Method for constructing animal model of paternal origin non-alcoholic fatty liver disease, intervention target and application thereof

Abstract

The application discloses a method for constructing a paternal non-alcoholic fatty liver disease (NAFLD) animal model, an intervention target and application thereof. The paternal NAFLD animal model is obtained by the following steps: daily intragastric perfusion of male rodents (such as Wistar rats) with caffeine (15, 30, 60 mg / kg) for 8 weeks, then impregnating the female rats to obtain offspring, and establishing a paternal pre-pregnancy caffeine exposure model. The offspring are weaned at 4 weeks after birth, and are separated into female and male cages and fed until 32 weeks, and the male and female offspring both exhibit typical NAFLD phenomena. Based on the paternal pre-pregnancy caffeine exposure rat model, the male offspring at 8 weeks after birth are treated with an AVV8-miR-142-3p adeno-associated virus, and the NAFLD phenomena can be effectively corrected. The animal model established by the application is novel, reliable and simple, and miR-142-3p can be used as an intervention target of the paternal NAFLD.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal model construction, and particularly relates to a construction method of a paternal non-alcoholic fatty liver disease animal model, an intervention target and application thereof. BACKGROUND

[0002] Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver metabolic disease in clinical practice, which mainly includes simple fatty liver, non-alcoholic steatohepatitis (NASH) and related cirrhosis. Epidemiological survey reports show that about one-fourth of the world's population suffers from NAFLD [1] , which has become an important reason threatening human life and health. Therefore, revealing the pathogenesis of NAFLD and finding potential intervention targets have become urgent problems to be solved. The formation of gametes in early life and the development of embryos are affected by adverse environmental factors, which can cause metabolic adaptation and developmental programming changes in offspring fetuses, thereby increasing the risk of multiple diseases in offspring after birth. In recent years, a large number of studies have confirmed that adverse lifestyle and environmental exposure of the father before pregnancy are important independent risk factors that lead to developmental retardation and increased risk of developmentally derived diseases in offspring. Epidemiological surveys have found that fathers experiencing famine can increase the risk of obesity and diabetes in offspring [2] . At the same time, clinical and laboratory studies have found that adverse environmental exposure (such as smoking, high-fat diet, exposure to exogenous substances, chronic stress, etc.) of the father before pregnancy can cause intrauterine growth retardation (IUGR) and susceptibility to multiple metabolic diseases such as lipid metabolism dysfunction and NAFLD in offspring after birth [3,4] . This indicates that adverse environmental factor exposure of the father before pregnancy is an important risk factor for increased risk of lipid metabolism dysfunction and NAFLD in offspring. However, there is currently no animal model of NAFLD in offspring caused by adverse environmental factor exposure of the father before pregnancy. Therefore, establishing a stable paternal NAFLD animal model is of great significance for in-depth exploration of the pathogenesis of paternal NAFLD, reasonable avoidance of adverse environmental exposure of the father before pregnancy, and guidance of eugenics and improvement of population quality.

[0003] Currently, although diet-induced NAFLD animal models are widely used in preclinical experimental research, these models can only reflect the influence of postnatal life habits on the occurrence of NAFLD, ignore the influence of genetic factors caused by paternal pre-pregnancy environmental exposure, and often only explore the changes of a single gender (male or female), and the research on NAFLD in different genders is relatively lagging behind. Therefore, existing research urgently needs to establish a paternal NAFLD rat model that induces high incidence of both male and female, which is highly similar to human NAFLD, to further explore its pathogenesis, early prevention and treatment, and thus benefit mankind.

[0004] Caffeine, as a central stimulant and chronic stressor widely exists in coffee, tea and various functional beverages. Studies have confirmed that caffeine has reproductive and developmental toxicity, and long-term intake of caffeine and / or caffeine-containing foods can reduce sperm motility and affect post-fertilization embryo development [5] . Therefore, in this study, the paternal pre-pregnancy caffeine gavage simulates caffeine exposure in human daily life to establish a paternal NAFLD animal model, which has the advantages of simple operation, high modeling rate and stability.

[0005] Epigenetic modifications (such as DNA methylation, non-coding RNA, etc.) are an important way to mediate the inheritance of acquired traits. Epigenetic changes caused by parental exposure to adverse environments can be transmitted to offspring through germ cell reprogramming and can be passed down to multiple generations. Studies have found that sperm non-coding RNA (such as miRNA) can act as an epigenetic information carrier to transmit paternal acquired phenotypes to offspring. Sperm miRNAs can mediate the intergenerational transmission of paternal acquired traits, such as diabetes, NAFLD, and hypercholesterolemia. In addition, paternal exposure to adverse environments (such as alcohol consumption, high-fat diet, etc.) can change the expression profile of sperm miRNAs (such as miR-21, miR-30, let-7c, etc.) and transmit them to offspring, thereby leading to changes in the expression of key genes involved in liver lipid metabolism and changes in lipid metabolism function [6-8] . This indicates that miRNAs, as carriers of paternal genetic information transmission and important markers of epigenetic modification, play an important role in the occurrence of paternal metabolic diseases and can be used as potential intervention targets for paternal NAFLD. The present invention successfully establishes a paternal NAFLD rat model for the first time, identifies miR-142-3p as an early intervention target for paternal NAFLD through bioinformatics analysis, screening and verification, and confirms that overexpression of miR-142-3p can effectively correct the paternal NAFLD phenomenon, which is of great significance for the development of therapeutic drugs and clinical prevention and treatment of paternal NAFLD.

[0006] Main references:

[0007] [1] Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease - Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 2016;64:73-84.

[0008] [2] Yan S, Hou W, Wu H, Jiang W, Li Y, Zhang Y, Li H, et al. Prenatal exposure to the Chinese famine and the risk of metabolic syndrome in adulthood across consecutive generations. Eur J Clin Nutr 2020;74:1229-1236.

[0009] [3] Chang RC, Thomas KN, Bedi YS, Golding MC. Programmed increases in LXRalpha induced by paternal alcohol use enhance offspring metabolic adaptation to high-fat diet induced obesity. Mol Metab 2019;30:161-172.

[0010] [4] De Jesus DF, Orime K, Kaminska D, Kimura T, Basile G, Wang CH, Haertle L, et al. Parental metabolic syndrome epigenetically reprograms offspring hepatic lipid metabolism in mice. J Clin Invest 2020;130:2391-2407.

[0011] [5] Jensen TK, Swan SH, Skakkebaek NE, Rasmussen S, Jorgensen N. Caffeine intake and semen quality in a population of 2,554 young Danish men. Am J Epidemiol 2010; 171: 883-891.

[0012] [6] Wu L, Lu Y, Jiao Y, Liu B, Li S, Li Y, Xing F, et al. Paternal psychological stress reprograms hepatic gluconeogenesis in offspring. Cell Metab 2016; 23: 735-743.

[0013] [7] T de Castro Barbosa T, Ingerslev LR, Alm PS, Versteyhe S, Massart J, Rasmussen M, Donkin I, et al. High-fat diet reprograms the epigenome of rat spermatozoa and transgenerationally affects metabolism of the offspring. Mol Metab 2016; 5: 184-197.

[0014] [8] Rodgers AB, Morgan CP, Leu NA, Bale TL. Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress. Proc Natl Acad Sci U S A 2015; 112: 13699-13704. SUMMARY

[0015] The technical problem solved by the present application is to provide a method for constructing a parent-derived non-alcoholic fatty liver disease (NAFLD) animal model with high success rate, effectiveness, reliability, repeatability, and simplicity.

[0016] To solve the above technical problems, the technical solutions of the present application are as follows:

[0017] In a first aspect, the present application provides a method for constructing a paternal non-alcoholic fatty liver disease animal model, characterized by comprising the following steps:

[0018] S1: selecting healthy male rodents, and intragastrically perfusing the rodents with 15, 30 and 60 mg / kg of caffeine every day, and allowing the male rodents to freely eat and drink;

[0019] S2: mating the male rodents with normal female rodents after intragastrically perfusing the male rodents with different doses of caffeine every day for 8 consecutive weeks;

[0020] S3: taking the fetal rodent livers from part of the pregnant rodents at 20 days of pregnancy, and allowing part of the pregnant rodents to naturally give birth to F1 offspring, taking the birth day of the offspring as postnatal day 0, selecting litters with 12-14 offspring at postnatal day 1, adjusting the number of male and female offspring in each litter to 6, and feeding the offspring, weaning the offspring at 4 weeks of age, and separating the male and female offspring, and continuing to normally feed part of the male offspring until the age of (8 / 32) weeks to take blood and livers;

[0021] S4: after the above steps are completed, detecting liver metabolism-related indexes of the offspring at different time points (20 days of pregnancy and 32 weeks of postnatal age) to comprehensively determine NAFLD, and finally obtaining a paternal NAFLD animal model.

[0022] As a preferred solution, in the step S1, the rodents are rats or mice.

[0023] Further, in the step S3, the normal diet is the same as the formula feed for mice and rats specified in the National Standard of the People's Republic of China GB14924.3-2001.

[0024] Further, in the step S4, the liver metabolism change detection-related indexes are the activities of serum glutathione and glutamic-oxaloacetic transaminase, liver triglyceride content, hematoxylin-eosin staining, oil red O staining, Masson staining and Sirius red staining.

