Application of vitamin A in preparation of medicine for treating or preventing hepatitis

Abstract

The invention discloses application of vitamin A in preparation of a medicine for treating or preventing hepatitis, and belongs to the technical field of biological medicine. Experiments find that vitamin A promotes mitochondrial biosynthesis and functions by activating a p38MAPK / PGC-1alpha signal channel, further improves the oxidative phosphorylation level, induces interferon to stimulate gene expression and inhibits hepatitis B virus replication so as to treat or prevent chronic hepatitis B, and a new thought is provided for treatment of chronic hepatitis B.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of vitamin A in the preparation of a drug for treating or preventing hepatitis. BACKGROUND

[0002] Chronic hepatitis B (CHB) has been listed as a global public health threat that needs to be prevented and controlled by the World Health Organization. The existing antiviral drugs are mainly nucleoside analogues and interferons. Nucleoside analogues can only inhibit the DNA replication of hepatitis B virus (HBV), but cannot eliminate hepatitis B virus surface antigen (HBsAg); the efficacy of interferon is greatly different among individuals, and only a small number of patients achieve HBsAg seroconversion. HBV infection is closely related to mitochondrial dysfunction of hepatocytes, and the virus can directly affect the mitochondrial-related membrane structure and function of infected hepatocytes. However, there is currently no anti-HBV therapy targeting mitochondrial function repair, and there is a lack of targeted intervention strategies in this field. SUMMARY

[0003] The purpose of the present application is to provide the application of vitamin A in the preparation of a drug for treating or preventing hepatitis. Vitamin A can promote the biosynthesis and function of mitochondria in HBV-infected hepatocytes through the p38 MAPK / PGC-1α signaling pathway to inhibit HBV replication, thereby providing a new treatment option for CHB patients.

[0004] Therefore, the present application provides the application of vitamin A in the preparation of a drug for treating or preventing hepatitis.

[0005] Preferably, the hepatitis is hepatitis B.

[0006] Preferably, the hepatitis B is chronic hepatitis B.

[0007] Preferably, the chronic hepatitis B is a disease caused by hepatitis B virus infection, accompanied by mitochondrial dysfunction.

[0008] Preferably, vitamin A promotes mitochondrial biosynthesis and function by activating the p38 MAPK / PGC-1α signaling pathway, thereby increasing the level of oxidative phosphorylation and inducing interferon-stimulated gene expression to inhibit hepatitis B virus replication for treating or preventing chronic hepatitis B.

[0009] The present application also provides a drug for treating or preventing hepatitis, comprising vitamin A and a drug carrier.

[0010] Preferably, the drug carrier comprises one of DMSO and PEG300.

[0011] The present application has the following beneficial effects:

[0012] The application provides application of vitamin A in preparation of a medicine for treating or preventing hepatitis, vitamin A can promote biosynthesis and function of mitochondria in HBV infected hepatocytes through a p38MAPK / PGC-1a signal pathway, thereby enhancing a level of mitochondrial oxidative phosphorylation (OXPHOS), and providing a theoretical basis and experimental basis for inhibiting HBV replication by vitamin A; meanwhile, a new thought is provided for CHB treatment, and can be used as a diagnosis and treatment index of CHB.

[0013] The technical solutions of the application are further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is a serum fat-soluble vitamin spectrum of chronic HBV infected persons at different natural history stages in the embodiment 1 of the application;

[0015] Figure 2 is a correlation between serum vitamin A and HBsAg, HBV DNA, ALT and AST of CHB patients in the embodiment 1 of the application;

[0016] Figure 3 is a change in HBV infection related indexes of HepG2.2.15 cells after being treated with different concentrations of vitamin A in the embodiment 2 of the application;

[0017] Figure 4 is a change in HBV infection related indexes in serum of HBV persistent infection mice in the experimental group and the control group in the embodiment 3 of the application;

