Application of rosmarinic acid in treatment of viral myocarditis caused by group B type 3 coxsackie virus

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of rosmarinic acid (RA) in treatment of viral myocarditis caused by group B type 3 coxsackie virus (CVB3). Mouse animal model research finds that the rosmarinic acid can significantly reduce the virus load in CVB3 infected mouse cardiac muscle tissue, and proves that the rosmarinic acid has the effect of inhibiting CVB3 virus replication; meanwhile, the treatment can obviously improve cardiac function impairment caused by CVB3 virus infection and relieve inflammatory response of cardiac muscle tissue, and the release of key inflammatory factors and myocardial injury factors is reduced. The invention provides an experimental basis for treating viral myocarditis by using rosmarinic acid as an anti-CVB3 virus drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of rosmarinic acid (RA) in the treatment of viral myocarditis caused by Coxsackievirus type B3 (CVB3). Background Technology

[0002] Viral myocarditis (VMC) is a localized or diffuse inflammatory disease of the myocardium caused directly by viral infection or through immune-mediated mechanisms. Among numerous pathogens, Coxsackievirus B (CVB), especially CVB3, is one of the most prevalent causes of viral myocarditis in humans. CVB3 infection can lead to myocardial cell degeneration and necrosis, and trigger intense inflammatory infiltration and oxidative stress damage. In severe cases, it can develop into dilated cardiomyopathy, heart failure, or even sudden death, posing a serious threat to the patient's life and health.

[0003] Currently, there are no specific and highly effective drugs for the treatment of viral myocarditis in clinical practice. Conventional treatment mainly focuses on symptomatic and supportive care and comprehensive management, including: Antiviral therapy: such as interferon, ribavirin and other broad-spectrum antiviral drugs, but their efficacy in the treatment of myocarditis is limited, and there are problems such as the virus easily developing drug resistance and obvious side effects.

[0004] Immunomodulation and anti-inflammatory therapy: such as intravenous immunoglobulin (IVIG) and glucocorticoids, are used to control excessive immune responses and cytokine storms. However, the timing and dosage of immunosuppressants are difficult to control; premature or excessive use may suppress necessary antiviral immunity, thereby exacerbating viral replication.

[0005] Symptomatic and supportive treatments, including rest, myocardial nutrition, antiarrhythmics, and correction of heart failure, can alleviate symptoms but cannot fundamentally inhibit viral replication or block the process of myocardial damage.

[0006] Antioxidant therapy: Given the important role of oxidative stress in myocardial injury, some antioxidants (such as coenzyme Q10) have been tried as adjunctive therapy, but their efficacy when used alone is uncertain and they lack specificity against CVB3.

[0007] Therefore, developing novel multi-target therapeutic drugs that can effectively inhibit CVB3 virus replication while reducing myocardial inflammation and oxidative damage has become a key scientific problem and clinical need that urgently needs to be solved in this field.

[0008] Rosmarinic acid (RA) is a naturally occurring, water-soluble phenolic acid compound widely found in various plants belonging to the Lamiaceae and Boraginaceae families. Numerous studies have demonstrated that rosmarinic acid possesses diverse pharmacological activities, including antioxidant, anti-inflammatory, antitumor, antibacterial, and neuroprotective effects. Particularly in the cardiovascular system, research suggests that rosmarinic acid may exert a protective effect against ischemia-reperfusion injury and atherosclerosis models through its antioxidant and anti-inflammatory properties. Furthermore, a few studies have reported that rosmarinic acid exhibits inhibitory activity against certain viruses, such as influenza virus and herpes simplex virus.

[0009] However, to date: No published literature or patents report that rosmarinic acid has anti-CVB3 virus activity.

[0010] No research has been found on the use of rosmarinic acid for the prevention or treatment of viral myocarditis caused by CVB3 infection.

[0011] Although rosmarinic acid is known to have anti-inflammatory and antioxidant activities, it remains unknown whether and how it exerts synergistic antiviral, anti-inflammatory, and cardioprotective effects in the CVB3 myocarditis model by regulating specific host cell signaling pathways (such as the SIRT1 / AMPK / Nrf2 pathway). Summary of the Invention

[0012] The purpose of this invention is to provide the use of rosmarinic acid (RA) in the treatment of viral myocarditis caused by Coxsackievirus type B3 (CVB3).

