Use of plin5 as a target in developing or screening or preparing a drug for promoting liver regeneration

Abstract

The application discloses application of PLIN5 as a target point in development, screening or preparation of a drug for promoting liver regeneration, and the development or screening comprises the following steps: S1, contacting a candidate compound with a cell expressing PLIN5 or a system containing PLIN5 protein; S2, detecting the influence of the candidate compound on the expression level or biological activity of PLIN5; S3, selecting the candidate compound capable of up-regulating the expression level of PLIN5 or enhancing the activity thereof. The application firstly finds that PLIN5 is a key positive regulation target point of liver regeneration, fills the research blank of PLIN5 in the field of liver regeneration regulation in the prior art, and provides a new theoretical basis for the molecular mechanism research of liver regeneration. The up-regulator based on PLIN5 has a clear promoting effect on liver regeneration, can be used for improving the liver function recovery after liver injury, and provides a new drug research and development direction for prevention of postoperative complications of liver resection, treatment of liver fibrosis, alcoholic liver and other diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of PLIN5 as a target in the development, screening, or preparation of drugs for promoting liver regeneration. Background Technology

[0002] Vertebrates, including humans, can live for decades, relying on their ability to maintain normal homeostasis and efficiently recover homeostasis after various injuries. The mammalian liver is one of the most regenerative tissues in the body. Under normal physiological homeostasis, the liver's turnover rate is slow, but after injury, the liver can proliferate a large number of hepatocytes, exhibiting significant proliferative potential to promote homeostasis recovery. The most classic research model is partial hepatectomy (PHx) (removal of a liver lobe), after which the liver can rapidly recover to its original volume and restore functional integrity.

[0003] Despite the liver's powerful intrinsic regenerative capacity, various pathological conditions, including drug abuse, viral infections, and malignant tumors, can severely impair liver function and regenerative potential. Acetaminophen (APAP) is one of the most widely used antipyretic analgesics globally, but overdose often leads to severe liver damage and even acute liver failure (ALF). Statistics show that APAP-related liver injury accounts for approximately half of all acute liver failure cases in the United States each year. Furthermore, liver regeneration is also impaired in some anticancer chemotherapy drugs, metabolic dysfunction-associated steatohepatitis (MASH), and metabolic diseases such as type 2 diabetes (T2D). For patients undergoing aggressive anticancer treatments such as surgical resection, radiotherapy, and chemotherapy, homeostasis imbalance and impaired regenerative capacity can be life-threatening.

[0004] Liver regeneration involves a complex network of molecular regulations, in which lipid metabolism plays a crucial role. The accumulation of lipid droplets (LDs) not only provides energy for physiological processes but also mitigates cell damage caused by excess free fatty acids. Perilipins (PLINs) are a constitutive family of lipid droplet-associated proteins, comprising five members (PLIN1–PLIN5). Previous studies have shown that PLIN5 enrichment in the liver and skeletal muscle promotes lipid droplet formation and improves insulin resistance. However, the specific role and mechanism of PLIN5 in liver regeneration remain unreported.

[0005] This invention, through the establishment of a 70% partial hepatectomy model, a PLIN5 gene knockdown mouse model, and an acute liver injury model, combined with molecular biology, cellular metabolic function detection, and histopathological analysis, has for the first time discovered that PLIN5 is a key positive regulator of liver regeneration, and elucidated its molecular mechanism of promoting hepatocyte proliferation by regulating mitochondrial biogenesis and function and influencing FGF21 expression. Based on this, this invention provides the application of PLIN5 as a target in promoting liver regeneration, which has significant scientific value and clinical translational prospects. Summary of the Invention

[0006] This invention aims to solve the above problems and provides the following technical solution: In a first aspect, the present invention provides the use of perilipin 5 (PLIN5) as a target in the development, screening or preparation of drugs for promoting liver regeneration.

[0007] Furthermore, the development or screening includes the following steps: S1: Contact the candidate compound with cells expressing PLIN5 or a system containing the PLIN5 protein; S2: Detect the effect of candidate compounds on PLIN5 expression levels or biological activity; S3: Select candidate compounds that can upregulate PLIN5 expression levels or enhance its activity.

[0008] Furthermore, the drug comprises a PLIN5 upregulator, which is selected from any of the following: (1) Nucleic acid molecules that promote the transcription or expression of the PLIN5 gene; (2) Recombinant expression vectors carrying the PLIN5 coding sequence; (3) PLIN5 protein or its biologically active fragments; (4) Small molecule compounds that can activate the PLIN5 signaling pathway; (5) Preparations that can stabilize PLIN5 protein expression or delay its degradation.

[0009] Furthermore, the promotion of liver regeneration includes any of the following: (1) Promote liver tissue repair and regeneration after partial hepatectomy; (2) Promotes liver tissue repair after chemical liver injury; (3) Improve liver regeneration disorders related to metabolic diseases; (4) Enhance the hepatocyte proliferation capacity after liver injury; (5) Improve mitochondrial function and promote the recovery of energy metabolism.

[0010] Furthermore, the drug also contains fibroblast growth factor 21 (FGF21) or an agonist thereof.

[0011] Furthermore, the screening adopts any of the following detection systems: (1) Cell model of PLIN5 overexpression; (2) Cell models of PLIN5 gene knockdown or knockout; (3) The binding system of PLIN5 protein to candidate compounds; (4) Reporter gene system based on PLIN5 promoter activity.

[0012] Secondly, the present invention also provides a method for screening candidate drugs that promote liver regeneration, comprising the following steps: (1) Contact the candidate compound with cells expressing PLIN5; (2) To detect the effects of candidate compounds on PLIN5 expression levels or mitochondrial function-related indicators; (3) Select candidate compounds that can upregulate PLIN5 expression levels, enhance mitochondrial respiratory function, or increase ATP production.

[0013] Furthermore, the mitochondrial function-related indicators include any one or more of the following: (1) Basal oxygen consumption (OCR); (2) ATP production capacity; (3) Maximum respiratory capacity; (4) Mitochondrial membrane potential; (5) Expression levels of genes related to mitochondrial biogenesis (PGC1α, Tfam, COX4).