[0018] Figure 5 is a heat map and principal component analysis diagram showing gene expression differences between the DMSO control group and the vitamin A experimental group, a volcano plot showing the number of differential genes between the DMSO control group and the vitamin A experimental group, and KOG, GO and KEGG analysis of signal pathway changes involved by the differential genes in the embodiment 4 of the application;

[0019] Figure 6 is a change in mitochondrial morphological structure, ROS expression level and ATP expression level of HepG2.2.15 cells after being treated with vitamin A in the experimental group and DMSO in the control group for 3 days in the embodiment 5 of the application;

[0020] Figure 7 is a change in OCR and mitochondrial DNA copy number of HepG2.2.15 cells, Hu7-1.3 HBV cells and HepG2-NTCP cells after being treated with vitamin A in the experimental group and DMSO in the control group for 1 day in the embodiment 5 of the application;

[0021] Figure 8The expression levels of ISGs mRNA and ISG20 protein in HepG2.2.15 cells after 1 day and 3 days of treatment in the vitamin A experimental group and DMSO control group of Example 6 of this invention are shown.

[0022] Figure 9 The levels of ATP, intracellular ISG20 mRNA, total RNA, and pgRNA in HepG2.2.15 cells after one day of treatment with oligomycin A and vitamin A in Example 7 of this invention are shown.

[0023] Figure 10 This refers to the expression levels of PGC-1α, NRF1, and TFAM mRNA in HepG2.2.15 cells one day after treatment with the Vitamin A experimental group and the DMSO control group in Example 8 of this invention.

[0024] Figure 11 This refers to the expression levels of PGC-1α and TFAM proteins in HepG2.2.15 cells treated for 3 days in the vitamin A experimental group and DMSO control group of Example 8 of the present invention, as well as the phosphorylation level of p38 MAPK at different time points after treatment;

[0025] Figure 12 This invention describes the changes in ATP, ROS, and mtDNA in HepG2.2.15 cells after 3 days of treatment with SB203580 and vitamin A in various treatment groups, as well as the expression levels of HBsAg, pgRNA, total RNA, and pgRNA in the cell supernatant. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0028] Example

[0029] experimental population

[0030] Chronic HBV-infected patients treated at the First Affiliated Hospital of Fujian Medical University from September 2022 to February 2025.

[0031] laboratory animals

[0032] Six-week-old SPF-grade C57BL / 6 female mice were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. to establish a chronic HBV infection model induced by rAAV8-1.3HBV.

[0033] drug

[0034] Vitamin A, with the aid of pharmaceutically acceptable carriers DMSO or PEG300.

[0035] Application mode

[0036] Directly acting on HBV infected cells in vitro, such as HepG2.2.15; in vivo, by gavage or feeding method to HBV infected animal models.

[0037] Detection index

[0038] HBV related markers: HBsAg, hepatitis B e antigen (HBeAg), HBV DNA, HBV RNA;

[0039] Mitochondrial function indicators: mitochondrial membrane potential MMP (JC-1 staining), reactive oxygen species ROS (DCFH-DA probe), ATP content (chemiluminescence method), OCR (Seahorse XF analyzer);

[0040] Signal pathway verification: p38 MAPK phosphorylation level, PGC-1α / NRF1 / TFAM expression (qPCR / WB).

[0041] Example 1

[0042] Experimental grouping: According to the disease stages of the above chronic HBV infected patients, they are divided into four subgroups, respectively, hepatitis B e antigen (HBeAg) positive chronic HBV infection group (IT), HBeAg positive CHB group (IA), HBeAg negative chronic HBV infection group (IC) and HBeAg negative CHB group (ENH), The number of experimental population in each group is 50, respectively, subgroup 1, subgroup 2, subgroup 3 and subgroup 4; 50 healthy people (HC) as control group.