[0025] In a second aspect, the present application provides an application of a paternal non-alcoholic fatty liver disease animal model in screening anti-paternal pre-pregnancy environmental interference substances or drugs, characterized in that the paternal NAFLD animal model is obtained by any of the above construction methods.

[0026] In a third aspect, the present application provides an application of a paternal non-alcoholic fatty liver disease animal model in screening a paternal non-alcoholic fatty liver disease early intervention target, characterized in that the paternal NAFLD animal model is obtained by any of the above construction methods.

[0027] In a fourth aspect, the present application provides a paternal non-alcoholic fatty liver disease early intervention target, and the intervention target is miR-142-3p.

[0028] In a fifth aspect, the present application provides a use of a paternal non-alcoholic fatty liver disease animal model in screening / preparing a therapeutic preparation for preventing and treating non-alcoholic fatty liver disease, characterized in that the paternal NAFLD animal model is obtained by any of the above methods.

[0029] Preferably, the paternal NAFLD therapeutic preparation comprises miR-142-3p adeno-associated virus, miR-142-3p lentivirus, etc.

[0030] The technical principle and research process of the present application are as follows:

[0031] In the present application, male rodents (such as Wistar rats) are given intragastric perfusion of caffeine (15, 30, 60 mg / kg) every day for 8 consecutive weeks, and then impregnated with female rats to obtain offspring, so as to establish a paternal pre-pregnancy caffeine exposure model. By detecting the activities of serum glutathione and glutamic-oxaloacetic transaminase of the offspring of paternal pre-pregnancy caffeine exposure, and observing liver hematoxylin-eosin staining, oil red O staining, Masson staining and Sirius red staining, a paternal NAFLD animal model is established. The model simulates the disease phenotype of the development of paternal NAFLD, and is of great significance for elucidating the mechanism of the development of paternal NAFLD and determining early intervention targets.

[0032] The advantages and beneficial effects of the present application are as follows:

[0033] 1. The present application is novel and has great practical significance. Caffeine, as a central stimulant widely existing in coffee, tea, soft drinks and some compound drugs, is widely used in daily life. Paternal pre-pregnancy caffeine exposure is a common social phenomenon. Therefore, the present application establishes a rat NAFLD model caused by paternal pre-pregnancy caffeine exposure, which is novel and can reflect the current situation of daily life, and has positive practical significance.

[0034] 2. The modeling method of the present application is simple, the detection indexes are stable and reliable, and the repeatability is strong. Different doses of caffeine are given to the paternal pre-pregnancy by intragastric perfusion, the offspring are weaned at 4 weeks after birth, and are fed with standard feed for 32 weeks. The increase of the activities of serum glutathione and glutamic-oxaloacetic transaminase, the content of liver triglyceride, and the histological staining (hematoxylin-eosin staining, oil red O staining, Masson staining and Sirius red staining) of liver tissue can simulate the characteristics of liver lipid metabolism function changes in NAFLD patients. The present model provides a reliable method for the construction of a paternal NAFLD animal model.

[0035] 3. The parent-derived NAFLD animal model constructed based on the application can be used for guiding the parent pre-pregnancy health life, exploring the mechanism of parent-derived NAFLD, and determining that miR-142-3p can be used as an early intervention target of parent-derived NAFLD. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 . Effect of PPCE of parent pre-pregnancy caffeine exposure on pre-and post-natal liver pathological changes and TG metabolism of male offspring.

[0037] Figure 1 Medium: (A): H&E staining and oil red O staining of GD20 male fetal liver; (B): GD20 male fetal liver steatosis score; (C): TG content of GD20 male fetal liver; (D, E): AST and ALT activity of PW32 male offspring serum; (F): Appearance and morphology of liver of PW32 male offspring; (G, H): Liver weight and liver index of PW32 male offspring; (I): H&E staining, oil red O staining, Masson staining and Sirius red staining of liver of PW32 male offspring; (J-N): NAS score and fibrosis score of liver of PW32 male offspring; O: TG content of liver tissue of PW32 male offspring. Compared with the control group CON, * P<0.05, ** P<0.01. TG: triglyceride; GD20: 20 days of pregnancy; H&E: hematoxylin-eosin; PW32: 32 weeks of postnatal age; AST: glutamic-oxaloacetic transaminase; ALT: glutamic-pyruvic transaminase; NAS: NAFLD activity score.

[0038] Figure 2 . Effect of PPCE of parent pre-pregnancy caffeine exposure on pre-and post-natal liver pathological changes and TG metabolism of female offspring.

[0039] Figure 2 Medium: (A): H&E staining and oil red O staining of GD20 female fetal liver; (B): TG content of GD20 female fetal liver; (C): H&E staining and oil red O staining of liver of PW32 female offspring; (D): TG content of liver tissue of PW32 female offspring. Compared with the control group CON, * P<0.05, ** P<0.01. TG: triglyceride; GD20: 20 days of pregnancy; H&E: hematoxylin-eosin; PW32: 32 weeks of postnatal age.

[0040] Figure 3 . Effect of PPCE of parent pre-pregnancy caffeine exposure on pre-and post-natal liver pathological changes and TG metabolism of female offspring.

[0041] Figure 3Fig. 6. Effects of PPCE on the expression of key genes and proteins involved in lipid metabolism in the GD20 male fetal livers. (A-C): Sequencing results of GD20 male fetal liver transcriptome; (D): Statistical chart of the expression levels of key genes involved in lipid metabolism in the GD20 male fetal livers; (E): Western blotting chart of the expression of key proteins involved in lipid metabolism in the GD20 male fetal livers; (F): Statistical chart of the expression levels of key proteins involved in lipid metabolism in the GD20 male fetal livers; (G): Statistical chart of the expression levels of key genes involved in lipid metabolism in the PW32 male fetal livers; (H): Western blotting chart of the expression of key proteins involved in lipid metabolism in the PW32 male fetal livers; (I): Statistical chart of the expression levels of key proteins involved in lipid metabolism in the PW32 male fetal livers. Compared with the control group CON, * P < 0.05, ** P < 0.01. GD20: 20 days of gestation; PW32: 32 weeks after birth.

[0042] Figure 4 Fig. 7. Sequencing screening of potential toxic targets of PPCE for the occurrence of NAFLD in male offspring rats.

[0043] Figure 4 Fig. 8. Effects of PPCE on the expression of differentially expressed miRNAs in the paternal sperm and fetal livers. (A): Heat map of differentially expressed miRNAs in the paternal sperm and fetal livers; (B): Wayne chart of differentially expressed miRNAs in the paternal sperm and fetal livers; (C): Expression of miRNAs in the GD20 offspring livers; (D): Expression level of miR-142-3p in the whole body of fetal rats detected by FISH; (E): Differentially expressed miRNAs in the PW32 offspring livers; (F, G): Expression levels of miR-142-3p in the GD20 and PW32 offspring livers. Compared with the control group CON, * P < 0.05, ** P < 0.01. NAFLD: non-alcoholic fatty liver disease; GD20: 20 days of gestation; FISH: fluorescence in situ hybridization; PW32: 32 weeks after birth.

[0044] Figure 5 Fig. 9. Effects of PPCE on the miR-142-3p / ACSL4 signaling pathway in the pre- and postnatal livers of male offspring.

[0045] Figure 5 Fig. 10. Effects of PPCE on the expression of miR-142-3p and target gene ACSL4. (A): Binding diagram of miR-142-3p and target gene ACSL4; (B): Expression level of ACSL4 mRNA in the GD20 male fetal livers; (C): Expression level of ACSL4 protein in the GD20 male offspring livers; (D): Statistical chart of the expression level of ACSL4 protein in the GD20 male offspring livers; (E): Expression level of ACSL4 mRNA in the GD20 male fetal livers; (F): Expression level of ACSL4 protein in the GD20 male offspring livers; (G): Statistical chart of the expression level of ACSL4 protein in the GD20 male offspring livers. Compared with the control group CON, *P<0.05, ** P<0.01. ACSL4: Acyl-CoA synthetase 4; GD20: 20 days of gestation; PW32: 32 weeks of age after birth.

[0046] Figure 6 Effects of miR-142-3p on lipid metabolism function of rat bone marrow mesenchymal stem cells (BMSCs) differentiated into hepatoid cells.

[0047] Figure 6 (A): Expression levels of genes related to lipid synthesis (ACSL4, SREBP1, FASN) and β-oxidation PPARα, CPT1α) in hepatoid differentiated BMSCs; (B): Expression levels of proteins related to lipid synthesis (ACSL4, SREBP1, FASN) and β-oxidation PPARα, CPT1α) in hepatoid differentiated BMSCs; (C): Statistical graph of lipid synthesis and β-oxidation-related gene expression levels in hepatoid differentiated BMSCs. Compared with the control group CON, * P<0.05, ** P<0.01. ACSL4: Acyl-CoA synthase 4; SREBP1: Sterol regulatory element-binding protein 1; FASN: Fatty acid synthase; PPARα: Peroxisome proliferator-activated receptor α; CPT1α: Carnitine palmitoyltransferase 1α.

[0048] Figure 7 miR-142-3p targets ACSL4 to regulate lipid metabolism in rat bone marrow mesenchymal stem cells (BMSCs) that differentiate into liver-like cells.

