Li Wang,#1Dan Wang,#2Xingan Wu,#3Rui Xu,1 and Yunlan Li4Author informationArticle notesCopyright and License informationDisclaimer
Associated Data
Supplementary MaterialsData Availability StatementGo to:
Abstract
Background
Carvacrol, as the major components of aromatic plants used for treating human skin diseases including origanum, Satureja, thymus, and coridothymus species, presented a kind of antiviral activity. To explore the mechanisms of carvacrol against herpes simplex virus (HSV) in vitro.
Method
The BSC-1 cells model of HSV infection was established, and from the two aspects of viral replication level and cell death pathway, the antiviral effects of carvacrol on HSV infected cells were also evaluated by plaque assay under the three modes including prevention, treatment, and direct inactivation.
Results
In the three ways, the half-maximal effective concentration (EC50) of 2% true carvacrol solution on HSV-2 infected cells were severally 0.43, 0.19 and 0.51 mmol/L, and the corresponding therapeutic index (TI) were 4.02, 9.11 and 3.39, respectively. It’s the opposite of the increased levels caused by HSV-2 infection, that both the expressions at the transcription genes and protein levels of virus own replication key factors (including ICP4, ICP27, VP16, gB, and UL30) and cytokines (including RIP3, TNF-α, and MLKL) of infected cells treated with carvacrol were dose-dependently inhibited. Besides, HSV-2 infection can cause the decrease of intracellular protein ubiquitination level, and carvacrol can reverse the ubiquitination decrease level caused by HSV-2 infection.
Conclusion
Carvacrol exhibits significant antiviral activity by inhibiting the HSV-2 proliferation process and HSV-2-induced TNF-α increasing levels, decreasing RIP3 and MLKL protein expressions through the intracellular RIP3-mediated programmed cell necrosis pathway. In addition, carvacrol also may exhibit anti-HSV-2 activity by reversing the ubiquitination decrease level caused by HSV-2 infection on the ubiquitin-proteasome system, which provides insights into the molecular mechanism.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12879-020-05556-9.Keywords: Carvacrol, Herpes simplex virus-2 (HSV-2), Antiviral activity, Programmed cell necrosis, Ubiquitin-proteasomeGo to:
Background
Herpes simplex virus (HSV) is enveloped, linear and double-stranded DNA virus, which belongs to the family herpesviridae, genus alphaherpesvirinae. It is one of the most common pathogenic agents in humans and divided into two types: HSV-1 and HSV-2 [1]. There is increasing awareness of the importance of skin infection disease caused by HSV infection. Several of emerging cases were found continuously in recent years [2, 3], which indicated an upward trend in HSV incidence. Extensive evidence proved that HSV viruses could typically cause severe afflictions, mainly because of the generation of genital lesions and severe infections like life-threatening encephalitis and disseminated infections in neonates [4–6]. The HSV is also associated with potentially fatal viral stromal keratitis, an ocular disease, which is a leading cause of cornea-derived blindness in developed countries [7]. Currently, the approved primary anti-HSV therapeutic drugs are acyclovir (ACV) and its derivatives, which interfere with viral DNA synthesis to reduce viral replication and transmission. But all mentioned nucleoside analogs drugs, including ACV, ganciclovir and penciclovir, were oriented on the same molecular mechanisms of action that hinders viral DNA synthesis by competitively inhibiting viral DNA polymerase or adding itself to viral DNA, and the multi-drug resistant HSV viral strains were starting to show up more and more with heavy use of nucleoside analogs agents. Besides, the disadvantages of their narrow antiviral spectrum and high costs gradually also aggravated people’s living burden [8–10], extremely in America [11]. For these reasons, there is a need for the development of novel antiherpes drugs which are safe and preferably inexpensive with limiting the primary infection and supporting further treatment.