[0014] Thirdly, based on the same invention, this invention also provides a kit for assessing liver regeneration capacity, comprising reagents for detecting PLIN5 expression levels, and instructions, which describe the operating method and judgment criteria for assessing liver regeneration capacity using PLIN5 expression levels.

[0015] Furthermore, the reagent for detecting PLIN5 expression levels includes: (1) Primer pairs and / or probes for detecting PLIN5 mRNA; or (2) Specific antibodies for detecting PLIN5 protein.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to discover that PLIN5 is a key positive regulatory target for liver regeneration, filling the research gap of PLIN5 in the field of liver regeneration regulation in the existing technology, and providing a new theoretical basis for the study of the molecular mechanism of liver regeneration.

[0017] 2. The PLIN5 upregulator of this invention has a clear effect on promoting liver regeneration and can specifically improve the recovery of liver function after liver injury, providing a new direction for drug development for the prevention of complications after clinical liver resection, the treatment of liver fibrosis, alcoholic liver and other diseases.

[0018] 3. The diagnostic reagent based on PLIN5 of this invention can rapidly and accurately assess liver regeneration capacity and the degree of liver damage, providing reliable molecular markers for the formulation of clinical treatment plans and prognostic assessment.

[0019] 4. The screening method provided by this invention can efficiently screen potential drugs targeting PLIN5, shorten the drug development cycle, and reduce development costs.

[0020] 5. This invention reveals the key role of the PLIN5-FGF21 regulatory axis in liver regeneration, providing a scientific basis for combination drug strategies. FGF21 can reverse regenerative impairment caused by PLIN5 deficiency and has the potential to synergistically promote liver regeneration. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other embodiments can be derived from the provided drawings without creative effort.

[0022] Figure 1The study identified PLIN5 as a liver regeneration enhancing factor in a 2 / 3 partial hepatectomy (PHx) mouse model through in vivo transcriptome screening. A shows a schematic diagram of the in vivo transcriptome screening experiment for liver regeneration after 2 / 3 partial hepatectomy; B shows the trend of liver-to-body ratio changes after 2 / 3 partial hepatectomy; C shows a bar chart showing the trend of serum alanine aminotransferase (ALT) levels after surgery; D shows representative images of hematoxylin-eosin (H&E) staining of mouse liver tissue at 2, 12, and 48 hours post-surgery; E shows histological scores; F shows a heatmap displaying the expression levels of PLIN family genes and the characteristics of Ki67-labeled hepatocyte proliferation stages; G and H show the mRNA expression levels of Ki67 and PLIN5 in mouse liver tissue at different post-surgery time points detected by real-time quantitative polymerase chain reaction (qPCR); and I shows the protein expression levels of PLIN5 and proliferating cell nuclear antigen (PCNA) in mouse liver tissue at 2 hours (acute injury phase) and 48 hours (peak proliferation phase) post-surgery using Western blotting. All data are expressed as mean ± standard error (mean ± SEM). Compared with preoperative values, *p < 0.05, **p < 0.01, ***p < 0.001; compared with 2 hours postoperatively, #p < 0.05, ##p < 0.01, ###p < 0.001 (n = 5).

[0023] Figure 2PLIN5 knockdown leads to impaired liver regeneration after partial 2 / 3 hepatectomy. A shows a schematic diagram of the in vivo PLIN5 knockdown experiment; B shows the PLIN5 protein expression level in hepatocytes after adeno-associated virus-mediated PLIN5 knockdown (AAV-shPLIN5) using Western blotting; C shows the relative mRNA expression levels of PLIN5 in normal liver tissue and the AAV-shPLIN5 group; D shows a bar chart of liver body ratio in each group 48 hours after partial 2 / 3 hepatectomy; E shows the survival rate curve of mice after partial 2 / 3 hepatectomy; F shows representative hematoxylin and eosin (H&E) staining images of liver tissue from the PLIN5 knockdown group and control group 48 hours after partial 2 / 3 hepatectomy (w / o indicates "none," i.e., control group); G shows the H&E staining in (F). Histological scoring analysis was performed on the &E stained images. H represents representative images of Ki67 immunohistochemical (IHC) staining of liver tissue from PLIN5 knockdown and control mice 48 hours after partial hepatectomy. I represents the number of Ki67-positive hepatocytes under a 20x microscope. J represents the expression levels of cyclin D1 (Cyclin D1) and p21 proteins in liver tissue from wild-type (WT) and PLIN5 knockdown (shPLIN5) mice 48 hours after partial hepatectomy, detected by Western blotting. (KL) represents the quantitative analysis of Cyclin D1 and p21 protein expression levels in (J). All data are expressed as mean ± standard error (mean ± SEM). Compared with the AAV-GFP control group, *p<0.05, **p<0.01, ***p<0.001 (n=5).

[0024] Figure 3Mitochondrial dysfunction is involved in impaired liver regeneration after PLIN5 deficiency-induced partial hepatectomy (2 / 3 hepatectomy). A shows the immunofluorescence staining analysis of CYP1A2 expression in liver tissues of wild-type (WT) and PLIN5 knockdown (shPLIN5) mice 48 hours after partial hepatectomy. B and C show the results of Western blotting. The expression level of CYP1A2 protein in the liver tissue of two groups of mice 48 hours after surgery was detected and quantitatively analyzed by blotting. D represents real-time live cell detection of primary hepatocytes isolated from regenerated liver tissue using a hippocampal XFe24 extracellular flow analyzer, and mitochondrial oxidative stress and energy metabolism status were analyzed based on oxygen consumption rate (OCR). E represents the relative mRNA expression level of mitochondrial oxidative phosphorylation (OXPHOS)-related genes. F and G represent the ATP levels of wild-type and PLIN5 knockdown hepatocytes and AML12 cell lines. H and I represent the expression levels and quantitative analysis of AMPK pathway and oxidation-related proteins. J represents representative transmission electron microscopy (TEM) images of mitochondrial morphology. K represents quantitative analysis of mitochondrial morphology in mouse liver tissue. L and M represent the Western blot detection and quantitative analysis of protein expression levels of mitochondrial biogenesis-related genes. All data are expressed as mean ± standard error (mean ± SEM). Compared with the negative control (AAV-shNT) group, *p<0.05, **p<0.01, ***p<0.001 (n=5).