[0043] The serum liposoluble vitamin spectrum of untreated chronic HBV infected patients was detected by UPLC-MS / MS technology, and the results are shown in Figure 1 , wherein Figure 1 (a) is the spectrum of fat-soluble vitamin A, (b) is the spectrum of fat-soluble vitamin D, (c) is the spectrum of fat-soluble vitamin E, and (d) is the spectrum of fat-soluble vitamin K. Figure 1 It can be seen that compared with the control group, the serum vitamin A level of the four subgroups of chronic HBV infected patients is significantly reduced, among which the reduction of HBeAg positive CHB patient group (IA) is the most significant; While the levels of other three kinds of fat-soluble vitamins D, vitamin E and vitamin K showed no statistical difference or insignificant difference among the groups.

[0044] The correlation between serum vitamin A and HBsAg, HBV DNA, ALT, AST of CHB patients is shown in Figure 2As shown, where Figure 2 In the table, (a) shows the correlation between vitamin A and HBsAg, (b) shows the correlation between vitamin A and HBV DNA, (c) shows the correlation between vitamin A and ALT, and (d) shows the correlation between vitamin A and AST. Figure 2 It can be seen that serum vitamin A levels in CHB patients are significantly negatively correlated with multiple clinical indicators.

[0045] These results indicate that decreased vitamin A levels are closely related to the progression of HBV infection and the degree of liver inflammation.

[0046] Example 2

[0047] HepG2.2.15 cells were treated with vitamin A at concentrations of 0 ng / mL, 200 ng / mL, 400 ng / mL, and 600 ng / mL, respectively. The expression levels of various markers in the cell supernatant and intracellular components were then measured. The results are as follows: Figure 3 As shown, where Figure 3 (a) shows the changes in HBsAg, HBeAg, HBV DNA, and HBV RNA in the cell supernatant of HepG2.2.15 cells after 3 days of vitamin A treatment. Figure 3 (b) shows the changes in intracellular HBsAg and HBeAg in HepG2.2.15 cells after 3 days of vitamin A treatment. Figure 3 (c) represents the changes in total RNA and pgRNA in HepG2.2.15 cells after one day of vitamin A treatment.

[0048] from Figure 3 As shown in (a), all indicators showed a significant decreasing trend under different concentrations of vitamin A, indicating that vitamin A has an inhibitory effect on HBV replication. Figure 3 As shown in (b) and (c), vitamin A can significantly reduce the levels of HBsAg and HBeAg in cells, while the levels of total RNA and pregenomic RNA (pgRNA) are also significantly reduced.

[0049] Treatment with 400 ng / mL vitamin A showed a significant inhibitory effect; therefore, 400 ng / mL was used as the final effective concentration of vitamin A in subsequent experiments.

[0050] Example 3

[0051] A mouse model of HBV infection was established, and two experimental strategies were used to evaluate the effect of vitamin A on HBV infection:

[0052] Strategy 1

[0053] The HBV persistently infected mice in the experimental group were orally gavaged with vitamin A (600 IU / 100 μL) every other day, denoted as the VA group; the HBV persistently infected mice in the control group were gavaged with the same volume of DMSO, denoted as the DMSO group; during the experiment, serum samples were collected every two weeks to detect the expression levels of the HBV infection-related indicators.

[0054] Strategy two

[0055] The HBV persistently infected mice in the experimental group were fed with vitamin A-deficient feed (0 IU / kg), denoted as the VAD group; and the HBV persistively infected mice in the control group were fed with the same volume of standard vitamin A-containing feed (4000 IU / kg), denoted as the VAC group; during the experiment, serum samples were collected every two weeks to detect the expression levels of the HBV infection-related indicators.

[0056] The results are shown in Figure 4 , wherein Figure 4 (a) is the changes in the serum HBV infection-related indicators of the HBV persistently infected mice after gavaging with DMSO and vitamin A, respectively, and (b) is the changes in the serum HBV infection-related indicators of the HBV persistently infected mice after feeding with normal vitamin A-containing feed and vitamin A-deficient feed, respectively.

[0057] As can be seen from Figure 4 (a), at week 8, the serum HBsAg and HBV DNA levels of the mice in the VA group were significantly lower than those in the DMSO group, indicating that vitamin A has an inhibitory effect on HBV replication.

