ABSTRACT
In this study, according to TCM theory of "liver qi stagnation forming fire", emotional stress mice model was employed to evaluate the protective effects of Qingre Xiaoyanning on herpes simplex virus type 1 (HSV-1) induced reactivation. The animal experimental protocol has been reviewed and approved by Laboratory Animal Ethics Committee of Jinan University, in compliance with the Institutional Animal Care Guidelines. BALB/c mice were divided into six groups, including mock group, HSV-1 latency group, HSV-1 reactivation group (HSV-1 latency + stress), low (0.658 g·kg-1·day-1) and high dose (1.316 g·kg-1·day-1) of Qingre Xiaoyanning groups and positive control group (acyclovir, 0.206 g·kg-1·day-1). Except for the normal group and HSV-1 latency group, all mice in other groups received a daily 12-h restraint stress for 4 days. After 7-day treatment of drugs, body weight and recurrent eye infections of mice were recorded. Brain tissues were harvested to monitor HSV-1 antigen distribution by immunohistochemical staining and detect virus titer by plaque assay. In the meantime, the mRNA and protein levels of infected cell polypeptide (ICP27) and glycoprotein B (gB) in the brain tissues were detected by RT-PCR and Western blot, respectively. The level of 4-hydroxynonenal (4-HNE) and expressions of ferroptosis-related proteins were measured by Western blot. The evaluation of malondialdehyde (MDA) content in the brain tissues was conducted by MDA assay commercial kit. The results showed that Qingre Xiaoyanning significantly retarded the decline of body weight of mice induced by HSV-1 reactivation, reduced the activation rate of HSV-1 and recurrent eye infections, declined virus titer of HSV-1, down-regulated gene and protein expressions of ICP27 and gB, and hindered the distribution of HSV-1 antigen in the brain of mice. Meanwhile, Qingre Xiaoyanning also decreased the protein expression of ferroptosis-related proteins, including DMT1, TFR1 and ALOX15 in the brain tissue of HSV-1 reactivated mice. The levels of lipid peroxidation products, 4-HNE and MDA, were also reduced by Qingre Xiaoyanning treatment. All the above results indicate that Qingre Xiaoyanning significantly inhibited HSV-1 reactivation by restraint stress, which might be related to the regulation of ferroptosis. Our findings provide a theoretical basis for the application of "clearing liver-fire" TCM on treatmenting HSV-1 reactivation-related symptoms.
ABSTRACT
In this study, emotional stress-induced herpes simplex virus type 1(HSV-1) susceptibility model was employed to simu-late the pathological state of " depression-induced liver fire", and the protection effect of Qingre Xiaoyanning(QX) in clearing liver fire was investigated. BALB/c mice were randomly divided into a normal group, a HSV-1 group, a restraint stress + HSV-1 group,low-(0. 658 g·kg~(-1)) and high-dose(1. 316 g·kg~(-1)) QX groups, and an acyclovir group. Except for the normal group and the HSV-1 group, the mice in other groups received daily restraint stress for 6 h from day 3 of medication. On day 9 of medication, mice were anesthetized by isoflurane and infected intranasally with HSV-1. Survival rate, weight change, encephalitis symptoms, and eye injury of mice were recorded for 14 d after virus infection. Hematoxylin-eosin(HE) staining and immunohistochemical staining were used to detect pathological changes and HSV-1 antigen distribution. Plaque assay was performed to detect the titer of HSV-1. The protein ex-pression of ICP27 in the mouse brain was detected by Western blot. The experimental results showed that QX could increase the survival rate of HSV-1-infected mice loaded with emotional stress(P<0. 001), reduce the titer of HSV-1 in the mouse brain(P<0. 01), relieve brain inflammation(P<0. 05) and eye injury(P<0. 05), down-regulate the expression of ICP27 related to HSV-1(P<0. 05), and decrease the distribution of HSV-1 antigen in the mouse brain. The results demonstrated that QX significantly reduced the susceptibility to HSV-1 induced by emotional stress, which is expected to provide a theoretical basis for the treatment and preven-tion of HSV-1 infection and promote the clinical development and application of Chinese medicine effective in clearing liver fire.