[0049] Figure 7 (A): Expression levels of genes related to lipid synthesis in hepatoid differentiated BMSCs, including ACSL4, SREBP1, FASN, and β-oxidized PPARα, CPT1α; (B, C): Expression levels of proteins related to lipid synthesis in hepatoid differentiated BMSCs, including ACSL4, SREBP1, FASN, and β-oxidized PPARα, CPT1α; (D): Immunofluorescence staining of proteins related to lipid synthesis in hepatoid differentiated BMSCs, including ACSL4, SREBP1, FASN, and β-oxidized PPARα, CPT1α; (E): Results of the miR-142-3p and ACSL4 dual-luciferase reporter gene assay. Compared with the control group CON, * P<0.05, ** P<0.01; compared with the PPCE group, # P<0.05, ##P<0.01. ACSL4: acyl-CoA synthetase 4; SREBP1: sterol regulatory element binding protein 1; FASN: fatty acid synthase; PPARa: peroxisome proliferator-activated receptor a; CPT1a: carnitine palmitoyltransferase 1a.

[0050] Figure 8 . Effects of liver overexpression of miR-142-3p on the occurrence of NAFLD in male offspring of paternal pre-pregnancy caffeine exposure PPCE.

[0051] Figure 8 Medium: (A): mRNA expression levels of liver lipid metabolism function genes in PW32 male offspring; (B): H&E staining and oil red O staining of liver in PW32 male offspring; (C): liver tissue score of PW32 male offspring; (D): TG content of liver tissue in PW32 male offspring. Compared with the control group CON, * P<0.05, ** P<0.01; compared with the PPCE group, # P<0.05, ## P<0.01. NAFLD: non-alcoholic fatty liver disease; PW32: 32 weeks of age after birth; H&E: hematoxylin-eosin; TG: triglyceride.

[0052] Figure 9 . Corticosterone but not caffeine mediates the high methylation and low expression of the miR-142-3p promoter region in spermatogonia.

[0053] Figure 9 Medium: (A): serum corticosterone content; (B): mRNA expression level of testicular GR; (C): methylation level of sperm miR-142-3p promoter region; (D, E): effects of caffeine and corticosterone on the mRNA expression of miR-142-3p in spermatogonia; (F, G): effects of corticosterone on the methylation of the miR-142-3p promoter region in spermatogonia. Compared with the control group CON, * P<0.05, ** P<0.01. GR: glucocorticoid receptor.

[0054] Figure 10 . RU486 intervention reverses the liver lipid metabolism disorder in male offspring rats before and after birth caused by paternal pre-pregnancy caffeine exposure PPCE.

[0055] Figure 10In: (A, B): Methylation level of miR-142-3p in the liver of GD20 offspring; (C, D): Expression levels of miR-142-3p, Acsl4, Srebp1, Fasn, Pparα, and Cpt1α mRNAs in the liver of GD20 offspring; (E, F): Expression levels of miR-142-3p, Acsl4, Srebp1, Fasn, Pparα, and Cpt1α mRNAs in the liver of PW32 offspring; (G): H&E and ORO staining at 400×; (H, I): TG content in the livers of GD20 and PW32 offspring. Compared with the control group CON, * P < 0.05, ** P < 0.01; compared with the PPCE group, # P < 0.05, ## P < 0.01. GD20: 20 days of gestation; Acsl4: acyl-CoA long-chain family member 4; Srebp1: sterol regulatory element-binding protein 1; Fasn: fatty acid synthase; Pparα: peroxisome proliferator-activated receptor α; Cpt1α: carnitine palmitoyltransferase 1α; PW32: 32 weeks after birth; H&E: hematoxylin-eosin; ORO: oil red O; TG: triglyceride. Detailed implementation manners

[0056] The technical content of the present invention will be further elaborated in detail below in combination with specific embodiments and drawings.

[0057]

Example 1

[0058] 1 Experimental animals

[0059] SPF-grade healthy Wistar rats were purchased from the Hubei Provincial Center for Disease Control and Prevention, and the animal license number was: SCXK(E)20200 - 2022. This study was approved by the Ethics Committee of the Medical Department of Wuhan University and was strictly carried out in accordance with the relevant treatment guidelines of the international experimental animal protection certification and evaluation agency.

[0060] The experimental animals were housed in a barrier environment at a temperature of 22 - 25°C, a humidity of 50%, and a 12-hour day-night cycle.

[0061] 2 Experimental methods

[0062] Male Wistar rats 60 (body weight 260-300g) after 2 weeks of adaptive feeding were randomly divided into control group, caffeine group. Caffeine group rats were given different doses of caffeine (15, 30, 60mg / kg) intragastric perfusion, the control group was given equal volume of normal saline intragastric perfusion. After 8 weeks of continuous administration, according to the male: female = 1: 2, the next morning, vaginal smear, determine the pregnant rats, recorded as pregnant 0 days. The rats in each group were free to normal diet. Feed purchased from Wuhan City Wanqianjiaxing Biological Technology Co., Ltd., license number: SCXK (E) 2011-0011. The feed formula is the same as the mouse and rat formula feed stipulated in the "People's Republic of China National Standard GB14924.3-2001".

[0063] The mother mouse naturally produces F1 generation, and the production day is taken as the 0th day after birth. On the 1st day after birth, 12-14 pups are selected from each group, and the male and female pups are adjusted to 6 each for lactation feeding to ensure balanced nutrition of the pups. The pups are weaned at 4 weeks after birth and separated by sex. 12 normal diet-fed rats are randomly selected from each group and fed until 12 weeks after birth. On the 2nd day after the completion of the above experiment, the animals are sacrificed under anesthesia.

[0064] 3. Test index and method

[0065] 3.1 Serum glutathione transaminase and glutathione transaminase activity detection

[0066] The glutathione transaminase and glutathione transaminase activity detection kit (item numbers: C009-2-1, C010-2-1, Nanjing Jiancheng) was used to detect the activity of glutathione transaminase and glutathione transaminase in the serum of each group of rats. After taking the serum sample from the-80℃ refrigerator, it was thawed on ice and mixed evenly. According to the operation steps in the kit instruction manual, the sample and the corresponding detection working solution were added in the 96-well plate, and the standard sample well and the blank well were set up, and mixed evenly. After the reaction, the absorbance value of each well at 510nm was measured by enzyme label instrument, and the activity of glutathione transaminase and glutathione transaminase was calculated by checking the standard curve.

[0067] 3.2 Triglyceride content detection

[0068] The content of triglyceride in the liver of each group of rats was detected by using a triglyceride content detection kit (A110-1, Nanjing Jiancheng). 50 mg of fresh liver tissue was added to physiological saline at a volume ratio of 1:9, homogenized under ice bath conditions, centrifuged at 2500 rpm for 10 min, and the supernatant was taken for detection. First, the content of triglyceride in the liver tissue homogenate of each group of rats was detected by using a triglyceride content detection kit, then the protein concentration in the liver tissue homogenate of each group of rats was detected by using a BCA protein concentration detection kit (P0012, Shanghai Biyun Tian), and finally the content of triglyceride corresponding to each g of protein in the liver tissue homogenate was used as the content of triglyceride in the liver tissue. That is, the content of triglyceride in the liver is mmol / g of protein.

[0069] 3.3 Hematoxylin-eosin staining

[0070] The liver tissue fixed by 10% formalin was embedded in a paraffin embedding machine after dehydration; the embedded tissue wax block was cut into 5 μm thin sections to prepare liver tissue sections; the sections were soaked in xylene solution for 5 min twice for deparaffinization; the sections were sequentially soaked in 100%, 95%, 85%, and 75% ethanol for 5 min, and washed with distilled water; the sections were soaked in hematoxylin-eosin staining solution for 30 s, washed with distilled water, then soaked in 1% hydrochloric acid alcohol and quickly taken out, and washed with distilled water; the sections were sequentially soaked in 75%, 85%, 95%, and 100% ethanol for 5 min, placed in a fume hood to dry the residual ethanol, then soaked in xylene solution for 5 min, and placed in a fume hood to dry the residual xylene; an appropriate amount of neutral gum was added for sealing; and the staining result was observed under a microscope.

[0071] 3.4 Oil red O staining

[0072] The liver tissue fixed by 10% formalin was cut into 6-8 μm thin sections by freezing sectioning, attached to a glass slide, and immediately fixed in 10% paraformaldehyde for 3 min, washed with distilled water, and stored at -20°C. When used, the frozen sections were taken out, placed at room temperature for 10 min, and soaked in 60% isopropanol solution for 2 min; the sections were stained in oil red O staining solution for 10 min, washed with 60% isopropanol solution to remove excess staining solution, and washed with distilled water; stained with hematoxylin solution for 30 s, and washed with distilled water; soaked in 1% hydrochloric acid alcohol and quickly taken out, and washed with distilled water; an appropriate amount of neutral gum was added for sealing; and the staining result was observed under a microscope.

[0073] 3.5 Masson staining

[0074] The liver tissue fixed by 10% formalin was dehydrated and embedded in a paraffin embedding machine. The embedded tissue wax block was cut into 5 μm thin sections to prepare liver tissue sections. The paraffin sections were immersed in xylene solution for 5 min x 2 times for deparaffinization. The sections were sequentially immersed in 100%, 95%, 85%, and 75% ethanol for 5 min, and cleaned with distilled water. Then, the sections were immersed in hematoxylin staining solution for 5 min, washed with distilled water, and then immersed in Masson staining solution for 5 min. Then, the sections were immersed in differentiation solution for 3 min. The sections were sequentially immersed in 75%, 85%, 95%, and 100% ethanol for 5 min, and then placed in a fume hood to evaporate the residual ethanol. Then, the sections were immersed in xylene solution for 5 min, and then placed in a fume hood to evaporate the residual xylene. An appropriate amount of neutral balsam was added for sealing. The staining results were observed under a microscope.