[0013] The technical solution adopted in this invention is as follows: The application of rosmarinic acid in the preparation of drugs for the treatment of viral myocarditis; Furthermore, the viral myocarditis is caused by Coxsackievirus B3.

[0014] A drug for treating viral myocarditis, the drug comprising rosmarinic acid as an active ingredient; Furthermore, the viral myocarditis is caused by Coxsackievirus B3; Furthermore, the dosage form of the drug includes granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or solutions.

[0015] Application of rosmarinic acid in the preparation of drugs against group B3 Coxsackievirus.

[0016] An anti-coxsackievirus B3 drug, wherein the anti-coxsackievirus B3 drug contains rosmarinic acid as an active ingredient; Furthermore, the dosage forms of the anti-group B3 Coxsackievirus drug include granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or solutions. Attached Figure Description

[0017] Figure 1 Effects of rosmarinic acid on the survival of CVB3-infected mice. (A) Schematic diagram of animal experimental procedure. (B) Weight change curves of mice in each group within 7 days after infection (n=5). (C) Survival rate curves of mice in each group within 7 days after infection (n=5).

[0018] Figure 2 : Pathological analysis of mouse myocardial tissue (HE staining, 400X).

[0019] Figure 3 Detection of serum pro-inflammatory cytokine levels in mice. The concentrations of (A) IFN-α, (B) IL-1β, (C) IL-6, and (D) TNF-α in serum were detected by qRT-PCR (n=5). Compared with the model group,* p <0.05, ** p <0.01.

[0020] Figure 4 Detection of serum myocardial injury markers in mice. The activities of (A) LDH and (B) CK-MB in serum were detected using a fully automated biochemical analyzer (n=5). Compared with the model group, * p <0.05, ** p <0.01.

[0021] Figure 5 Detection of CVB3 RNA expression levels in myocardial tissue. The relative expression levels of CVB3 RNA in myocardial tissue were detected by qRT-PCR (n=5). Compared with the model group, *** p <0.001.

[0022] Figure 6 Effects of rosmarinic acid on CVB3 replication and expression of key host signaling pathway proteins in myocardial tissue. Western blotting was used to detect the expression levels of CVB3 3D protein and key host signaling pathway proteins such as SIRT1, p-AMPK-α, AMPK-α, Nrf2, HO-1, and nuclear factor NF-κB P65 (nNF-κB P65) in myocardial tissue of mice in each group. GAPDH and Tubulin were used as internal controls. Detailed Implementation

[0023] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0024] The CAS number of rosmarinic acid (RA) involved in the embodiments of the present invention is 20283-92-5.

[0025] Example 1: Rosmarinic acid improves survival rate and body weight loss in mice infected with Coxsackievirus type 3 (C3) in group B. To evaluate the efficacy of rosmarinic acid (RA) in treating viral myocarditis induced by group B3 Coxsackievirus (CVB3), a mouse model was first established, and its basic survival indicators were observed. The specific methods were as follows: Mice were randomly divided into three groups: ① normal control group (Control / Mock); ② CVB3 infection model group (CVB3); ③ CVB3 infection and rosmarinic acid treatment group (CVB3 / RA). Except for the normal control group, all other groups of mice were intraperitoneally injected with 10 mg of rosmarinic acid on day 0. 6 TCID 50 A viral myocarditis model was established using CVB3 viral fluid. The CVB3 / RA group received daily intraperitoneal injections of 100 mg / kg rosmarinic acid starting 12 hours post-infection and continuing until the end of the experiment (day 7 post-infection). Body weight and survival rates were monitored and recorded daily during this period.