[0025] Figure 4Elevated FGF21 levels can exacerbate liver damage and promote liver regeneration. A shows a heatmap illustrating the relative mRNA expression level of FGF21 at different time points after partial hepatectomy (PHx); B shows plasma FGF21 levels at 24 and 48 hours after PHx; C shows Western blot analysis of FGF21 and PCNA protein expression in liver tissue of wild-type (WT) B6 mice at 0, 12, 24, and 48 hours after PHx; D is a schematic diagram of the experimental protocol for studying the effect of mFGF21 protein on liver regeneration after PHx; E shows the liver weight / body weight ratio; F shows representative H&E staining and Ki67 immunohistochemical staining; G is a schematic diagram of the experimental protocol for knocking down FGF21 by intraperitoneal injection of AAV-shFGF21; H shows Western blot analysis of FGF21 protein expression; and I shows the expression of FGF21 protein at 48 hours after PHx. h represents representative H&E staining and Ki67 immunohistochemical staining; J represents quantitative statistics of Ki67-positive hepatocytes under 20x magnification; K represents the effect of H&E staining on FGF21 knockdown on CCl4- or APAP-induced liver injury; L and O represent quantitative data on the area of ​​necrosis induced by CCl4 (L) or APAP (O) in H&E sections under 10x magnification; M and P are bar charts of serum ALT levels 24 h after CCl4 treatment (M) and APAP treatment (P). All data are expressed as **mean ± standard error (mean ± SEM)**. Compared with the PBS group, AAV-shNT group, or APAP / CCl4 group, *p<0.05, **p<0.01, ***p<0.001. (n=5) Figure 5 PLIN5 improves mitochondrial function and thus promotes liver regeneration by regulating FGF21; A shows plasma FGF21 levels 14 days after PLIN5 knockdown; B shows the mRNA expression level of FGF21 in liver tissue after liver-specific PLIN5 knockdown; C shows the PLIN5 knockdown effect after 2 / 3 partial hepatectomy. - / -The results of Western blot analysis of FGF21 protein expression level in mouse liver tissue are shown in Figure 1. D represents the quantitative analysis of grayscale values ​​corresponding to C. E is a schematic diagram of the experimental protocol for studying the effect of mFGF21 protein on liver regeneration after partial hepatectomy of 2 / 3 of the liver in PLIN5 knockout mice. F represents the liver weight / body weight ratio. G represents the results of representative H&E staining and Ki67 immunohistochemical staining. H represents the quantitative statistics of Ki67 positive hepatocytes under a 20x microscope. I represents the real-time live cell analysis of mitochondrial oxidative stress and energy metabolism in primary hepatocytes isolated from PLIN5 knockout regenerated liver tissue using a Seahorse XFe24 extracellular throughput analyzer based on oxygen consumption rate (OCR). J represents the Western blot results of key regulatory proteins of mitochondrial function and the corresponding quantitative analysis of proteins (K). Data description: All data are expressed as mean ± standard error (mean ± SEM); compared with the AAV-GFP group, *p<0.05, **p<0.01, ***p<0.001; sample size n=5. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. The following embodiments are only for illustrative purposes and are not intended to limit the scope of the present invention in any way. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are conventional methods. Unless otherwise specified, the materials and reagents used in the present invention are commercially available. Furthermore, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0027] Example 1: Dynamic changes in liver regeneration and PLIN family expression after 70% partial hepatectomy 1.1 Experimental Materials Experimental animals: Eight-week-old male C57BL / 6N mice, weighing 21.0±1.0g, were purchased from Henan Scobes Biotechnology Co., Ltd. All mice were housed at Liaocheng University in a temperature- and light-controlled environment (20-22°C; 10:14 hour light-dark cycle) with free access to water. The experimental procedures strictly followed the institutional animal care guidelines, and all procedures were approved by the Liaocheng University Institutional Animal Care and Use Committee. Mice were fed commercially available standard mouse pellet feed (Beijing Keao Xieli Feed Co., Ltd., Beijing, China).

[0028] Main reagents: ALT Activity Assay Kit (Nanjing Jiancheng, Catalog No. C009-2-1); Ki67 antibody (Proteintech, catalog number 28074-1-AP); PLIN5 antibody (Abcam, catalog number ab192876). PCNA antibody (CST, catalog number 13110). Trizol reagent (Invitrogen, catalog number 15596026); Reverse transcription kit (Novizan, catalog number R433); qPCR premix (Novazan, catalog number QN222).

[0029] 1.2 Experimental Methods 1.2.1 Establishment of a 70% Partial Hepatectomy Model Following previous experimental methods, a two-thirds partial hepatectomy was performed on mice by surgically removing the left lateral and middle lobes of the liver. The specific experimental method is as follows: (1) Anesthetize mice with 2% isoflurane gas; (2) After disinfecting the abdomen, make a vertical incision in the middle of the abdomen below the xiphoid process, 1.5cm-3cm in length. When opening the abdomen, be careful not to damage the abdominal organs and tissues. Cut open the epidermis, muscle layer and peritoneum in sequence, locate the liver, and use forceps to pull open and fix the abdominal muscles on both sides to fully expose the liver.

[0030] (3) Using ophthalmic scissors, dissect the ligaments related to the liver, ligate the middle lobe and left lateral lobe of the liver in sequence, and then remove them, taking care to preserve the gallbladder; when ligating, be close to the base of the liver lobe to minimize the residual liver lobe and damage to blood vessels, and be gentle when ligating to avoid tearing the blood vessels under the liver. However, it is worth noting that when ligating the middle lobe, the position of the suture knot should not be too close to the superior vena cava of the liver, otherwise it will lead to venous occlusion or stenosis, hindering the blood return from the remaining right lobe and caudate lobe, thereby leading to necrosis of liver tissue and failure of regeneration. Therefore, the position of the suture knot should be beyond the gallbladder, but the distance from the superior vena cava should not be less than 2 mm.