[0058] As can be seen from Figure 4 (b), the serum HBsAg, HBeAg and HBV DNA levels of the mice in the VAD group were significantly higher than those in the VAC group, indicating that vitamin A deficiency affects the infection and replication of HBV.

[0059] The above results show that vitamin A can inhibit HBV replication in vivo, providing new experimental evidence for potential intervention strategies for HBV infection.

[0060] Example 4

[0061] HepG2.2.15 cells were treated with vitamin A and DMSO, respectively, denoted as VA and DMSO, for eukaryotic transcriptome RNA sequencing, and the results are shown in Figure 5 , wherein Figure 5 (a) and (b) are heat maps and principal component analysis charts, respectively, showing the gene expression differences between the DMSO control group and the VA experimental group; Figure 5(c) in the figure is a volcano plot, showing the number of differentially expressed genes between the DMSO control group and the VA experimental group. The sequencing screening criteria were: *P < 0.05, > 1.2-fold Foldchange; Figure 5 (d) and (e) in the figure represent the changes in signaling pathways involved in differentially expressed genes analyzed by KOG, GO, and KEGG.

[0062] from Figure 5 As can be seen from (a) and (b) in the figures, there are significant differences in the gene expression profiles within HepG2.2.15 cells of the DMSO control group and the VA experimental group; from Figure 5 As shown in (c), compared to the DMSO control group, VA treatment resulted in the upregulation of 101 genes and the downregulation of 741 genes in HepG2.2.15 cells. These differentially expressed genes were further enriched using the KOG, GO, and KEGG gene sets. Figure 5 As can be seen from (d) and (e), energy metabolism and the mitochondrial OXPHOS signaling pathway were significantly upregulated in the VA experimental group.

[0063] The results show that vitamin A can upregulate the level of mitochondrial OXPHOS in HepG2.2.15 cells, providing experimental evidence for elucidating that vitamin A regulates mitochondrial OXPHOS and thus inhibits HBV replication in HepG2.2.15 cells.

[0064] Example 5

[0065] The morphology and structure of mitochondria in HepG2.2.15 cells were observed using transmission electron microscopy after treatment with vitamin A and DMSO for 3 days, and were denoted as VA and DMSO, respectively. The results are as follows: Figure 6 As shown in (a) in the figure, from Figure 6 As can be seen in (a) of the DMSO control group, the mitochondria showed obvious swelling, partial damage to the membrane structure, and sparse or even collapsed cristae. In contrast, the VA experimental group showed a more intact mitochondrial membrane structure, with significantly increased and tightly arranged cristae. This suggests that vitamin A may enhance mitochondrial function by improving mitochondrial quality control.

[0066] Excessive ROS accumulation may impair mitochondrial structure and function. Cellular ROS levels were detected using the DCFH-DA fluorescent probe, and the results are as follows: Figure 6 As shown in (b) in the figure, from Figure 6 As can be seen in (b), the ROS level of HepG2.2.15 cells in the VA experimental group decreased significantly, indicating that vitamin A improved electron transfer efficiency, reduced electron leakage, and thus reduced ROS generation.

[0067] The ATP levels in the VA experimental group and the DMSO control group were quantitatively determined using a luminescence method. The results are as follows:Figure 6 As shown in (c) in the figure, from Figure 6 As can be seen in (c), the ATP content in the VA experimental group was significantly higher than that in the control group, indicating that the mitochondria' ability to synthesize ATP was enhanced.

[0068] The oxygen consumption rate of cells was detected using a Seahorse XF analyzer, and the results are as follows: Figure 7 As shown in (a) and (b), where Figure 7 (a) shows the mitochondrial stress test curve of HepG2.215 cells, and (b) shows the key parameters of mitochondrial function. Figure 7 As shown in (a) and (b), compared with the control group, the vitamin A treatment group showed significantly increased basal respiration, ATP production, and proton leakage. This result, combined with the aforementioned experiments, ultimately demonstrates that vitamin A comprehensively enhances mitochondrial energy metabolism by increasing mitochondrial OXPHOS activity.

