Sarcandra glabra Extract Reduces the Susceptibility and Severity of Influenza in Restraint-Stressed Mice

Abstract

Sarcandra glabra, as a type of "antipyretic-detoxicate drugs", has always been widely used in traditional Chinese medicine (TCM). The Sarcandra glabra extract (SGE) is applied frequently as anti-inflammatory and anti-infectious drug in folk medicine. However, relative experiment data supporting this effective clinical consequence was limited. In order to mimic the physiological conditions of the susceptible population, we employed restraint stress mouse model to investigate the effect of SGE against influenza. Mice were infected with influenza virus three days after restraint, while SGE was orally administrated for 10 consecutive days. Body weight, morbidity, and mortality were recorded daily. Histopathologic changes, susceptibility genes expressions and inflammatory markers in lungs were determined. Our results showed that restraint stress significantly increased susceptibility and severity of influenza virus. However, oral administration of SGE could reduce morbidity, mortality and significantly prolonged survival time. The results further showed that SGE had a crucial effect on improving susceptibility markers levels to recover the balance of host defense system and inhibiting inflammatory cytokines levels through down-regulation of NF-κB protein expression to ameliorate the lung injury. These data showed that SGE reduced the susceptibility and severity of influenza.

Figure 1

Chemical fingerprint of SGE. Each peak of SGE in the HPLC fingerprint was identified by comparison of the retention times and UV spectra of chemically defined standard compounds. The standard compounds were isolated from SGE previously. Several batches of SGE were analyzed, and similar profiles were observed. HPLC condition was as follows: agilent series 1100 HPLC; column, Welch material XB-C18, 4.6 × 250 mm, particle size 5 mm; mobile phase A, H₂O with 0.2% HAc; mobile phase B, MeOH with 0.2% HAc; elution program: 20% B in 5 min, linear gradient from 20% B to 62% B in 55 min, 100% B in 12 min; flow rate at 0.80 mL/min; detection wavelength at 330 nm; injection volume in 10 μL and oven temperature at 35°C.

Figure 2

Effect of SGE on survival rates (a) and body weight changes (b) of restraint-stressed mice after infection. Three days before H1N1 virus infection, Kunming mice were fixed in a restraint cage for 18 h. Time course of mortality and survival days of each mouse were recorded until the 21st day after viral infection. Data were obtained from 9–11 animals in each group. **P < 0.01, *P < 0.05.

Figure 3

Mice showed few visible signs of external damage in lung lobes on day 5 postinfection. (a) Intact lungs. (b) Histopathologic changes in lung tissues collected at the 5th days postinfection. Representative histologic sections of lung tissues from experimental mice were stained by H&E, bar = 100 μm. (c) Lung index was calculated according to the following formula: lung index = lung weight (mg)/body weight (g). The results represented the mean ± SE of values obtained from 8 Kunming mice in each group. The significance of differences was from the normal control group at ^## P < 0.01 and from the model group at **P < 0.01, *P < 0.05.

Figure 4

Effect of SGE on susceptibility gene expressions in restraint-stressed mice. Viral NP, SP-A, SP-D, ICAM-1 and IFITM 3 mRNA expressions in lung tissues from 8 mice in each group were determined by RT-PCR and normalized by 18S. The significance of differences was from the normal control group at ^## P < 0.01, ^# P < 0.05, from virus control group at ^&& P < 0.01, ^& P < 0.05, and from the model group at **P < 0.01, *P < 0.05.

Figure 5

Effect of SGE on serum NO level in restraint-stressed mice after virus infection. Level of serum NO was determined by Griess test. The results represented the mean ± SE of values obtained from eight Kunming mice in each group. The significance of differences was from the normal control group at ^## P < 0.01 and from the model group at **P < 0.01, *P < 0.05.

Figure 6

Effect of SGE on mRNA expressions of proinflammatory cytokines of lung tissues in restraint-stressed mice postinfection. iNOs, TNF-α, and IL-1β mRNA expression in lung tissues from 8 mice in each group were determined by RT-PCR and normalized by 18S. The significance of differences were from the normal control group at ^## P < 0.01, from virus control group at ^&& P < 0.01, ^& P < 0.05, and from the model group at *P < 0.01, *P < 0.05.

Figure 7

Effect of SGE on expression of nuclear NF-κB p65 protein in restraint-stressed mice postinfection. Nuclear NF-κB p65 protein levels in lung tissues from 4 mice in each group were determined by western blotting and normalized by β-actin. The significance of differences was from the normal control group at (^##) P < 0.01 and from the model group at (**) P < 0.01.