[0075] 3.6 Sirius red staining

[0076] The liver tissue fixed by 10% formalin was dehydrated and embedded in a paraffin embedding machine. The embedded tissue wax block was cut into 5 μm thin sections to prepare liver tissue sections. The paraffin sections were immersed in xylene solution for 5 min x 2 times for deparaffinization. The sections were sequentially immersed in 100%, 95%, 85%, and 75% ethanol for 5 min, and cleaned with distilled water. Then, the sections were immersed in hematoxylin staining solution for 5 min, washed with distilled water, and then immersed in Masson staining solution for 5 min. Then, the sections were immersed in differentiation solution for 3 min. The sections were sequentially immersed in 75%, 85%, 95%, and 100% ethanol for 5 min, and then placed in a fume hood to evaporate the residual ethanol. Then, the sections were immersed in xylene solution for 5 min, and then placed in a fume hood to evaporate the residual xylene. An appropriate amount of neutral balsam was added for sealing. The staining results were observed under a microscope.

[0077] 3.7 Real-time fluorescent quantitative PCR experiment

[0078] The RNA in the liver tissue of each group of mice was extracted by using Trizol reagent, and cDNA was obtained after reverse transcription. The cDNA, upper and lower primers of each gene, SYBR Green I fluorescent dye Mix, and other reagents were uniformly mixed and placed in an RT-qPCR instrument for reaction. The RT-qPCR reaction conditions were as follows (the reaction system was 10 μL): 95°C pre-denaturation for 2 min; 95°C denaturation for 10 s, 62°C annealing for 30 s; 72°C extension for 15 s, a total of 40 cycles. GAPDH was used as an internal reference, and the relative expression amount of the target gene mRNA was calculated by using 2 -△△Ct Method.

[0079] 3.8 Western blot experiment

[0080] Liver tissue protein extraction: Approximately 50 mg of liver tissue was collected from each group of rats and placed in a 1.5 mL EP tube. 400 μL of protein lysis buffer (a mixture of mammalian protein extraction reagent, phosphatase inhibitor, and protease inhibitor at a volume ratio of 98:1:1) was added. The liver tissue was thoroughly lysed using an ultrasonic homogenizer and then placed on ice for 30 min. The mixture was then centrifuged at 12000 rpm at 4℃ for 15 min. The supernatant was transferred to another EP tube to obtain the liver tissue protein extract. An appropriate amount of the protein extract was diluted with distilled water, and the protein concentration was determined according to the BCA protein content assay method. The remaining liver tissue protein extract was added to 5× protein loading buffer at a volume ratio of 4:1 and denatured at 95℃ for 5 min to obtain the protein samples for each group. After cooling at room temperature, the samples were stored at -20℃ for later use.

[0081] Western Blotting: Separating and stacking gels were prepared according to the SDS-PAGE gel kit instructions and incubated at room temperature for 30–40 min. 100 μg of protein samples from each group were taken for electrophoresis. After electrophoresis, the protein samples from the SDS-PAGE gel were transferred to a PVDF (0.45 μm) membrane using a semi-dry method at 25 V / 1.0 A for 25 min. The PVDF membrane containing the protein samples was blocked with 5% skim milk for 1 h. The target gene antibody was diluted with 5% BSA solution according to the primary antibody dilution ratio and incubated overnight at 4°C, followed by washing with 1×TBST for 10 min × 3 times. The membrane was then incubated with secondary antibody at room temperature for 1 h, followed by washing with 1×TBST for 10 min × 3 times. After ECL luminescence development, bands were obtained using a G:BOX imaging system, with β-actin as an internal control.

[0082] 4 Experimental Results

[0083] 4.1 Effects of PPCE on prenatal and postnatal liver pathological changes and TG metabolism in male offspring

[0084] The results are as follows Figure 1 As shown. H&E and ORO staining results showed that, compared with the control group, the liver pathology of the PPCE group at GD20 showed obvious fatty vacuolar degeneration, fatty degeneration, and lipid accumulation. Figure 1 (A). Meanwhile, the PPCE group's liver steatosis score ( Figure 1 B) and TG content ( Figure 1 Serum C levels were significantly elevated. At PW32, serum liver function enzyme (AST, ALT) activities were significantly elevated in the PPCE group. Figure 1 (D, E) The liver appears light brown, and liver weight and liver index are significantly elevated. Figure 1(FH). H&E, ORO, Masson, and Sirius red staining results showed that, compared with the control group, the livers of male PPCE offspring rats at PW32 exhibited typical NAFLD histological features, including marked vacuolar steatosis, inflammatory infiltration (indicated by green arrows), and collagen fiber deposition (indicated by black and red arrows). Figure 1 (I), liver NAS score ( Figure 1 The contents of JN and TG were significantly increased ( Figure 1 (O). Experimental results show that PPCE can cause pathological changes in the liver before and after birth in male offspring rats, and lead to NAFLD in adulthood.

[0085] 4.2 Effects of PPCE on prenatal and postnatal liver pathological changes and TG metabolism in female offspring

[0086] The results are as follows Figure 2 As shown. Compared with the control group, PPCE can cause obvious fat vacuolation in some hepatocytes of the fetal liver of female offspring, and Oil Red O staining shows obvious lipid accumulation. Figure 2 In the middle A), the fetal liver triglyceride content was significantly increased ( Figure 2 (B) Compared with the control group, the livers of female PW32 offspring of PPCE showed extensive vacuolar steatosis with excessive lipid deposition. Figure 2 In the middle C), the liver triglyceride content was significantly increased ( Figure 2 (D). The experimental results show that PPCE can cause pathological changes in the liver before and after birth in female offspring rats, and lead to NAFLD in adulthood. However, it is worth noting that the degree of pathological changes in the liver of female offspring is significantly milder than that of male offspring, showing a sex difference.

[0087] 4.3 Effects of PPCE on changes in hepatic lipid metabolism function before and after birth in male offspring

[0088] The results are as follows Figure 3 As shown. Transcriptome sequencing results revealed that PPCE caused significant changes in the expression of numerous genes in the fetal liver of male offspring, among which the differentially expressed genes were closely related to lipid metabolism. Figure 3 (AC). RT-qPCR and Western blot results showed that, compared with the control group, PPCE significantly increased the mRNA and protein expression levels of fatty acid synthesis-related genes and significantly decreased the level of β-oxidation-related proteins in the fetal liver of male offspring. Figure 3 (D-DF); PPCE significantly increased the mRNA and protein expression levels of genes related to fatty acid synthesis in the liver of PW32 male offspring, and significantly decreased the level of β-oxidation-related proteins. Figure 3 (Medium GI). Experimental results show that PPCE can cause changes in liver lipid metabolism function in male offspring before and after birth, mainly manifested as enhanced fatty acid synthesis and weakened β oxidation.

[0089] In summary, the method of the present application is to give male rats pre-pregnancy caffeine for 8 weeks, and the offspring are weaned at 4 weeks after birth and continue to be fed with regular diet until 32 weeks. It is found that the male and female offspring of the father pre-pregnancy caffeine exposure male rats all have typical manifestations of NAFLD, which indicates that the father-derived non-alcoholic fatty liver disease (NAFLD) model is successfully established. The modeling method of the present application is simple, and the serum liver function enzyme activity of the model rats is increased, the liver triglyceride content is significantly increased, the liver fatty degeneration is obvious, and the lipid metabolism function gene expression is changed, which indicates that the modeling method of the present application is stable, effective, reliable and highly reproducible.

[0090]

Example 2

[0091] 1 Experimental animals

[0092] SPF healthy Wistar rats were purchased from Hubei Provincial Center for Disease Control and Prevention, animal license number: SCXK (E) 2020-2022. This study was approved by the Ethics Committee of Wuhan University Medical School, and was strictly in accordance with the relevant handling guidelines of the International Experimental Animal Protection Certification Evaluation Agency. Experimental animals were raised in a barrier environment with a temperature of 22-25℃, humidity of 50%, and 12h of alternating day and night.

[0093] 2 Experimental animal treatment

[0094] After 2 weeks of adaptive feeding, 30 male Wistar rats (body weight 260-300g) were randomly divided into control group, caffeine group (15, 30, 60mg / kg·d). The rats in the caffeine group were given different doses of caffeine by intragastric perfusion every day, and the rats in the control group were given the same volume of normal saline by intragastric perfusion. After 8 weeks of continuous administration, normal female Wistar rats were caged according to the ratio of male to female 1:2, and the next morning vaginal smears were taken to determine the pregnant rats, which were recorded as pregnant 0 days. The rats in each group were fed with normal diet. The feed was purchased from Wuhan Wanqianjiaxing Biological Technology Co., Ltd., license number: SCXK (E) 2011-0011. The feed formula is the same as the mouse and rat formula feed stipulated in the "National Standard of the People's Republic of China GB14924.3-2001".