[0031] (4) Return the remaining liver tissue and abdominal contents to the incision, suture the abdomen closed in sequence, and disinfect the incision again after suturing.

[0032] (5) Postoperative fasting and water restriction for 6 hours. Subcutaneous injection of 5% glucose solution to replace intraoperative fluid loss due to evaporation, bleeding and fluid displacement, and for nutrition. Place in a 37℃ incubator for 6 hours for rewarming.

[0033] (6) With the end of the operation as 0h, the animals were sacrificed at 24h, 72h and 7d after the operation according to the group time requirements. The weight of the regenerated liver in each group was weighed and recorded, and samples were taken. The liver tissue was quickly stored in a -80℃ low temperature freezer after collection.

[0034] 1.2.2 Liver function tests Blood was centrifuged at 8000 rpm for 15 minutes to obtain serum. Following the instructions of the ALT activity assay kit, the ALT activity in the serum was determined by measuring the colorimetric product produced by the enzymatic reaction.

[0035] 1.2.3 Histological analysis Liver tissue was fixed overnight in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 5 μm. After routine HE staining, morphological changes were observed under an optical microscope.

[0036] 1.2.4 Immunohistochemical staining The steps are briefly as follows: Sections were dewaxed with xylene, rehydrated with graded ethanol, and washed with PBS. They were then boiled in 10 mM sodium citrate buffer (pH 6.0) for 10 minutes for antigen retrieval, and cooled to room temperature. After blocking with 10% goat serum, specific primary antibody was added, and the sections were incubated overnight at 4°C. The sections were then incubated with 3% hydrogen peroxide for 10 minutes to remove endogenous peroxidase activity, followed by the addition of the appropriate secondary antibody and incubation at room temperature for 60 minutes. Finally, they were counterstained with hematoxylin. Microscopic observation and image acquisition were performed, and quantitative analysis was conducted using ImageJ software.

[0037] 1.2.5 Protein extraction and Western blot Samples were lysed using ice-cold RIPA lysis buffer, and protein concentration was determined by the BCA method. After separation by SDS-PAGE, proteins were transferred to nitrocellulose membranes, blocked with 5% skim milk, and incubated overnight at 4°C with primary antibodies (PLIN5 1:1000, PCNA 1:2000). The corresponding secondary antibodies were then incubated at room temperature for 1 hour. Immunoreactive bands were detected using Super Pico ECL chemiluminescence buffer, and results were recorded using a Licor imaging system. Ponceau S S staining served as an internal control.

[0038] 1.3 Experimental Results and Analysis like Figure 1 As shown in Figure B, the liver-to-body ratio shows a gradual recovery trend during liver regeneration. The activity of ALT, a marker of liver injury, rapidly increases postoperatively, peaking in the early stages (1-6 hours postoperatively) and then gradually decreasing, suggesting that liver injury occurs immediately after hepatectomy and is gradually repaired during liver regeneration. Figure 1 C).

[0039] Histological analysis results showed ( Figure 1(D, E) The preoperative liver histology score remained at a low level, and the score (liver edema) increased significantly 2 hours after surgery and remained at a specific level within 48 hours after surgery, indicating that liver resection surgery can lead to damage to liver tissue structure, and that this structural damage is gradually repaired during liver regeneration.

[0040] The scoring criteria are the scoring standards for liver inflammation activity, as detailed in the table below:

[0041] To systematically evaluate hepatocyte proliferative activity, this invention further detected key proliferation-related indicators. The detection of proliferation-related indicators showed ( Figure 1 (F, G) Compared with preoperative levels, the mRNA expression level of the key proliferation marker Ki67 was significantly upregulated at 2 hours and 48 hours postoperatively (4.2 times and 5.8 times that of preoperative levels, respectively).

[0042] PLIN family member expression analysis Figure 1 (F, H) showed that the mRNA expression level of PLIN5 changed significantly during liver regeneration, starting to rise 2 hours post-surgery and peaking at 48 hours (6.5 times the pre-surgery level). Simultaneously, the protein expression trend of another proliferation-related protein—proliferating cell nuclear antigen (PCNA)—was highly consistent with that of PLIN5. Figure 1 The expression of PLIN1, PLIN2, PLIN3, and PLIN4 showed no significant regular fluctuations. These results suggest that PLIN5 may be involved in the regulation of liver regeneration.

[0043] Example 2: PLIN5 knockdown inhibits liver regeneration and reduces survival rate after partial hepatectomy (2 / 3 of the liver). 2.1 Experimental Materials Adeno-associated virus: AAV8.TBG-shPLIN5, AAV8.TBG-GFP, constructed and packaged by Gemma Gene, with a titer of 4 × 10⁻⁶. 12 Genome particles / mL.

[0044] Main antibodies: CCND1 antibody (CST, catalog number 2978), p21 antibody (Abcam, catalog number ab188224), GAPDH antibody (Proteintech, catalog number 60004-1-Ig).

[0045] 2.2 Experimental Methods 2.2.1 In vivo AAV-mediated PLIN5 knockdown 4×10 11The AAV8.TBG-shPLIN5 (experimental group) or AAV8.TBG-GFP (control group) genome particles were dissolved in 200 μl PBS and injected into 8-week-old male C57BL / 6N mice (n=30 / group) via tail vein. 14 days after injection, 6 mice / group were randomly selected to test the PLIN5 knockdown efficiency, and the remaining mice underwent 70% partial hepatectomy.

[0046] 2.2.2 Survival Rate Analysis After PHx surgery, the survival status of mice was continuously observed for 72 hours, mortality was recorded, and survival rate curves were plotted.

[0047] 2.2.3 Liver-to-body ratio and histological analysis Mice were sacrificed at 0h, 24h, 48h, 7d, and 14d after PHx surgery (n=6 / time point / group), and the regenerated liver weight and body weight were measured to calculate the liver-to-body ratio (liver weight / body weight × 100%).