[0026] The effect of rosmarinic acid on the survival status of CVB3-infected mice is as follows: Figure 1As shown in the figure, the normal control group mice showed steady weight gain throughout the 7-day observation period. In stark contrast, the CVB3 model group mice experienced a rapid and sustained weight loss starting from day 2 post-infection until the end of the experiment, indicating that the viral infection led to a severe wasting pathological state. The weight change trend of the rosmarinic acid treatment group mice was intermediate between the two: they experienced some weight loss in the early stages of infection (days 1-3), but the decline was significantly less than that in the CVB3 model group; from day 4 onwards, the weight loss trend was curbed and began to gradually recover, with their weight significantly higher than that of the CVB3 model group by day 7. This indicates that rosmarinic acid treatment can effectively alleviate weight loss caused by viral infection and promote the body's compensation and recovery. All normal control group mice survived, with a survival rate of 100%. The survival condition of the CVB3 model group mice was severe; they began to die from day 3 post-infection, and the survival rate was significantly reduced by the end of the experiment on day 7, demonstrating the high lethality of CVB3 infection. In contrast, the survival rate of CVB3 / RA mice treated with rosmarinic acid was significantly higher than that of the CVB3 model group throughout the observation period. Treatment not only delayed the onset of death but also significantly reduced the cumulative number of deaths, directly demonstrating that rosmarinic acid can significantly improve the survival rate of CVB3-infected mice and has a clear protective effect. This lays a solid foundation for further in-depth research into the specific molecular mechanisms of rosmarinic acid in cardioprotection, anti-inflammation, and antiviral effects, providing a comprehensive therapeutic basis.

[0027] Example 2: Rosmarinic acid improves myocardial tissue pathological damage caused by CVB3 infection To evaluate the protective effect of rosmarinic acid against myocardial tissue pathological damage caused by CVB3 infection, based on Example 1, heart tissues of mice in each group were collected on day 7 post-infection and histological analysis was performed by hematoxylin-eosin (HE) staining.

[0028] Pathological analysis of mouse myocardial tissue, such as Figure 2 As shown, the cardiomyocytes of the normal control group mice were structurally intact and neatly arranged, with no obvious inflammatory cell infiltration or necrosis areas. In contrast, the myocardial tissue of the CVB3 model group mice exhibited typical severe pathological changes of viral myocarditis, including diffuse infiltration of a large number of inflammatory cells (mainly lymphocytes), myocardial fiber rupture and dissolution, and obvious focal necrosis areas. After treatment with rosmarinic acid (CVB3 / RA), the pathological damage of the mouse myocardial tissue was significantly improved, with a significant reduction in the extent and degree of inflammatory cell infiltration, and more regular arrangement of myocardial fibers and a reduction in necrotic areas. These histopathological results indicate that rosmarinic acid can effectively reduce the direct pathological damage to myocardial tissue caused by CVB3 infection and has a significant protective effect on cardiomyocytes.

[0029] Example 3: Rosmarinic acid reduces the level of pro-inflammatory cytokines in the serum of CVB3-infected mice. To investigate the inhibitory effect of rosmarinic acid on systemic inflammatory response induced by CVB3 infection, serum from mice in each group was collected on day 7 post-infection, based on Example 1, and the levels of key pro-inflammatory cytokines were detected by real-time quantitative PCR (qRT-PCR).

[0030] Results of mouse serum pro-inflammatory cytokine level detection: Figure 3 As shown in the figure, compared with the normal control group, the serum concentrations of pro-inflammatory cytokines, including interferon-α (IFN-α), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), in the CVB3 model group mice were significantly increased, indicating that viral infection triggered a severe systemic inflammatory response. However, after treatment with rosmarinic acid, the levels of all the above-mentioned pro-inflammatory cytokines in the mouse serum decreased significantly. Although they did not completely return to normal levels, they were significantly controlled compared with the CVB3 model group. This result indicates that rosmarinic acid can effectively inhibit the systemic pro-inflammatory cytokine release storm caused by CVB3 infection. This is consistent with the pathological result of reducing inflammatory cell infiltration in myocardial tissue, jointly demonstrating that rosmarinic acid has a significant anti-inflammatory effect, which may be one of the important mechanisms for its treatment of viral myocarditis.

[0031] Example 4: Rosmarinic acid reduces the level of myocardial injury markers in the serum of CVB3-infected mice. To evaluate the protective effect of rosmarinic acid against cardiomyocyte damage caused by CVB3 infection, based on Example 1, serum from mice in each group was collected on day 7 post-infection, and key enzymatic markers of myocardial injury were detected using a fully automated biochemical analyzer.

[0032] Results of mouse serum myocardial injury marker detection: Figure 4 As shown in the figure, compared with the normal control group, the activities of creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) in the serum of mice in the CVB3 model group were significantly increased, indicating clear cardiomyocyte damage and necrosis. After treatment with rosmarinic acid, the activities of CK-MB and LDH in the serum of mice significantly decreased, indicating that myocardial damage was effectively contained. This result indicates that rosmarinic acid can effectively reduce the direct damage to cardiomyocytes caused by CVB3 infection and protect the integrity of cardiomyocytes. This discovery based on serum enzymatic markers, together with the damage reduction and inhibition of inflammatory response observed in histopathology, demonstrates the multiple therapeutic effects of rosmarinic acid on viral myocarditis.