[0095] Sperm samples of the parent rats were collected. Part of the pregnant 20-day female mice were sacrificed after anesthesia, and the male offspring fetal rat liver samples were collected. Part of the female mice naturally gave birth to F1 generation, and the day of birth was taken as the 0th day after birth. On the 1st day after birth, 12-14 pups were selected from each group, and the male and female pups were adjusted to 6 each for lactation feeding to ensure balanced nutrition. The pups were weaned at 4 weeks after birth and separated by sex, and 12 normal diet-fed rats were randomly selected from each group and fed until 32 weeks after birth. Then the animals were sacrificed after anesthesia, and the 32-week-old male offspring liver samples were collected.

[0096] 3 Rat bone marrow mesenchymal stem cells (BMSCs) extraction, culture, hepatocyte-like cell differentiation and transfection

[0097] 3.1 Extraction of BMSCs

[0098] (1) The ultraclean bench and the cell laboratory environment were sterilized by ultraviolet irradiation for more than 30 min, and the reagents and consumables required for the extraction and culture of BMSCs cells were placed in the ultraclean bench and sterilized by ultraviolet irradiation for more than 30 min.

[0099] (2) The 3-week-old Wistar male rats were sacrificed by cervical dislocation, and then soaked in 75% ethanol for 20 min.

[0100] (3) The bilateral femurs and tibias of Wistar male rats were separated in the ultraclean bench, and the muscles, fasciae and other tissues attached to the bone surface were removed, and then washed with PBS and removed the dry marrow end of the bilateral femurs or tibias.

[0101] (4) The syringe was used to suck the α-MEM culture medium without serum to fully flush the bone marrow cavity of the femur and tibia, and repeated 3-5 times to ensure sufficient cells.

[0102] (5) The cell suspension containing cells was mixed uniformly by gentle blowing, so that it became a uniform liquid without large pieces of bone marrow, and then filtered with a cell screen with a pore size of 70 μm.

[0103] (6) The filtered cell suspension was centrifuged at 1500 rpm at room temperature for 5 min, and then the supernatant was discarded, and the lower cell precipitate was resuspended with α-MEM complete medium containing 10% FBS, 100 mg / mL streptomycin, and 100 U / mL penicillin (hereinafter referred to as "culture medium").

[0104] (7) The cell number of the cell suspension was counted by using a cell counter, and then the cell density was adjusted to about 1×10 6 cells / mL, and inoculated in a T25 cell culture bottle, and cultured in a constant temperature cell incubator at 5% CO2, 37°C, and the growth state of the cells was observed every day.

[0105] 3.2 Passage and plating of BMSCs

[0106] (1) The growth state of BMSCs was observed under a microscope, and when the adherent cells grew to about 90% density, the culture medium in the culture bottle was discarded.

[0107] (2) After washing with PBS for 3 times, add 1 mL trypsin digestion solution, and put into the cell culture box for 1-2 min. When the cells are observed to be detached under the microscope, add 3 mL complete medium to terminate the digestion.

[0108] (3) Absorb the medium and blow the cells gently. Then transfer the cell medium to a centrifuge tube and centrifuge at 1000 rpm for 5 min.

[0109] (4) After centrifugation, discard the cell supernatant and add 1 mL complete medium to resuspend the cell precipitate.

[0110] (5) Seed the cell suspension in the culture plate or subculture in the culture bottle at a density of 1 x 10 6 cells / mL.

[0111] 3.3 BMSCs hepatocyte-like cell differentiation

[0112] When the cell density in the culture plate reaches about 80%, replace the medium with IMEM medium containing 1% FBS, 100 U / mL streptomycin, 100 U / mL penicillin, 20 ng / mL hepatocyte growth factor (HGF), 2 ng / mL epidermal growth factor (EGF), 50 nmol / L dexamethasone, and 50 mg / mL insulin transferrin-sodium selenite (ITS) (hepatocyte-like cell differentiation medium) and continue to culture for 14 days. During this period, observe the growth state every day, and replace the differentiation medium every 3-4 days.

[0113] 3.4 Transfection of BMSCs hepatocyte-like differentiated cells

[0114] Rat-miR-142-3p inhibitor, Rat-miR-142-3p mimics, microRNA inhibitor NC, microRNA mimics NC and Rat-ACSL4 overexpression plasmid were synthesized by GenScript. Before use, the dry powder siRNA was centrifuged, and 250 μL DEPC water was added to each 1 OD microRNA inhibitor or mimics according to the instructions to make a 20 μM solution, which was stored at -20°C. The transfection steps were as follows: first, add 200 μL serum-free opti-DMEM, 10 μL Rat-miR-142-3p inhibitor or microRNA inhibitor NC (final concentration 200 pM) to a sterile RNase-free EP tube, tube ①; then take another sterile EP tube, add 200 μL serum-free opti-DMEM and 5 μL Lipo3000, tube ②. Mix gently respectively and stand for 5 min, then slowly drop tube ② into tube ①, stand at room temperature for 20 min. Replace the culture medium in the six-well plate with fresh serum-free differentiation medium (1.6 mL of culture medium per well), then add 400 μL of the mixture of tube ① and tube ② to the cell wells. Shake the culture plate gently to mix the liquid thoroughly, then place it in the cell culture incubator for 24 h before replacing the culture medium or performing subsequent related experiments. ACSL4 overexpression plasmid transfection was performed according to the same method as above.

[0115] 4. Test index and method

[0116] 4.1 miRNAs sequencing analysis

[0117] (1) Sample preparation: about 100 mg of fetal liver tissue was taken and placed in a centrifuge tube, and stored at -80°C for standby use. Rat sperm samples were taken from the rat epididymis and placed in a centrifuge tube, and stored at -80°C for standby use.

[0118] (2) Quality control detection: total RNA was extracted from the sample, and the concentration, purity and integrity of the total RNA were detected to ensure that the sample quality was qualified.

[0119] (3) RNA library construction: mRNA of eukaryotes was enriched by magnetic beads with Oligo-dT, and the first cDNA strand was synthesized with mRNA as template, followed by the addition of buffer, dNTPs, enzyme-free water and DNA synthesis enzyme and other reagents to synthesize the second cDNA strand. Then, after cDNA purification and end repair, the cDNA library was obtained by RT-qPCR method.

[0120] (4) RNA library quality control: After the construction of the cDNA library, the concentration and insert size of the library were detected, and finally the effective concentration of the library was accurately quantified by RT-qPCR method.

[0121] (5) Sequencing analysis: After the RNA library quality control detection is qualified, NovaSeq 6000 system is used for high-throughput sequencing.

[0122] (6) Subsequently, the high-throughput sequencing data is processed and analyzed, including basic analysis (sequencing data quality control, sequence comparison analysis, transcript assembly), miRNAs identification and prediction, expression analysis (expression details, expression distribution, sample relationship analysis), expression difference analysis (expression difference details, difference difference statistics, expression difference visualization analysis), miRNAs target gene prediction, gene set analysis (Venn analysis, clustering analysis, functional annotation analysis, functional enrichment analysis, expression correlation analysis) and the like.

[0123] 4.2 Real-time fluorescent quantitative PCR experiment

[0124] The RNA in the liver tissue of each group of mice was extracted by using Trizol reagent, and the cDNA was obtained after reverse transcription. The cDNA, each gene upstream and downstream primer, labeled SYBR Green I fluorescent dye Mix and other reagents were mixed uniformly and placed in the RT-qPCR instrument for reaction. The RT-qPCR reaction conditions are as follows (the reaction system is 10 μL): 95 ℃ pre-denaturation for 2 min; 95 ℃ denaturation for 10 s, 62 ℃ annealing for 30 s; 72 ℃ extension for 15 s, a total of 40 cycles. GAPDH was used as an internal reference, and the relative expression amount of the target gene mRNA was calculated by using 2 -△△Ct Method. The primer sequences are as follows in Table 1:

[0125] Table 1. Primer sequence.

[0126]

[0127] 4.3 Western blot experiment

[0128] Liver tissue protein extraction: Take the liver tissue of each group of rats, cut about 50 mg of liver tissue into a 1.5 mL EP tube, add 400 μL of protein lysate (mammalian protein extraction reagent, phosphatase inhibitor mixture, proteinase inhibitor mixture mixed in a volume ratio of 98:1:1), and use an ultrasonic crusher to fully crush the liver tissue and then place it on ice for lysis for 30 min. Centrifuge at 12000 r / min, 4°C for 15 min, and take the supernatant and place it in another EP tube to obtain the liver tissue protein extract. Take an appropriate amount of protein extract and dilute it with distilled water, then measure the protein concentration according to the BCA protein content determination method. The remaining liver tissue protein extract is added to 5x protein loading buffer at a volume ratio of 4:1, denatured at 95°C for 5 min, and then cooled at room temperature, and stored at -20°C for use.

[0129] Western Blotting: Prepare the separation gel and the concentrated gel according to the SDS-PAGE gel kit instructions, and let it stand at room temperature for 30-40 min; take 100 μg of protein sample from each group for electrophoresis; after electrophoresis, use a nitrocellulose membrane semi-dry method to transfer the protein sample in the SDS-PAGE gel to a PVDF (0.45 μm) membrane under the conditions of 25V / 1.0A, 25 min; block the PVDF membrane loaded with the protein sample with 5% skim milk for 1 h; dilute the target gene antibody with 5% BSA solution according to the dilution ratio of the primary antibody, and incubate at 4°C overnight, wash with 1x TBST for 10 min x 3 times; incubate the secondary antibody at room temperature for 1 h, wash with 1x TBST for 10 min x 3 times; develop the color using the ECL luminescence method, and obtain the band using the G:BOX imaging analysis system, with β-actin as the internal reference.