[0048] 2.2.4 Cell proliferation analysis Liver tissue sections were subjected to Ki67 immunohistochemical staining 48 hours after surgery. Ten fields of view (200×) were randomly selected under a microscope to count the number of Ki67 positive hepatocyte nuclei and the total number of hepatocyte nuclei, and the positive rate (%) was calculated.

[0049] 2.2.5 Western blot detection of cell cycle-related proteins Total protein was extracted from liver tissue 48 hours post-surgery. Western blot was used to detect the expression of CCND1 and p21 proteins, with GAPDH as an internal control. ImageJ software was used for grayscale quantitative analysis.

[0050] 2.3 Experimental Results and Analysis Using Western blot, Figure 2 B) and real-time quantitative polymerase chain reaction (qPCR), Figure 2 C) Verification of PLIN5 knockdown efficiency showed that the protein and mRNA expression levels of PLIN5 in the AAV-shPLIN5 group were significantly reduced.

[0051] As an indicator for evaluating liver regeneration, the liver-to-body ratio in the AAV-shPLIN5 group was significantly lower than that in the control group at all postoperative time points. Figure 2 D), suggesting that PLIN5 knockdown can significantly weaken liver regeneration capacity.

[0052] Survival analysis showed that after partial hepatectomy (2 / 3 of the liver), the survival rate of mice in the AAV-shPLIN5 group was significantly lower than that in the AAV-GFP control group 3 days post-surgery. Figure 2E), indicating that PLIN5 knockdown increases mortality in mice after hepatectomy.

[0053] Histopathological examination results ( Figure 2 (F, G) showed that the liver histological score of the AAV-shPLIN5 group was significantly higher than that of the control group 48 hours after surgery, suggesting that PLIN5 knockdown aggravates liver tissue damage after hepatectomy.

[0054] Proliferative capacity analysis showed that the proportion of Ki67-positive hepatocytes in the AAV-shPLIN5 group was significantly lower than that in the control group 48 hours after surgery. Figure 2 H, I). Meanwhile, the expression of CCND1, a key protein promoting cell cycle progression, was significantly downregulated in the AAV-shPLIN5 group (H, I). Figure 2 J, K); Conversely, the protein expression of cell cycle repressor p21 was significantly upregulated in the AAV-shPLIN5 group ( Figure 2 J, L).

[0055] The above results further confirm that PLIN5 knockdown can inhibit hepatocyte proliferation by regulating the expression of cell cycle-related proteins, thereby impairing liver regeneration function, thus clarifying the positive regulatory role of PLIN5 in the process of liver regeneration.

[0056] Example 3: PLIN5 knockdown impairs liver metabolic function and mitochondrial function during liver regeneration. 3.1 Experimental Materials Cell line: AML12 mouse hepatocytes (ATCC) Lentivirals: shPLIN5 lentivirus and shNT control lentivirus, constructed and packaged by Gemma Genetics. Main reagents: CYP1A2 antibody (Proteintech, catalog number 19910-1-AP); COX4 antibody (CST, catalog number 4850); p-AMPK antibody (CST, catalog number 2535); PGC1α antibody (Abcam, catalog number ab191838); Tfam antibody (Abcam, catalog number ab131607); Seahorse XF cell mitochondrial stress assay kit (Agilent, catalog number 103015-100); ATP assay kit (Beyotime, catalog number S0026). 3.2 Experimental Methods 3.2.1 Immunohistochemistry and Western blot detection of metabolic enzyme expression Liver tissue sections were subjected to CYP1A2 immunohistochemical staining 48 hours postoperatively, using the same method as in Example 1. CYP1A2 protein expression was detected by Western blot, with GAPDH used as an internal control.

[0057] 3.2.2 Establishment of PLIN5 knockdown model in AML12 cells (1) The target vector plasmid containing shPLIN5, the packaging plasmid psPAX2 and the envelope plasmid pMD2.G were co-transfected into 293FT cells; (2) Collect the virus-containing culture medium 48 hours after transfection; (3) AML12 cells were transduced with the virus at 37°C for 8 hours in the presence of 8 μg / ml hexamethylene bromide; (4) After screening with 2 μg / mL puromycin for 72 hours, AML12-shPLIN5 cells with stable PLIN5 knockdown and control AML12-shNT cells were obtained.

[0058] 3.2.3 Seahorse XF Cell Mitochondrial Stress Test (1) AML12-shNT and AML12-shPLIN5 cells were mixed at a concentration of 1×10⁻⁶. 5 / wells were inoculated into 24-well Seahorse assay plates and incubated overnight; (2) Before the test, wash the cells with PBS and replace them with Seahorse XF RPMI medium containing 20 mM glucose, 2 mM L-glutamine and 1 mM sodium pyruvate. (3) Add oligomycin (1.5 μM), FCCP (1 μM), and rotenone / antimycin A (0.5 μM) in sequence, and monitor oxygen consumption rate (OCR) in real time. (4) Import the data into the Seahorse XF Mitochondrial Stress Test Report Generator to calculate parameters such as basal respiration, ATP production, and maximum respiration.

[0059] 3.2.4 Detection of intracellular ATP levels Follow the instructions for the ATP assay kit: (1) Lyse the cells, centrifuge at 12000g for 5 minutes at 4℃, and collect the supernatant; (2) Add the ATP detection working solution, mix well, and then measure the RLU value; (3) The protein concentration was standardized by BCA method.

[0060] 3.2.5 Observation of mitochondrial morphology by transmission electron microscopy (1) The liver tissue was cut into 1 mm³ pieces 48 hours after the operation; (2) Fixation with 2.5% glutaraldehyde, followed by fixation with 1% osmium tetroxide; (3) Gradient ethanol dehydration, epoxy resin embedding; (4) Ultrathin sections (70 nm) were stained with uranium acetate and lead citrate; (5) Observe with transmission electron microscopy and randomly select 10 fields of view to count the total number of mitochondria and the number of damaged mitochondria (mitochondrial swelling, cristae breakage, vacuolization).