[0033] Example 5: Rosmarinic acid inhibits CVB3 RNA replication in myocardial tissue To verify the inhibitory effect of rosmarinic acid on CVB3 at the molecular level, based on Example 1, total RNA was extracted from the myocardial tissue of mice in each group on day 7 post-infection, and the expression level of CVB3 RNA was detected by real-time quantitative PCR (qRT-PCR).

[0034] Results of CVB3 RNA expression level detection in myocardial tissue are as follows Figure 5 As shown in the figure, qRT-PCR analysis revealed that, compared with the CVB3 model group, mice treated with rosmarinic acid exhibited significantly reduced CVB3 RNA expression in their myocardial tissue. No CVB3 RNA was detected in the myocardial tissue of the normal control group (Control). This result directly confirms at the gene transcription level that rosmarinic acid can effectively inhibit CVB3 replication in myocardial tissue. This is highly consistent with the aforementioned results showing that rosmarinic acid reduces serum myocardial injury markers and alleviates myocardial pathological damage, jointly indicating that inhibiting viral replication is one of the core mechanisms of action of rosmarinic acid in treating viral myocarditis.

[0035] Example 6: Rosmarinic acid inhibits CVB3 replication and inflammatory response by regulating the SIRT1 / AMPK / Nrf2 signaling pathway. To elucidate the underlying mechanism of rosmarinic acid (RA) in treating CVB3 viral myocarditis, focusing on its regulatory role on key viral replication proteins and host signaling pathways, based on Example 1, total protein was extracted from the myocardial tissue of mice in each group on day 7 post-infection, and the expression level of CVB3 3D protein was detected by Western blotting. Western blotting analysis directly confirmed (… Figure 6 Rosmarinic acid treatment significantly reduced the protein expression level of 3D polymerase, essential for CVB3 replication, in myocardial tissue. This confirmed its potent direct antiviral effect at the functional protein level, providing conclusive evidence for the previously observed decrease in viral RNA levels. This strong antiviral effect is inseparable from rosmarinic acid's regulation of the host cellular environment. Further studies showed that rosmarinic acid effectively activated the SIRT1 / AMPK / Nrf2 signaling pathway, manifested as a significant upregulation of SIRT1 and Nrf2 protein expression and AMPK-α phosphorylation levels. Experimental results showed that rosmarinic acid promoted the stabilization and nuclear translocation of the transcription factor Nrf2 and induced the upregulation of its target gene heme oxygenase-1 (HO-1), alleviating virus-induced myocardial oxidative stress damage. Furthermore, the level of NF-κB p65 was significantly reduced in the rosmarinic acid treatment group, which is consistent with the previous findings of a significant decrease in the levels of serum pro-inflammatory factors (TNF-α, IL-6, IL-1β), clearly elucidating the mechanism by which rosmarinic acid inhibits myocardial inflammation.

[0036] In summary, this study found that rosmarinic acid synergistically enhances Nrf2-mediated antioxidant defense and inhibits NF-κB-mediated inflammatory responses by activating the SIRT1-AMPK signaling hub. At the same time, the improvement of these host cell pathways creates a microenvironment unfavorable to viral replication, ultimately leading to the inhibition of CVB3 replication.

Claims

1. The application of rosmarinic acid in the preparation of drugs for the treatment of viral myocarditis.

2. The application according to claim 1, characterized in that: The viral myocarditis was caused by Coxsackievirus B3.

3. A drug for treating viral myocarditis, characterized in that: The drug contains rosmarinic acid as its active ingredient.

4. The drug according to claim 3, characterized in that: The viral myocarditis was caused by Coxsackievirus B3.

5. The drug according to claim 3, characterized in that: The dosage forms of the drug include granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or solutions.

6. Application of rosmarinic acid in the preparation of drugs against group B3 Coxsackievirus.

7. A drug for treating Coxsackievirus type B3, characterized in that: The drug against Coxsackievirus B3 contains rosmarinic acid as its active ingredient.

8. The anti-coxsackievirus B3 drug according to claim 7, characterized in that: The dosage forms of the anti-group B3 Coxsackievirus drug include granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or solutions.

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