[0069] Increased mitochondrial DNA content typically reflects mitochondrial biosynthesis and replication processes. The mtDNA content in HepG2.2.15 cells stably transfected with HBV, Huh7 cells transiently transfected with 1.3HBV plasmid, and HepG2-NTCP cells simulating natural infection conditions was detected by RT-PCR. qPCR analysis yielded the following results: Figure 7 As shown in (c), (d), and (e), where Figure 7 In the table, (c) represents the mtDNA content in HepG2.2.15 cells, (d) represents the mtDNA content in Huh7 cells transiently transfected with the 1.3HBV plasmid, and (e) represents the mtDNA content in HepG2-NTCP cells. Figure 7 As can be seen from (c), (d), and (e), the mtDNA copy number was significantly increased in the VA experimental group, indicating that vitamin A not only enhances mitochondrial function but also promotes mitochondrial replication to enhance the cell's metabolic capacity.

[0070] The above results indicate that vitamin A plays a role in pathological conditions such as HBV infection by enhancing mitochondrial OXPHOS activity and optimizing mitochondrial function.

[0071] Example 6

[0072] Interferon-induced genes (ISGs) play a crucial role in the host's resistance to viral infection. Dozens of common ISGs were detected in HepG2.2.15 cells treated with VA and DMSO for one day using qPCR. The results are as follows: Figure 8 As shown in (a) in the figure, from Figure 8As shown in (a), vitamin A treatment can increase the mRNA levels of HepG2.2.15 interferon-stimulated gene 20 (ISG20), ISG15, tripartite motif-containing protein 5 (TRIM5), and 2'-5'-oligoadenylate synthetase 1 (OAS1) genes, with the ISG20 gene showing the most significant upregulation and the largest fold difference.

[0073] Western blot was used to detect the expression level of ISG20 protein in HepG2.2.15 and Huh7-1.3HBV cells 3 days after treatment with VA experimental group and DMSO control group. The results are as follows: Figure 8 As shown in (b) in the figure, from Figure 8 As can be seen from (b) in the figure, vitamin A can increase the expression level of ISG20 protein in HepG2.2.15 cells.

[0074] The above results indicate that ISG20 can serve as a key effector molecule for vitamin A regulation of antiviral responses, playing a crucial role in vitamin A's inhibition of HBV replication.

[0075] Example 7

[0076] Oligomycin A is an inhibitor of oxidative phosphorylation. After treating HepG2.2.15 cells with DMSO, oligomycin A, and vitamin A for one day, the changes in intracellular ATP, ISG20 mRNA, total RNA, and pgRNA levels in each treatment group were detected. The results are as follows: Figure 9 As shown, where Figure 9 In the table, (a) represents the expression level of ATP, (b) represents the expression level of intracellular ISG20 mRNA in each treatment, (c) represents the expression level of intracellular pgRNA in each treatment group, and (d) represents the expression level of intracellular total RNA in each treatment group. Figure 9 As can be seen from (a), oligomycin A can inhibit OXPHOS; from Figure 9 As can be seen from (b) above, compared with vitamin A alone, the addition of oligomycin A significantly inhibited the expression of the ISG20 gene; from Figure 9 As can be seen from (c) and (d), oligomycin A also antagonizes the effect of vitamin A in inhibiting pgRNA and total RNA.

[0077] The above results indicate that vitamin A can enhance OXPHOS, thereby promoting the production of ISG20 and thus playing an anti-HBV role.

[0078] Example 8

[0079] Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) / nuclear respiratory factor 1 (NRF1) / mitochondrial transcription factor A (TFAM) signaling pathway is a classical pathway for regulating mitochondrial gene transcription and mitochondrial replication.