[0061] 3.2.6 Western blot detection of mitochondrial function-related proteins Total protein was extracted from liver tissue 48 hours after surgery. Western blot was used to detect the expression of COX4, p-AMPK, PGC1α, and Tfam proteins, with GAPDH used as an internal control.

[0062] 3.3 Experimental Results and Analysis First, the expression of the key liver metabolic enzyme cytochrome P450 1A2 (CYP1A2) was detected. Immunohistochemical staining results showed that the expression level of CYP1A2 in liver tissue of the AAV-shPLIN5 group was decreased. Figure 3 A) suggests that PLIN5 knockdown can impair metabolic function during liver regeneration; Further verification using Western blotting showed that CYP1A2 protein expression levels in this group were significantly lower than those in the negative control (AAV-shNT) group 48 hours post-surgery. Figure 3 B, C).

[0063] To clarify the changes in mitochondrial function, this invention performed oxygen consumption rate (OCR) detection on AML12 hepatocytes. The results showed that under basal respiration, ATP production (rotenone / antimycin A stimulation), and maximal respiration (uncoupling agent FCCP stimulation), the oxygen consumption rate of hepatocytes in the AAV-shPLIN5 group was significantly lower than that in the AAV-shNT group. Figure 3 D), and the relative intracellular ATP level was also significantly lower than that of the control group ( Figure 3 (F, G) indicates that PLIN5 knockdown can inhibit mitochondrial oxidative phosphorylation and reduce ATP production.

[0064] Liver tissue mitochondrial counting results showed that 48 hours post-surgery, the AAV-shPLIN5 group had significantly fewer mitochondria than the control group, and the proportion of damaged mitochondria was increased. Figure 3 J, K); Further analysis revealed that the protein expression levels of the mitochondrial markers cytochrome c oxidase subunit 4 (COX4) and phosphorylated adenosine monophosphate activated protein kinase (p-AMPK) were significantly downregulated in this group. Figure 3 H, I).

[0065] The expression levels of genes related to mitochondrial biogenesis were detected. The results showed that the protein expression levels of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) and mitochondrial transcription factor A (Tfam), key regulators of mitochondrial biogenesis, were significantly lower in the AAV-shPLIN5 group than in the control group. Figure 3 L, M).

[0066] The above results suggest that PLIN5 knockdown can impair mitochondrial biogenesis and function, leading to insufficient energy supply, which may be one of the mechanisms by which PLIN5 regulates liver regeneration.

[0067] Example 4: Increased FGF21 expression can promote liver damage repair and regeneration 4.1 Experimental Materials Recombinant protein: FGF21 recombinant protein (Novazia, catalog number C04D) Adeno-associated virus: AAV8.TBG-shFGF21, AAV8.TBG-GFP (Germazon gene) ELISA Kit: Mouse FGF21 ELISA Kit (Solarbio, catalog number SEKM-0078) Chemical inducing agents: Carbon tetrachloride (CCL4, Sigma, catalog number 270652); Acetaminophen (APAP, Sigma, catalog number A7085) 4.2 Experimental Methods 4.2.1 Analysis of Regenerated Datasets Liver tissues from mice were collected at different time points (0h, 6h, 12h, 24h, 48h, 7d) after PHx surgery, and the expression changes of FGF21 at different stages of liver regeneration were analyzed.

[0068] 4.2.2 FGF21 Expression Detection Plasma and liver tissue were collected at different time points after PHx surgery (0h, 6h, 12h, 24h, 48h, 7d). Plasma FGF21 level was detected by ELISA, and FGF21 protein expression in liver tissue was detected by Western blot.

[0069] 4.2.3 FGF21 exogenous supplementation experiment (1) Mice were randomly divided into a control group (PBS) and an FGF21 group (n=12 / group); (2) The FGF21 group received intraperitoneal injection of FGF21 (15 mg / kg / d) for 9 consecutive days; (3) A 70% partial hepatectomy was performed on the 7th day; (4) Mice were sacrificed at 24h, 36h and 48h after surgery (n=6 / time point / group) and liver-to-body ratio, HE staining and Ki67 immunohistochemistry were detected.

[0070] 4.2.4 Hepatocyte-specific FGF21 knockout (1) Tail vein injection of AAV8-TBG-shFGF21 or AAV8-TBG-GFP (4×10¹¹ genome particles / animal); (2) 14 days after injection, Western blot was used to verify the FGF21 knockout efficiency; (3) Perform 70% partial hepatectomy and detect the proportion of Ki67 positive cells 48 hours after the operation.

[0071] 4.2.5 Acute Liver Injury Model APAP model: (1) Mice were fasted for 16 hours; (2) Intraperitoneal injection of APAP (300 mg / kg, dissolved in PBS); (3) After APAP treatment, the animals were allowed to resume eating 6 hours later and sacrificed 24 hours later. Serum ALT and liver tissue necrosis area were measured.

[0072] CCL4 model: (1) CCL4 and corn oil are diluted at a ratio of 1:10; (2) Intraperitoneal injection of CCL4 (1 mL / kg body weight); (3) The animals were sacrificed after 48 hours, and serum ALT and the area of ​​liver tissue necrosis were measured.

[0073] 4.3 Experimental Results and Analysis like Figure 4 As shown in Figure A, FGF21 expression decreases during the initiation / inflammatory phase, increases during the regeneration phase, and returns to baseline levels after regeneration terminates. ELISA and Western blot confirmed that plasma FGF21 levels and protein expression both increase during liver regeneration, reaching a peak at 48 hours post-surgery (see Figure A). Figure 4 B and Figure 4 C).

[0074] To verify the positive regulatory effect of FGF21 on liver regeneration, mice were first injected with FGF21 intraperitoneally for 9 days, followed by 2 / 3 hepatectomy. Surprisingly, the mice injected with FGF21 had a higher liver-to-body ratio, suggesting that FGF21 administration may accelerate liver growth.

[0075] Subsequently, the histological repair and proliferative capacity of the liver were examined. The results showed that FGF21 intake helped repair abnormal liver tissue, manifested as a reduction in the number of swollen hepatocytes (see...). Figure 4(F). Consistent with this, at 24 and 36 hours post-hepatectomy, the proportion of Ki67-positive hepatocytes in the FGF21 group was significantly higher than that in the control group.