A large number of herbs and aromatic plants are frequently used for treating human skin diseases, especially from the family of Lamiaceae including origanum, satureja, thymus, and coridothymus species [12]. Carvacrol, a monoterpene phenol that is also known as 2-methyl-5-(1- methyl ethyl)-phenol, is one of the significant components of oregano essential oils, and presents a wide diversity of biological activities, such as antiviral [13–15], anticancer [12], antimicrobial [16], antioxidant and anti-inflammatory [17, 18]. Besides, carvacrol also has been identified as a natural, economical food preservative. Currently, the carvacrol-related health products, including 60 soft gels and 60 vegetarian capsules, are available for antioxidant treatments on the market. The relevant literature have also indicated that carvacrol has an safety and tolerability effect on healthy volunteers through a phaseIclinical trial and possible therapeutic effect on asthmatic patients through a phase II clinical trial in recent years [19, 20]. Carvacrol could exert antiviral activity by preventing the death of cells infected with HSV, but the specific mechanism of it against HSV virus has not been reported up to now [21, 22]. As a new alternative energy, carvacrol provides a new possibility for the development of HSV treatment and preventive health care drugs with advantages of great source, safety, low toxicity, and nature.
So the purpose of this paper was to explore the antiviral activity of carvacrol against HSV in vitro by plaque assay. The possible mechanisms of carvacrol’s antiviral effect on HSV-2 infected BSC-1 cells were studied from two aspects of viral replication level and cell death pathway through molecular biological techniques, which can provide adequate theoretical supports for the discovery of new antiviral drugs and alternative energy.Go to:
Methods
Carvacrol, cells, virus strains and major reagents
Carvacrol (Oregano oil; Purity: 99.8%) and 2% carvacrol true solution, prepared by dissolving 2 mL carvacrol with 33% sulfur-β-paste in distilled water at 100 mL, were kindly provided by prof. S. W from the air force medical university in China. Vero cells and HSV laboratory standard virus strain (HSV-1-F strain and HSV-2-G strain) were kindly gifted by prof. X. A from the air force medical university. BSC-1 cells were kindly donated by prof. Z. Q from Wuhan University. Vero and BSC-1 cells were incubated under Dulbecco-modified eagle’s medium (DMEM, high glucose) with 10% fetal bovine serum (FBS) at 37 °C in the atmosphere containing 5% CO2. HSV strains were grown for 3 ~ 4 days on cells in an atmosphere of 5% CO2 at 37, and the virus stock solution was stored at − 80 °C until use. Whereafter, the plaque assay [23] was performed to determinate viral multiplicity of infection (MOI) on cells. Of which, high glucose DMEM medium, FBS and cell counting CCK8 kits were purchased from Shanghai sangon biological engineering co. Ltd. The following antibodies were used: anti-ICP4 polyclonal antibody (Abcam; ab96432), anti-ICP27 monoclonal antibody (Abcam; ab31631), anti-VP16 monoclonal antibody (Abcam; ab110226), anti-gB monoclonal antibody (Abcam; ab6506), anti-Caspase-3 antibody (Abcam; ab 90,437), anti-Ub antibody (Abcam; ab7780), anti-RIP3 (Abcam; ab56164), anti-MLKL antibody (Abcam; ab184718), anti-Caspase-1 (Proteintech; 22,915–1-AP), anti-TNF-α (Proteintech; 60,291–1-lg). Goat anti-rabbit infrared secondary antibody (IR Dye800CW, 926–32,211) and goat anti-mouse infrared secondary (IR Dye680RD; 926–68,070) were purchased from American LICOR biosciences. Secondary antibody binding to Alexa Fluor 488 or Cy3 was purchased from Xi’an Zhuangzhi biotechnology co.Ltd. Fast 1000 total RNA rapid extraction kit, 5 × PrimeScript RT Master Mix (TakaRa), RNase Free dH2O (TakaRa) and TB GreenTM Premix Ex TaqTM II (TakaRa) were obtained from Xi’an kehao biological engineering co. Ltd.
You can read the full article at NCBI https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7661259/