[0130] 4.4 Cell immunofluorescence staining

[0131] First, wash the cell slides with pre-cooled PBS for 2-3 times, then fix them with 4% formaldehyde fixing solution at room temperature for 30 min, and then wash them with PBS for 5 min x 3 times; then, put them in sodium citrate antigen repair solution at pH 6.0, heat them in a microwave oven for 5 min for antigen repair, cool them naturally, and then wash them with PBS for 5 min x 3 times; then, block them with 5% bovine serum albumin (BSA) solution prepared with PBS at room temperature for 30 min; then, add the primary antibody working solution evenly to the sample, incubate it at 4°C overnight, wash it with PBS for 5 min x 3 times after the incubation of the primary antibody; further, add the fluorescently labeled secondary antibody working solution to the sample, incubate it at room temperature in the dark for 1 h, and wash it with PBS for 5 min x 3 times after the incubation of the secondary antibody; further, immerse the cell nucleus in DAPI staining solution in the dark for 5 min, and then add an appropriate amount of fluorescent anti-quenching agent for mounting; finally, observe and take pictures under a laser confocal microscope or a fluorescence microscope.

[0132] 5 Experimental Results

[0133] 5.1 Sequencing to screen potential toxic targets for PPCE-induced NAFLD in male offspring rats

[0134] The results are as follows Figure 4 As shown. Compared with the control group, the expression of multiple miRNAs in paternal sperm and offspring fetal liver was altered in the PPCE group. Figure 4 Among them, there are 10 miRNAs that are commonly differentially expressed (A). Figure 4 The changes were most significant in miR-142-3p (part of the B group). Figure 4 (C). Fluorescence in situ hybridization results showed that the expression of miR-142-3p in the fetal liver was much higher than that in other organs, exhibiting good organ specificity. Figure 4 (Middle D). Meanwhile, sequencing analysis indicated that the most significant difference in miR-142-3p was observed in the liver of the PPCE group at PW32. Figure 4 (E). RT-qPCR results showed that, compared with the control group, the expression of liver miR-142-3p was significantly reduced in the PPCE group at GD20 and PW32 ( Figure 4 (F, G). This indicates that PPCE can reduce miR-142-3p expression in paternal sperm and this reduction can extend to the liver of male offspring, leading to decreased miR-142-3p expression in the liver before and after birth. The experimental results suggest that miR-142-3p may be a potential toxic target mediating PPCE-induced NAFLD in male offspring.

[0135] 5.2 Effects of PPCE on the liver miR-142-3p / ACSL4 signaling pathway in male offspring before and after birth

[0136] The results are as follows Figure 5 As shown. Analysis using target prediction databases (TargetScan, miRDB, and miRcode) revealed an interaction between long-chain acyl-CoA synthase 4 (ACSL4) and miR-142-3p. Figure 5 (A). Subsequently, this invention examined the effects of PPCE on the mRNA and protein expression of ACSL4 in the liver of male offspring before and after birth. RT-qPCR and Western blot results showed that, compared with the control group, PPCE significantly increased the mRNA and protein expression levels of ACSL4 in the fetal liver of male offspring and the liver of PW32 offspring. Figure 5 (BG). The experimental results show that PPCE can cause changes in the miR-142-3p / ACSL4 signaling pathway in the liver of male offspring.

[0137] Effects of 5.3 miR-142-3p on lipid metabolism function of rat bone marrow mesenchymal stem cells (BMSCs) in hepatoid differentiation

[0138] In a BMSCs-mediated hepatoid cell model, transfection with miR-142-3p inhibitor and miR-142-3pmimics was used to observe the effect of miR-142-3p on the expression of key genes related to lipid metabolism in BMSCs-mediated hepatoid cells. Results are as follows: Figure 6 As shown. Compared with the control group, transfection with miR-142-3p inhibitor significantly increased the mRNA and protein expression levels of key lipid synthesis factors such as ACSL4, SREBP1, FASN, ACC, and ACLY in BMSCs differentiated into hepatocyte-like cells. Figure 6 In AC), the mRNA and protein expression levels of PPARα and CPT1α were significantly reduced. Figure 6 (AC). Conversely, miR-142-3pmimics transfection significantly reduced the mRNA and protein expression levels of key lipid synthesis genes such as ACSL4, SREBP-1, FASN, ACC, and ACLY in hepatocytes. Figure 6 In AC), and significantly increased the expression levels of PPARα, CPT1α mRNA and protein ( Figure 6 (AC). Experimental results show that miR-142-3p has a regulatory effect on lipid metabolism in hepatocytes.

[0139] 5.4 miR-142-3p targets ACSL4 to regulate lipid metabolism in rat BMSCs hepatoid differentiation cells.

[0140] In a hepatoid cell model of directed differentiation of BMSCs, miR-142-3p mimics were transfected and ACSL4 was overexpressed to observe its effect on the expression of key genes in lipid metabolism. Results are as follows: Figure 7 As shown. Compared with the control group, overexpression of ACSL4 significantly increased the expression levels of key lipid synthesis factors such as SREBP1, FASN, and ACC in hepatocytes differentiated from BMSCs. Figure 7 (AC), while significantly reducing the expression levels of PPARα and CPT1α ( Figure 7 AC; however, treatment with miR-142-3 pmimics significantly reversed the altered lipid metabolism function of BMSCs induced by ACSL4 overexpression in directed differentiation into hepatoid cells. Figure 7 (middle AC). Immunofluorescence staining yielded the same results ( Figure 7 (D). Dual-luciferase reporter gene assay results showed that miR-142-3p and ACSL4 interact ( Figure 7Fig. 6. The experimental results show that miR-142-3p can regulate the lipid metabolism of BMSCs hepatocyte-like differentiation cells by targeting ACSL4.

[0141] In summary, the method of the present application is screened and verified by bioinformatics analysis, and it is found that miR-142-3p can regulate the lipid metabolism of hepatocytes by targeting ACSL4, which can be used as an early intervention target for paternal NAFLD.

[0142]

Example 3

[0143] 1 Experimental animals

[0144] SPF healthy Wistar rats were purchased from Hubei Center for Disease Control and Prevention, animal license number: SCXK (E) 2020-2022. This study was approved by the Ethics Committee of Wuhan University Medical School, and was strictly in accordance with the relevant handling guidelines of the International Experimental Animal Protection Certification Evaluation Agency. Experimental animals were raised in a barrier environment with a temperature of 22-25°C, humidity of 50%, and 12h alternating day and night.

[0145] 2 Experimental methods

[0146] After 2 weeks of adaptive feeding, 30 male Wistar rats (body weight 260-300g) were randomly divided into control group, caffeine group (15, 30, 60mg / kg·d). The rats in the caffeine group were given different doses of caffeine by intragastric perfusion every day, and the control group was given equal volume of normal saline by intragastric perfusion. After 8 weeks of continuous administration, normal female Wistar rats were caged according to the ratio of male to female 1:2, and the next morning vaginal smears were taken to determine pregnant rats, recorded as pregnant 0 days. The rats in each group were free to eat normally. The feed was purchased from Wuhan Wanqianjiaxing Biological Technology Co., Ltd., license number: SCXK (E) 2011-0011. The feed formula is the same as the mouse and rat formula feed stipulated in the "People's Republic of China National Standard GB14924.3-2001".

[0147] The mother rats were naturally produced, and F1 generation was obtained, with the production day as the 0th day after birth. On the 1st day after birth, 12-14 pups were selected from each group, and the number of male and female pups was adjusted to 6 each for lactation feeding to ensure balanced nutrition of the pups. The pups were weaned at 4 weeks after birth and separated by sex, and 16 normal diet-fed rats were randomly selected from each group and fed until 8 weeks after birth. AVV8-miR-142-3p overexpression adeno-associated virus was injected into the tail vein; the male rats treated with AVV8-miR-142-3p were randomly divided into 2 groups, 8 rats in each group, and one group was fed until 12 weeks after birth, and the other group was fed until 32 weeks after birth. The animals were sacrificed 2 days after the above experiment was completed.

[0148] 3. Detection index and method

[0149] 3.1 Triglyceride content detection

[0150] The triglyceride content detection kit (A110-1, Nanjing Jiancheng) was used to detect the triglyceride content in the liver of rats in each group. 50 mg of fresh liver tissue was added to physiological saline at a volume ratio of 1:9, homogenized in an ice bath, centrifuged at 2500 rpm for 10 min, and the supernatant was used for detection. First, the triglyceride content detection kit was used to detect the triglyceride content in the liver tissue homogenate of rats in each group, then the BCA protein concentration detection kit (P0012, Shanghai Biyun Tian) was used to detect the protein concentration in the liver tissue homogenate of rats in each group, and finally the triglyceride content corresponding to each g of protein in the liver tissue homogenate was used as the triglyceride content in the liver tissue. That is, the triglyceride content in the liver = mmol / g protein.

