCmimics and NC inhibitors) were collected and used for experiments with RIPA lysis buffer (Beyotime Biotechnology Co. Ltd., Shanghai, China) supplemented with protease and phosphatase inhibitors to fully extract proteins. The supernatant was collected after centrifugation and the protein concentration was measured using a BCA protein assay kit. After heating the samples at 100°C for 6 min, 5× loading buffer (Beyotime Biotechnology Co. Ltd., Shanghai, China) was added. Then, the protein samples were separated using SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes by electrophoresis. The membranes were further blocked using protein-free fast blocking buffer (1×) (Shanghai Epizyme Biopharmaceutical Technology Co., Ltd., China) and then incubated with primary antibodies including anti-IL-17, anti-HSP90, and anti-GAPDH overnight at 4°C. The membranes were then washed three times with TBST and incubated with HRP-conjugated secondary antibodies for 1 h. Protein bands were visualized using the Omni-ECL™ Femto Light Chemiluminescence Kit (Shanghai Epizyme Biomedical Technology Co., Ltd., China). The bands were then quantified using ImageJ software (National Institutes of Health, USA) for comparison.

[0067] 2 Experimental Results 2.1 In vitro safety evaluation of pab-miR396a-5p In Example 1, we demonstrated that PLEVPs can deliver pab-miR396a-5p, target HSP90 and regulate the IL-17 signaling pathway. To further verify the role of pab-miR396a-5p, we synthesized its mimics and inhibitors and transfected them into HaCaT cells. CCK-8 assay and live / dead cell staining confirmed that pab-miR396a-5p mimics and inhibitors did not cause significant cytotoxicity ( Figure 3 AC).

[0068] 2.2 Effect of pab-miR396a-5p on genes related to IL-17 signaling pathway qPCR analysis showed that ( Figure 4 ), the expression of pab-miR396a-5p increased by 147.7 times in the mimic group, while it decreased by 60% in the inhibitor group. HSP90 was significantly downregulated in the mimic group, while IL-17 pathway genes such as Il17ra, Il17rc and Il6 were significantly reduced in the mimic group, while the expression in the inhibitor group did not change significantly. NF-κB and JAK-STAT pathway-related genes such as Ikbkb, Rela, Nfkb1, Jak2 and Stat3 were significantly downregulated in the mimic group ( Figure 5 ). Subsequent IF staining confirmed these findings. The expression of IL-17, HSP90, p-p65, and p-STAT3 in the mimic group was reduced, while the inhibitor group showed slightly increased or similar expression compared with the negative control ( Figure 6 ), and finally, Western blot analysis confirmed ( Figure 7 ), in the mimic group, the key protein levels of HSP90, IL-17, p-p65, and p-STAT3 decreased significantly, indicating that pab-miR396a-5p inhibited IL-17 signaling pathway by inhibiting NF-κB and JAK-STAT signaling by targeting HSP90.

[0069] 2.3 Effect of pab-miR396a-5p on the expression of related inflammatory factors ELISA analysis of cell supernatants showed ( Figure 8 ), inflammatory cytokines such as IL-17A, IL-6, IL-1β, and CXCL10 were significantly decreased in the mimetic group but increased in the inhibitor group.

[0070] Example 3: Lipid nanoparticle-encapsulated Pab-miR396a-5p mimics effectively prevent imiquimod (IMQ)-induced psoriasis

[0071] 1 Experimental methods

[0072] 1.1 Preparation and characterization of miRNA-LNPs To evaluate the therapeutic effect of pab-miR396a-5p, LNPs (lipid nanoparticles) were first prepared using microfluidic chip technology. In the experiment, pab-miR396a-5p mimics (mimics) or negative controls (NC mimics) and lipid mixtures consisting of cationic lipids, cholesterol and PEG-modified lipids were injected into the microfluidic chip channel for high-speed mixing as two components. Through the precise control of the microfluidic chip, efficient encapsulation of miRNA and lipid materials was achieved to form uniform and stable miRNA@LNPs. Subsequently, dynamic light scattering (DLS) was used to characterize the particle size and distribution of the nanoparticles. The morphology of LNPs was further observed by transmission electron microscopy (TEM). In addition, fluorescence labeling or ultraviolet spectroscopy was used to detect the encapsulation efficiency.