[0076] Given that FGF21 supplementation can enhance hepatocyte proliferation, this invention further investigated whether inhibiting FGF21 expression would inhibit the repair process. As previously described, mice were injected with AAV8-TBG-GFP or AAV8-TBG-shGFP for 14 days to induce hepatocyte-specific FGF21 ablation (see...). Figure 4 G and Figure 4 H). Subsequently, a two-thirds hepatectomy was performed. Forty-eight hours post-hepatectomy, liver histology in FGF21 knockout mice showed mild abnormalities, but the proportion of Ki-67-positive proliferating hepatocytes was significantly suppressed compared to the GFP group (see H). Figure 4 I and Figure 4 J).

[0077] To further elucidate the role of FGF21 in liver homeostasis and injury repair, this invention employs two classic acute liver injury models: an acetaminophen (APAP) overdose model and a carbon tetrachloride (CCL4)-induced model. Figure 4 K and Figure 4 As shown in N, FGF21 intake can alleviate APAP or CCL4-induced liver injury, but this protective effect disappears when FGF21 is ablated, specifically manifested in changes in the proportion of necrotic areas in liver tissue and plasma alanine aminotransferase (ALT) levels (see N). Figure 4 L and Figure 4 O).

[0078] In summary, these data indicate that FGF21 overexpression contributes to enhanced liver growth after injury, while FGF21 deficiency weakens the regenerative and repair capabilities of hepatocytes.

[0079] Example 5: PLIN5 deficiency impairs liver regeneration and mitochondrial function; FGF21 can reverse this damage. 5.1 Experimental Methods 5.1.1 Effect of PLIN5 knockdown on FGF21 expression (1) Mice were treated with AAV-shPLIN5 and AAV-GFP (n=12 / group), using the same method as in Example 2; (2) Plasma and liver tissue were collected 48 hours after PHx surgery; (3) ELISA was used to detect plasma FGF21 levels; (4) qPCR detection of FGF21 mRNA expression in liver tissue; (5) Western blot detection of FGF21 protein expression in liver tissue.

[0080] 5.1.2 FGF21 Compensation Experiment Design (1) Mice treated with AAV-shPLIN5 were randomly divided into two groups: PLIN5 KD group and PLIN5 KD+FGF21 group (n=12 / group); mice treated with AAV-shNT served as the control group (n=12). (2) The PLIN5 KD+FGF21 group received intraperitoneal injection of FGF21 (15mg / kg / d), starting 3 days before PHx surgery and continuing until euthanasia; (3) 48 hours after PHx surgery, 6 mice in each group were sacrificed to detect Ki67 positivity rate and mitochondrial function; (4) Fourteen days after PHx surgery, the remaining 6 mice in each group were sacrificed and the liver-to-body ratio was calculated.

[0081] 5.1.3 Mitochondrial function testing (1) Primary hepatocytes were isolated 48 hours after the operation; (2) OCR detection using Seahorse XF24, the method is the same as in Example 3; (3) Western blot was used to detect the expression of PGC1α, Sirt1 and COX4 proteins, with Tubulin as an internal control.

[0082] 5.1.4 Isolation of primary mouse hepatocytes A two-step collagenase perfusion method was used: (1) Anesthetize the mice and open the abdomen to expose the portal vein; (2) Insert the perfusion needle and first infuse the perfusion solution containing EGTA (10 mL / min, 5 min). (3) Infuse with digestive solution containing collagenase (5 mL / min, 8-10 min). (4) Hepatocytes were isolated, washed with PBS, and tested for cell viability by trypan blue staining (>85% were used for subsequent experiments).

[0083] 5.2 Experimental Results and Analysis To investigate the potential regulatory role of PLIN5 on FGF21 during liver regeneration, the expression of fibroblast growth factor 21 (FGF21) in mice with 70% partial hepatectomy (PHx) was first examined.

[0084] Enzyme-linked immunosorbent assay (ELISA) results showed that 48 hours after partial hepatectomy, serum FGF21 protein levels were significantly reduced in PLIN5-deficient mice. Figure 5 A). Consistent with ELISA results, compared to the control AAV-GFP group, the relative mRNA expression level of FGF21 in the liver of mice treated with AAV-shPLIN5 was significantly downregulated. Figure 5 B). Western blot analysis further confirmed that FGF21 protein expression was decreased in the liver tissue of mice treated with AAV-shPLIN5, and Coomassie Brilliant Blue (Ponceau S) staining was used as an internal control. Figure 5 CD).

[0085] Furthermore, this invention evaluated the effects of PLIN5 deficiency and FGF21 supplementation on liver regeneration. Liver-to-body ratio results showed that 14 days after partial hepatectomy, the liver-to-body ratio in the AAV-shPLIN5-treated group was significantly lower than that in the control group; however, treatment with FGF21 restored the liver-to-body ratio in mice to a level comparable to that of the control group. Figure 5 E, F).

[0086] Hematoxylin-eosin (HE) staining of liver tissue further confirmed that PLIN5 deficiency impairs liver regeneration in mice, while FGF21 can reverse this damaging effect. Figure 5 G, top row). Immunohistochemical staining results for the cell proliferation marker Ki67 showed that the proportion of Ki67-positive hepatocytes was significantly reduced in PLIN5-deficient mice during the early stages of liver regeneration; while FGF21 supplementation effectively reversed this proliferation defect, manifested as an increase in the number of Ki67-positive cells. Figure 5 G, bottom row and Figure 5 H).

[0087] Given the crucial role of mitochondrial function in liver regeneration, this invention assessed mitochondrial respiratory function in liver tissue by detecting oxygen consumption rate (OCR). Compared with the AAV-shNT (non-targeting shRNA) control group, mice treated with AAV-shPLIN5 showed a significantly reduced basal oxygen consumption rate; treatment with FGF21 restored basal mitochondrial respiratory function in PLIN5-deficient livers. PLIN5-deficient mice exhibited diminished responses to mitochondrial inhibitors (oligomycin, rotenone / antimycin A [Rot / AA]) and uncoupling agents (FCCP), indicating impaired mitochondrial oxidative phosphorylation (OXPHOS); supplementation with FGF21 also reversed this damage. Figure 5 I).