[0151] 3.2 Hematoxylin-eosin staining

[0152] The liver tissue fixed with 10% formalin was dehydrated and embedded in a paraffin embedding machine; the embedded tissue wax block was cut into 5 μm thin sections to prepare liver tissue sections; the sections were soaked in xylene solution for 5 min x 2 times for deparaffinization; the sections were sequentially soaked in 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol for 5 min, and washed with distilled water; the sections were soaked in hematoxylin-eosin staining solution for 30 s, washed with distilled water, then soaked in 1% hydrochloric acid alcohol and quickly taken out, washed with distilled water; the sections were sequentially soaked in 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol for 5 min, placed in a fume hood to dry the residual ethanol, then soaked in xylene solution for 5 min, placed in a fume hood to dry the residual xylene; add an appropriate amount of neutral gum to seal the section; observe the staining results under a microscope.

[0153] 3.3 Oil red O staining

[0154] The liver tissue fixed with 10% formalin was frozen sectioned to cut the mouse liver tissue sample into 6-8 μm thin sections, which were attached to a glass slide and immediately fixed in 10% paraformaldehyde for 3 min, then washed with distilled water and stored at -20°C. When used, the frozen sections were taken out and placed at room temperature for 10 min, then soaked in 60% isopropanol solution for 2 min; the sections were stained with oil red O staining solution for 10 min, washed with 60% isopropanol solution to remove excess staining solution, and washed with distilled water; stained with hematoxylin solution for 30 s, and washed with distilled water; soaked in 1% hydrochloric acid alcohol and quickly taken out, washed with distilled water; add an appropriate amount of neutral gum to seal the section; observe the staining results under a microscope.

[0155] 3.4 Real-time fluorescent quantitative PCR experiment

[0156] The RNA in the liver tissue of each group of mice was extracted by using Trizol reagent, and cDNA was obtained after reverse transcription. The cDNA, each gene upper and lower stream primer, labeled SYBR Green I fluorescent dye Mix and other reagents were mixed uniformly and then placed in an RT-qPCR instrument for reaction. The RT-qPCR reaction conditions were as follows (the reaction system was 10 μL): 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 62℃ annealing for 30 s; 72℃ extension for 15 s, a total of 40 cycles. GAPDH was used as an internal reference, and the relative expression amount of the target gene mRNA was calculated by using 2 -△△Ct Method The relative expression amount of the target gene mRNA was calculated by using 2

[0157] 4 Experimental results

[0158] 4.1 Effect of liver overexpression of miR-142-3p on the occurrence of NAFLD in PPCE male offspring

[0159] The results are shown in Table 1. Figure 8 Compared with the control group, the overexpression intervention of AVV8-miR-142-3p can reduce the expression of ACSL4 in the liver of the PW32 male offspring of PPCE, inhibit the expression of the genes related to the de novo synthesis of fatty acids in liver cells (ACSL4, SREBP1, FASN and ACC), and increase the expression level of the genes related to lipid β oxidation (PPARα and CPT1α) in the liver of the PW32 male offspring of PPCE (Table 1, middle A). Figure 8 Compared with the control group, the liver of the PW32 male offspring of PPCE rats showed obvious hepatocyte steatosis and a large amount of lipid accumulation, the Kleiner score and the TG content in the liver were significantly increased (Table 1, middle B-D), while after the intervention of AVV8-miR-142-3p, the liver of the PW32 male offspring of PPCE rats showed no obvious steatosis and lipid accumulation, the Kleiner score and the TG content in the liver were significantly reduced (Table 1, middle B-D). Figure 8 Figure 8 The experimental results show that the overexpression of miR-142-3p in the liver can reverse the occurrence of NAFLD in the adult male offspring of PPCE.

[0160] In summary, the modeling method of the present application can correct the liver lipid metabolism disorder of the male offspring of the paternal pre-pregnancy caffeine exposure and inhibit the occurrence of long-term NAFLD after the targeted treatment of miR-142-3p, which shows that the model of the present application can be used for screening the treatment drugs and treatment methods of paternal NAFLD, and it is confirmed that the overexpression of miR-142-3p can treat paternal NAFLD, which has a positive effect on the development and prevention of the treatment drugs for the clinical paternal NAFLD.​

[0161] Example 4: Investigation of the predisposition of paternal non-alcoholic fatty liver disease using the model of the present application

[0162] 1 Experimental animals

[0163] SPF level healthy Wistar rats were purchased from Hubei Center for Disease Control and Prevention, Animal License No: SCXK(E)2020-2022. This study was approved by the Ethics Committee of Wuhan University Medical School and was strictly in accordance with the relevant handling guidelines of the International Laboratory Animal Protection Certification Assessment Agency. Experimental animals were raised in a barrier environment with a temperature of 22-25°C, humidity of 50%, and 12h light-dark alternation.

[0164] 2 Experimental animal treatment

[0165] After 2 weeks of adaptive feeding, 30 male Wistar rats (body weight 260-300g) were randomly divided into control group, PPCE group, RU486 group (RU) and PPCE+RU486 group (PPCE+RU). The rats in the PPCE group were given 60mg / kg of caffeine intragastrically, the rats in the RU group were given 1.0mg / kg of RU486 intragastrically, the rats in the PPCE+RU group were given 60mg / kg of caffeine and 1.0mg / kg of RU486 intragastrically, and the control group was given the same volume of normal saline intragastrically. After 8 weeks of continuous administration, normal female Wistar rats were caged according to the ratio of male to female 1:2, and the next morning vaginal smears were taken to determine pregnant rats, recorded as pregnant 0 days. The rats in each group were free to eat normally. The feed was purchased from Wuhan Wanqianjiaxing Biological Technology Co., Ltd., License No: SCXK(E)2011-0011. The feed formula was the same as the mouse and rat formula feed stipulated in the National Standard of the People's Republic of China GB14924.3-2001.

[0166] Some pregnant 20-day female mice were sacrificed after anesthesia, and male offspring fetal rat liver samples were collected. Some female mice naturally gave birth to F1 generation, and the day of birth was taken as postnatal day 0. On postnatal day 1, 12-14 pups were selected from each group, and the number of male and female pups was adjusted to 6 each for lactation feeding to ensure balanced nutrition of the pups. The pups were weaned at 4 weeks of age and separated by sex, and 12 normal diet-fed rats were randomly selected from each group and raised until 32 weeks of age. Then the animals were sacrificed under anesthesia, and the 32-week-old male offspring liver samples were collected.

[0167] 3 Mouse spermatogonial cell culture and treatment

[0168] The mouse spermatogonial cells GC-1 were cultured in a cell culture box at 37℃ and 5% CO2 using DMEM medium containing 10% FBS and 1% penicillin-streptomycin double antibody (hereinafter referred to as "medium"). The GC-1 cells in the logarithmic growth phase were taken, the original culture medium in the culture bottle was discarded, and then 5 mL of 37℃ preheated PBS buffer was used to clean the cells twice, and then 5 mL of medium containing different final concentrations of caffeine (0, 0.1, 1, 10 μM) or corticosterone (0, 125, 250, 500 nM) was added, and then the cells were placed in a cell culture box at 37℃ and 5% CO2 for 24 h. Then the cells were collected to detect the expression level of miR-142-3p and the methylation level of the promoter region. The effects of different concentrations of caffeine and corticosterone on the expression of spermatogonial cells miR-142-3p were investigated.

[0169] 4. Test index and method

[0170] 4.1 Detection of methylation level of miR-142-3p promoter region

[0171] The bisulfite sequencing (BSP) method was used to detect the methylation level of the miR-142-3p promoter region, and the specific operation method is as follows.

[0172] (1) The genomic DNA of each sample was extracted using a cell / tissue genomic DNA extraction kit.

[0173] (2) 5 μg of the extracted sample genomic DNA was dissolved with enzyme-free water and diluted to a volume of 50 μL, and then NaOH solution was added and mixed uniformly, and denatured at 42℃ water bath for 30 min. Then, 30 μL of 10 mM hydroquinone, 520 μL of 3M sodium bisulfite were added to the sample in turn, and mixed uniformly. Finally, 200 μL of paraffin oil was added to each sample, and incubated at 50℃ in the dark for 16 h.

[0174] (3) The purified kit was used for column purification, and the modified DNA was recovered and subjected to PCR amplification.

[0175] (4) The product obtained by PCR amplification was subjected to agarose gel electrophoresis, and the amplified fragment was recovered. Then, the amplified fragment (target gene) was recombined with a cloning vector.

[0176] (5) The competent cells were thawed on ice; 10 μL of the recombined product was added to the competent cells, mixed gently, and then incubated on ice for 30 min; then the competent cells and the recombined product mixture were immediately placed in a 42℃ constant temperature water bath for 90 s, and then immediately placed on ice for 2-3 min. Finally, 500 μL of LB culture medium without antibiotics was added, and the culture was incubated at 37℃ and 250 rpm for 45 min.

[0177] (6)Centrifuge at 3000 rpm for 2 min, discard the supernatant.

[0178] (7) Mix the bacteria solution gently by blowing, then add it to the culture plate containing ampicillin, evenly spread it with a sterile swab, and then invert it in a 37°C constant temperature incubator for overnight culture.

[0179] (8) Select 3-5 clones of transformants for colony PCR, and then confirm the positive clones for sequencing identification.