[0073] 1.2 Animals Male Balb / c mice (4–6 weeks), weighing approximately 18–22 g, were provided by GemPhar-matech (Nanjing, China). All animals were housed at room temperature (25 °C) and 40–70% humidity. All animal procedures were in accordance with the guidelines of the Jiangsu Institute of Traditional Chinese Medicine (Approval No. SYXK(SU)2021-0025).

[0074] 1.3 Verification of the therapeutic effect of miRNA-LNPs in vivo In this experiment, the 2.5 × 2.5 cm area on the back of the mouse was first depilated one day before the establishment of the psoriasis mouse skin model. Subsequently, 62.5 mg of IMQ was evenly applied to the depilated area for 7 consecutive days. Starting from the third day of IMQ administration, the treatment group used the prepared miRNA@LNPs in the IMQ-induced psoriasis mouse model to evaluate its therapeutic effect in psoriasis skin inflammation. Body weight, total skin thickness and PASI score were monitored daily.

[0075] 1.4 Back lesion score and spleen index measurement The severity of psoriasis-like inflammation in mice was assessed using the Psoriasis Area and Severity Index (PASI) scoring system. The principle is as follows: The severity of skin inflammation was assessed daily using a 5-point scale (0-4) to evaluate erythema, desquamation, and thickening. Each parameter was scored according to severity from 0 to 4: 0: none; 1: mild; 2: moderate; 3: severe; 4: very severe. The sum of the three parameter scores represents the severity of psoriatic dermatitis (0-12 points). The spleens of the mice were also collected, measured, and weighed after the mice were sacrificed. The spleen index of each group was calculated using the formula: spleen index = spleen weight (g) / mouse body weight (g) × 100.

[0076] 1.5 ELISA test ELISA is used to detect the levels of inflammatory factors such as IL-6, IL-17A, IL-1β, and CCL2 in animal serum. The treated cell culture supernatant or animal sample supernatant is added to the wells of the enzyme-labeled plate pre-coated with specific antibodies, incubated at 37°C for 1-2 hours, and then washed to remove unbound substances. Then add the enzyme-labeled secondary antibody, incubate at 37°C for 1 hour and wash again. After adding the substrate colorimetric solution TMB to color at room temperature in the dark for 10-20 minutes, add the stop solution to terminate the reaction, and immediately measure the absorbance at a wavelength of 450nm. Calculate the concentration of inflammatory factors in the sample according to the standard curve, and analyze and compare the differences in treatment among the groups.

[0077] 1.6 H&E, immunofluorescence and immunohistochemistry All dorsal skins of mice were collected and fixed with 4% paraformaldehyde, then embedded in paraffin and cut into 4 μm thick sections. For histological analysis, skin sections were stained with hematoxylin and eosin (H&E), and immunohistochemistry (IHC) staining was used to evaluate the expression of Ki67, IL-17, and IL-22. Immunofluorescence (IF) staining was used to detect the expression of Caspase3, CD45, and CD4. All stained tissues were photographed under a digital slide scanner (Pannoramic MIDI, 3DHISTECH, Hungary). Meanwhile, in the in vivo biocompatibility study, the main organs of mice were subjected to the same scanning and observation procedures.

[0078] 2 Experimental Results 2.1 Preparation of miRNA-LNPs and characterization of their physicochemical properties To further explore the therapeutic effect of pab-miR396a-5p, we used a microfluidic system to prepare lipid nanoparticles (LNPs) encapsulating pab-miR396a-5p mimics (mimics) or negative controls (NC mimics).

[0079] Dynamic light scattering (DLS) measurements showed that the particle size of miRNA mimics@LNPs was approximately 82.65 nm (PDI=0.172), and the particle size of NC mimics@LNPs was approximately 79.74 nm (PDI=0.102), indicating that the nanoparticles were evenly distributed ( Fig. 9 and Table 2). Transmission electron microscopy (TEM) observations confirmed that both nanoparticles had a distinct spherical structure ( Fig.10 ), and the encapsulation efficiencies of the two miRNAs reached 98.37% and 98.62%, respectively (Table 2).

[0080] Table 2. Characteristics of LNPs encapsulating pab-miR396a-5p mimics and NC mimics.