[0088] Western blot analysis showed that the protein expression levels of key genes related to mitochondrial biogenesis and oxidative phosphorylation (including PGC1α, Sirt1, and COX4) were significantly downregulated in PLIN5-deficient liver tissue. Figure 5 JK). Using tubulin as an internal control, treatment with FGF21 restored the expression levels of these proteins to levels close to those of the control group. Figure 5 JK).

[0089] Example 6: Validation of a screening method for PLIN5-targeted drugs (exemplary method) 6.1 Experimental Methods 6.1.1 Establishment of the screening system (1) Constructing an AML12 cell line overexpressing PLIN5 (AML12-PLIN5-OE) and control cells; (2) Seed cells in 96-well plates (1×10⁻⁶ cells per well). 4 / hole); (3) Add to the candidate compound library (containing 50 small molecule compounds known to have hepatoprotective effects) at a final concentration of 10 μM; (4) After culturing for 24 hours, collect cells and extract RNA and protein.

[0090] 6.1.2 Detection Indicators (1) qPCR detection of PLIN5 mRNA expression; (2) Western blot detection of PLIN5 protein expression; (3) Seahorse assay for mitochondrial function (selecting the top 5 compounds that upregulate PLIN5 expression); (4) CCK-8 assay for cell proliferation capacity.

[0091] 6.1.3 Validation of positive compounds (1) The selected positive compounds were injected intraperitoneally into normal C57BL / 6N mice (n=6 / group) at a dose of 20 mg / kg / d for 7 consecutive days; (2) Perform a 70% partial hepatectomy; (3) The liver-to-body ratio, Ki67 positivity rate and mitochondrial function were measured 48 hours after surgery.

[0092] 6.2 Experimental Results 6.2.1 Screening Results Three compounds (Comp#12, Comp#28, and Comp#35) were screened from 50 candidate compounds and significantly upregulated PLIN5 mRNA expression (3.2-fold, 2.8-fold, and 4.5-fold, respectively, compared to the control group) and protein expression (2.5-fold, 2.2-fold, and 3.8-fold, respectively, compared to the control group).

[0093] 6.2.2 Verification of Mitochondrial Function The basal respiration, ATP production, and maximum respiration of AML12 cells treated with the three compounds were significantly higher than those of the control group (for example, Comp#35: basal respiration 185 vs 145 pmol / min; ATP production 145 vs 112 pmol / min; maximum respiration 280 vs 235 pmol / min).

[0094] 6.2.3 In vivo validation 48 hours after PHx surgery, the liver-to-body ratio of mice in the Comp#35 treatment group was significantly higher than that in the control group (3.2% vs 2.7%), the Ki67 positivity rate was significantly increased (28.5% vs 21.3%), and mitochondrial function-related indicators (OCR, ATP level) were significantly improved.

[0095] This screening system can be used to efficiently screen candidate drugs that target PLIN5 to promote liver regeneration.

[0096] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. Application of PLIN5 as a target in the development, screening, or preparation of drugs to promote liver regeneration.

2. The application according to claim 1, characterized in that, The development or screening includes the following steps: S1: Contact the candidate compound with cells expressing PLIN5 or a system containing the PLIN5 protein; S2: Detect the effect of candidate compounds on PLIN5 expression levels or biological activity; S3: Select candidate compounds that can upregulate PLIN5 expression levels or enhance its activity.

3. The application according to claim 1, characterized in that, The drug contains a PLIN5 upregulator, which is selected from any of the following: (1) Nucleic acid molecules that promote the transcription or expression of the PLIN5 gene; (2) Recombinant expression vectors carrying the PLIN5 coding sequence; (3) PLIN5 protein or its biologically active fragments; (4) Small molecule compounds that can activate the PLIN5 signaling pathway; (5) Preparations that can stabilize PLIN5 protein expression or delay its degradation.

4. The application according to claim 1, characterized in that, The promotion of liver regeneration includes any of the following: (1) Promote liver tissue repair and regeneration after partial hepatectomy; (2) Promotes liver tissue repair after chemical liver injury; (3) Improve liver regeneration disorders related to metabolic diseases; (4) Enhance the hepatocyte proliferation capacity after liver injury; (5) Improve mitochondrial function and promote the recovery of energy metabolism.

5. The application according to claim 1, characterized in that, The drug also contains fibroblast growth factor 21 or an agonist thereof.

6. The application according to claim 1, characterized in that, The screening process employs any of the following detection systems: (1) Cell model of PLIN5 overexpression; (2) Cell models of PLIN5 gene knockdown or knockout; (3) The binding system of PLIN5 protein to candidate compounds; (4) Reporter gene system based on PLIN5 promoter activity.

7. A method for screening candidate drugs that promote liver regeneration, characterized in that, Includes the following steps: (1) Contact the candidate compound with cells expressing PLIN5; (2) To detect the effects of candidate compounds on PLIN5 expression levels or mitochondrial function-related indicators; (3) Select candidate compounds that can upregulate PLIN5 expression levels, enhance mitochondrial respiratory function, or increase ATP production.

8. The method according to claim 7, characterized in that, The mitochondrial function-related indicators include one or more of the following: (1) Basal oxygen consumption (OCR); (2) ATP production capacity; (3) Maximum respiratory capacity; (4) Mitochondrial membrane potential; (5) Expression levels of genes related to mitochondrial biogenesis (PGC1α, Tfam, COX4).

9. A kit for assessing liver regenerative capacity, characterized in that, It includes reagents for detecting PLIN5 expression levels, as well as instructions for use, which describe the procedures and criteria for assessing liver regeneration capacity using PLIN5 expression levels.

10. The reagent kit according to claim 9, characterized in that, The reagents used to detect PLIN5 expression levels include: (1) Primer pairs and / or probes for detecting PLIN5 mRNA; or (2) Specific antibodies for detecting PLIN5 protein.

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