[0080] The mRNA levels of PGC-1α, NRF1 and TFAM after vitamin A acting on HepG2.2.15 cells for 1 day were detected by RT-PCR, and the results are shown in Figure 10 , wherein Figure 10 (a) is the mRNA expression level of PGC-1α, (b) is the mRNA expression level of NRF1, and (c) is the mRNA expression level of TFAM. Figure 10 It can be seen from (a) that, compared with the control group, the expression of PGC-1α mRNA gene was significantly up-regulated in the vitamin A treatment group.

[0081] The protein expression levels of PGC-1α and TFAM after vitamin A acting on HepG2.2.15 cells for 3 days were detected by RT-PCR, and the results are shown in Figure 11 (a). Figure 11 It can be seen from (a) that vitamin A can also up-regulate the expression levels of PGC-1α and TFAM proteins.

[0082] The phosphorylation levels of p38 MAPK after vitamin A treating HepG2.2.15 cells for 10 min, 30 min and 60 min were detected, and the results are shown in Figure 11 (b). Figure 11 It can be seen from (b) that the phosphorylation of p38 MAPK is most significant at 30 min.

[0083] Example 9

[0084] The changes of ATP, ROS, mtDNA in each treatment group after 3 days of treatment of HepG2.2.15 cells by SB203580 and vitamin A, the expression levels of HBsAg and pgRNA in the supernatant of cells, and the expression levels of total RNA and pgRNA in the cells after 1 day of treatment of HepG2.2.15 cells by SB203580 and vitamin A are measured, and the results are shown in Figure 12 wherein Figure 12 (a) is the change of ATP in each treatment group, (b) is the change of ROS in each treatment group, (c) is the change of mtDNA in each treatment group, (d) is the expression level of HBsAg in the supernatant of cells, (e) is the expression level of pgRNA in the supernatant of cells, (f) is the expression level of pgRNA in the cells of each treatment group, and (g) is the expression level of total RNA in the cells of each treatment group.

[0085] As can be seen from (a), (b) and (c) in Figure 12 compared with the treatment of vitamin A alone, the treatment of SB203580 significantly inhibited the ATP production and mtDNA increase induced by vitamin A, and also blocked the effect of vitamin A on reducing ROS. As can be seen from (d), (e), (f) and (g) in Figure 12 the ability of vitamin A to inhibit HBsAg and pgRNA in the supernatant of HepG2.2.15 cells and total RNA and pgRNA in the cells was also antagonized after the addition of the inhibitor. The above results show that vitamin A can indeed improve mitochondrial function and synthesis through p38 MAPK / PGC-1α, and thus inhibit HBV replication.

[0086] Therefore, the present application provides the use of vitamin A in the preparation of a drug for treating or preventing hepatitis. Vitamin A can promote the biosynthesis and function of mitochondria in HBV-infected hepatocytes through the p38 MAPK / PGC-1α signaling pathway to inhibit HBV replication, thereby providing a new treatment option for CHB patients.

[0087] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit them. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can still be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. Use of vitamin A in the preparation of a drug for treating or preventing hepatitis.

2. Use of vitamin A according to claim 1 for the preparation of a medicament for the treatment or prevention of hepatitis, characterized in that: The hepatitis is hepatitis B.

3. Use of vitamin A according to claim 2 for the preparation of a medicament for the treatment or prevention of hepatitis, characterized in that: The hepatitis B is chronic hepatitis B.

4. Use of vitamin A according to claim 3 for the preparation of a medicament for the treatment or prevention of hepatitis, characterized in that: The chronic hepatitis B is a disease caused by hepatitis B virus infection, accompanied by mitochondrial dysfunction.

5. Use of vitamin A according to claim 4 for the preparation of a medicament for the treatment or prevention of hepatitis, characterized in that: Vitamin A promotes mitochondrial biogenesis and function by activating the p38 MAPK / PGC-1α signaling pathway, thereby increasing the level of oxidative phosphorylation and inducing interferon-stimulated gene expression, and inhibiting hepatitis B virus replication to treat or prevent chronic hepatitis B.

6. A medicament for treating or preventing hepatitis, characterized by comprising the compound of claim 1. The drug comprises vitamin A and a pharmaceutical carrier.

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