[0081]

[0082] 2.2 Evaluation of the therapeutic effect of IMQ-induced psoriasis mouse model In an in vivo experiment, we used an IMQ-induced psoriasis mouse model to evaluate the therapeutic efficacy of the above-mentioned LNPs and compared them with PLEVPs and tacrolimus. Fig.11 ) found that the skin condition of the PLEVPs group was the best and closest to the normal control group; the miRNA mimics group also showed significant improvement, while the NC mimics group was similar to the model group, with obvious scaling and erythema. Fig.12 ) Consistent with the skin observation, both miRNA mimics and PLEVPs treatment significantly reduced the enlargement of the spleen in mice. In addition, we monitored the body weight, PASI score, skin thickness, and spleen index of mice ( Fig.13 ). The miRNA mimics group showed significant weight recovery, and the PASI score and skin thickness were significantly reduced, with similar effects to PLEVPs and tacrolimus. It is worth noting that the PASI score in the PLEVPs group improved most significantly, even better than tacrolimus; while the spleen index did not reach a statistically significant difference, it showed a downward trend in all treatment groups.

[0083] 2.4 Histopathological analysis (H&E staining) H&E staining results ( Fig.14 ) further supported the above conclusion: the model group and NC mimics group showed obvious epidermal thickening and scaling, while the epidermal thickness and inflammation of the miRNA mimics group were alleviated. Although the effect was slightly inferior to PLEVPs, it still showed a significant role in regulating keratinocyte function.

[0084] 2.5 ELISA detection of inflammatory factor levels ELISA test results ( Fig.15 ) showed that inflammatory factors such as IL-6, IL-17a and CCL2 were significantly reduced in the miRNA mimics and PLEVPs treatment groups, similar to the therapeutic effect of tacrolimus. Interestingly, the level of inflammatory factors in the miRNA mimics group was even slightly lower than that in the PLEVPs group, although the difference did not reach statistical significance, which was related to the targeted high expression of pab-miR396a-5p. In addition, the inflammatory factors in the NC mimics group were also slightly reduced, which may be attributed to the anti-inflammatory effect of LNPs themselves, which has been reported in previous studies.

[0085] 2.6 IF and IHC verification of key signaling pathways and target protein expression levels In immunofluorescence (IF) and immunohistochemistry (IHC) analysis ( Fig.16 ), we observed significant downregulation of p-p65 and p-STAT3 in the miRNA mimics group and the PLEVPs group, which is consistent with previous in vitro results and confirms the mechanism of inhibiting the activity of the IL-17 signaling pathway by inhibiting these upstream pathways. IHC results further confirmed that the expression of IL-17 and HSP90 in the miRNA mimics group was significantly reduced, indicating successful regulation of target genes and inhibition of psoriasis-related inflammation.

[0086] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. An exosome-like nanovesicle functional miRNA derived from perilla leaves, characterized in that: The functional miRNA is pab-miR396a-5p, as shown in SEQ ID NO.

1.

2. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: the pab-miR396a-5p as described in claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition further comprises a delivery vector, wherein the delivery vector contains the pab-miR396a-5p.

4. The pharmaceutical composition according to claim 3, characterized in that The delivery carrier is one of exosomes, lipid nanoparticles, polymer nanocarriers, inorganic nanocarriers or protein carriers.

5. The pharmaceutical composition according to claim 3 or 4, characterized in that The delivery vehicle is a lipid nanoparticle.

6. Use of the exosome-like nanovesicle functional miRNA derived from perilla leaves as claimed in claim 1 as an active ingredient or the pharmaceutical composition as claimed in claims 2-5 for preparing a preventive and / or therapeutic anti-inflammatory drug.

7. The use according to claim 6, characterized in that Used to treat skin inflammation, immune-mediated skin diseases, allergic diseases or chronic inflammatory diseases.

8. The use according to claim 7, characterized in that Used for the treatment of psoriasis.

9. The use according to any one of claims 6 to 8, characterized in that The pab-miR396a-5p can indirectly inhibit the activation of NF-κB and JAK-STAT pathways by targeting and inhibiting the 3′-UTR of HSP90, thereby inhibiting inflammatory factors in the IL-17 signaling pathway and alleviating immune-mediated skin inflammation.

10. The use according to claim 9, characterized in that The inflammatory factor is one or more of IL-6, IL-17a, IL-1β or CCL2.

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