[0180] 4.2 Triglyceride content detection

[0181] The triglyceride content detection kit (A110-1, Nanjing Jiancheng) was used to detect the triglyceride content in the liver of each group of rats. 50 mg of fresh liver tissue was added to physiological saline at a volume ratio of 1:9, homogenized under ice bath conditions, centrifuged at 2500 rpm / min for 10 min, and the supernatant was used for detection. First, the triglyceride content detection kit was used to detect the triglyceride content in the liver tissue homogenate of each group of rats, then the BCA protein concentration detection kit (P0012, Shanghai Biyun Tian) was used to detect the protein concentration in the liver tissue homogenate of each group of rats, and finally the triglyceride content corresponding to each g of protein in the liver tissue homogenate was used as the triglyceride content in the liver tissue. That is, the triglyceride content in the liver = mmol / g protein.

[0182] 4.3 Hematoxylin-eosin staining

[0183] The liver tissue fixed with 10% formalin was dehydrated and embedded in a paraffin embedding machine; the embedded tissue wax block was cut into 5 μm thin sections to prepare liver tissue sections; the sections were soaked in xylene solution for 5 min twice for deparaffinization; the sections were sequentially soaked in 100%, 95%, 85%, 75% ethanol for 5 min, and then washed with distilled water; the sections were soaked in hematoxylin-eosin staining solution for 30 s, washed with distilled water, then soaked in 1% hydrochloric acid alcohol and quickly taken out, and washed with distilled water; the sections were sequentially soaked in 75%, 85%, 95%, 100% ethanol for 5 min, placed in a fume hood to dry the residual ethanol, then soaked in xylene solution for 5 min, and placed in a fume hood to dry the residual xylene; add an appropriate amount of neutral gum to seal the section; observe the staining results under a microscope.

[0184] 4.4 Oil red O staining

[0185] The liver tissue fixed by 10% formalin was cut into 6-8 μm slices by frozen section, and the mouse liver tissue samples were pasted on glass slides and immediately placed in 10% paraformaldehyde for 3 min, washed with distilled water and stored at -20°C. When used, the frozen sections were taken out, placed at room temperature for 10 min, soaked in 60% isopropanol solution for 2 min; the sections were stained in oil red O staining solution for 10 min, washed with 60% isopropanol solution to remove excess staining solution, washed with distilled water; stained with hematoxylin solution for 30 s, washed with distilled water; soaked in 1% hydrochloric acid alcohol and quickly taken out, washed with distilled water; add appropriate amount of neutral gum to seal the slide; observe the staining results under a microscope.

[0186] 4.5 Real-time fluorescent quantitative PCR experiment

[0187] Trizol reagent was used to extract RNA from the liver tissues of mice in each group, and cDNA was obtained after reverse transcription. The cDNA, upstream and downstream primers of each gene, SYBR Green I fluorescent dye Mix and other reagents were mixed uniformly and placed in an RT-qPCR instrument for reaction. The RT-qPCR reaction conditions are as follows (the reaction system is 10 μL): 95°C pre-denaturation for 2 min; 95°C denaturation for 10 s, 62°C annealing for 30 s; 72°C extension for 15 s, a total of 40 cycles. GAPDH was used as an internal reference, and the relative expression amount of the target gene mRNA was calculated by 2 -△△Ct Method.

[0188] 5 Experimental results

[0189] 5.1 Corticosterone (but not caffeine) mediates high methylation and low expression of spermatogonial cell miR-142-3p promoter region

[0190] MiRNAs and their methylation modification are key epigenetic markers in the process of germ cell reprogramming, which can transmit the "imprint" of the father's experience of adverse environment to the offspring. Studies have found that paternal chronic stress can cause changes in sperm epigenetic modification through high glucose corticosteroids, thereby programming abnormal liver glucose metabolism in offspring, suggesting that high glucose corticosteroids may be an important mechanism for the occurrence of paternal metabolic diseases. Therefore, in this study, the inventors first detected the serum corticosterone content, testicular glucocorticoid receptor (GR) expression level and sperm miR-142-3p promoter region methylation level of the parental male rats. The results are shown in Figure 9 Compared with the control group, the serum corticosterone content and testicular GR mRNA expression level of the PPCE group were significantly increased Figure 9 A, B), and the sperm miR-142-3p promoter region methylation level was significantly increased Figure 9(C). Furthermore, this invention treated mouse spermatogonia with different concentrations of caffeine (0, 0.1, 1, 10 μM) or corticosterone (0, 125, 250, 500 nM) in vitro to determine the cause of decreased sperm miR-142-3p expression. The results showed that, compared with the CON group, different concentrations of caffeine treatment had no significant effect on spermatogonia miR-142-3p expression. Figure 9 Treatment with different concentrations of corticosterone significantly reduced the expression level of miR-142-3p in spermatogonia. Figure 9 (E), and increases the methylation level of the miR-142-3p promoter region ( Figure 9 (F, G). Experimental results showed that corticosterone (but not caffeine) programmed the hypermethylation of miR-142-3p in PPCE paternal sperm.

[0191] 5.2 RU486 intervention reversed PPCE-induced hepatic lipid metabolism disorders before and after birth in male offspring rats.

[0192] Furthermore, this invention simultaneously intervened in PPCE rats at the whole-animal level using the GR antagonist RU486 to confirm the programming mechanism of paternal hyperglycemia-mediated changes in hepatic lipid metabolism in male PPCE offspring. The results are as follows: Figure 10 As shown. Compared with the control group, at GD20 and PW32, the PPCE group showed increased methylation and decreased expression of the liver miR-142-3p promoter region. Figure 10 In moderate AD, the expression of genes involved in liver lipid synthesis is increased while the expression of genes involved in β-oxidation is decreased. Figure 10 E, F), while liver lipid accumulation and TG content increased ( Figure 10 (Medium GI). However, RU486 intervention could significantly reverse the aforementioned changes in male offspring before and after birth induced by PPCE ( Figure 10 The experimental results indicate that high glucocorticoid exposure mediates PPCE-induced changes in hepatic lipid metabolism in male offspring by activating GR.

[0193] In summary, the method of this invention confirms that corticosterone (but not caffeine) can lead to hypermethylation and low expression of sperm miR-142-3p, which in turn leads to hypermethylation and reduced expression of miR-142-3p in the offspring liver, causing hepatic lipid metabolism dysfunction and ultimately resulting in adult NAFLD. Therefore, applying the model described in this invention to elucidate the causes of paternal non-alcoholic fatty liver disease (NAFLD) has significant practical implications for understanding the impact of paternal preconception chronic stress on the long-term metabolic health of offspring and for guiding men of reproductive age to live healthy lives during their reproductive years.

Claims

1. A method for constructing an animal model of paternal non-alcoholic fatty liver disease, characterized in that: Includes the following steps: S1: Several healthy male rodents were selected and divided into a control group and a caffeine group. The caffeine group was divided into three groups according to different doses of 15, 30 and 60 mg / kg caffeine, and was given intragastric gavage daily. The male rodents had free access to food and water. S2: Male rodents were administered different doses of caffeine into their stomachs daily for 8 consecutive weeks and then mated with normal female mice to conceive. S3: In step S2 above, some pregnant mice were given fetal livers on day 20 of pregnancy; some pregnant mice gave birth naturally to obtain F1 offspring. The day of birth was taken as day 0 after birth. One day after birth, litters with 12 to 14 offspring were selected, and each litter was adjusted to have 6 male and 6 female offspring for nursing. The offspring were weaned 4 weeks after birth and the males and females were separated into different cages. Some male offspring continued to be fed normally until 32 weeks of age, at which time blood and liver were collected. S4: After completing the above steps, detect liver lipid metabolism-related indicators at different time points, namely 20 days of gestation and 32 weeks after birth, to comprehensively determine non-alcoholic fatty liver disease; finally, obtain a paternal non-alcoholic fatty liver disease animal model.

2. The method for constructing an animal model of paternal non-alcoholic fatty liver disease according to claim 1, characterized in that: In S1, the rodents are rats and mice.

3. The method for constructing an animal model of paternal non-alcoholic fatty liver disease according to claim 1 or 2, characterized in that: In S3, the normal diet is formulated in the same way as the mouse and rat feed formula specified in the National Standard of the People's Republic of China GB14924.3-2001.

4. The method for constructing an animal model of paternal non-alcoholic fatty liver disease according to claim 3, characterized in that: In S4, the liver lipid metabolism-related indicators are: serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, liver triglyceride content, hematoxylin-eosin staining, Oil Red O staining, Masson staining, and Sirius red staining.

5. The application of a paternal non-alcoholic fatty liver disease animal model in screening for paternal potential developmental toxicity environmental disruptors or drugs, characterized in that: The animal model is obtained by the construction method as described in any one of claims 1 to 4.

6. The application of a paternal non-alcoholic fatty liver disease (NAFLD) animal model in screening early intervention targets for paternal NAFLD, characterized in that: The animal model is obtained by the construction method as described in any one of claims 1 to 4.

7. An early intervention target for paternal non-alcoholic fatty liver disease, characterized in that: The intervention target is miR-142-3p.

8. The application of a paternal non-alcoholic fatty liver disease animal model in screening / preparing therapeutic agents for the prevention and treatment of non-alcoholic fatty liver disease, characterized in that: The paternal non-alcoholic fatty liver disease animal model is obtained by the construction method as described in any one of claims 1 to 4.

9. The application according to claim 8, characterized in that: The paternal non-alcoholic fatty liver disease treatment agents include: adenovirus containing miR-142-3p and lentivirus containing miR-142-3p.

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