| BackgroundSevere acute respiratory syndrome (SARS) is a novel fatal infectious disease to hit the international community in the early 21st century while SARS shocked the whole world because of its spreading quickly, higher mortality rate and lacking a rapid reliable diagnostic test, effective specific therapy and vaccination. By the concerted efforts of all medical fields, it has been found that the pathogen of SARS is a novel mutant of an animal coronavirus, called SARS-CoV, which has probably jumped from an animal host to humans. So far, many questions about SARS remain unanswered, including the origin, pathogenesis, natural history, specific agents against SARS-CoV and so on. Therefore, the studies on virology, epidemiology, pathogenesis, diagnosis, treatment, community prevention of SARS disease have been being the hot spots in our country and the worldwide.Because the vaccines and micromolecule compounds against SARS-CoV virus have been created not yet, it would be a shortcut to find some Chinese herbal medicines for treating severe acute respiratory syndrome. Chinese herbal medicines have been used for infectious diseases for thousand years, about 189 kinds of the traditional Chinese medicines with antiviral activity and 273 kinds of those with immunological regulating functions have been found. The traditional Chinese medicines are low price and little side-effects, and have exhibited the unique advantages in inhibiting viral proliferation, regulating host immune functions and improving the pathological damage of infectious diseases.ObjectiveTo choice a kind of murine coronavirus (murine hepatitis virus strain 3, MHV-3) which has a higher homology feature with SARS-CoV and establish a infective cell model and the fulminant hepatitis mouse model for studying the effect of the injections of shuang-huang-lian,herba houttuyniae and allitridin on the action of inhibiting MHV-3 replication in the cell model and treatment and prevention of MHV-3 disease in the animal model, which would supply the experimental evidence to exploring anti-SARS-CoV agents.MethodsThe appropriate MOI was determined by means of plaque test. Then, MHV-3 infected L2 cell model for useful screening drugs against MHV-3 had been established. The maximum tolerance concentration (MTC) and median cytotoxic concentration (TC50) of L2 cells for shuang-huang-lian, herba houttuyniae and allitridin were confirmed by methyl thiazolyl tetrazolium (MTT) colorimetric assay. The inhibiting activities of the three drugs against MHV-3 were represented by IC50, which were measured by plaque-reduction assay. The serial concentrations of each drug for tests depended on its MTC and IC50. The efficacy of the drugs against MHV-3 viruses and their dosage-potency relationship were assessed. According to MTC of drugs, large, medium and small doses were determined by equimultiple dilution from MTC. Based on the virus replication cycle, three modes of drug treatment were used and testes were divided following four experimental groups, including①infected controls;②preventing group: Cells were treated with drug for 6h in advance, then infected with viruses and did not treated with drug any more;③counteracting group: Cells were added with viruses and drug at the same time. After finishing the viral absorption, the drug was removed;④treating group: Cells were treated with drug after the absorption of viruses. At 12h after finishing viral absorption and adding cultural media with or without drug, both cells and cultural supernatant were collected, melted by freezing for three times and centrifugated, of which 100μl of supernatant were further used for measuring viral titter.BALB/c mice aged 8 months were injected intraperitoneally with MHV-3 (100PFU/mouse) and animal models of fulminant virus hepatitis were established. The dosage of shuang-huang-lian (750mg/kg), herba houttuyniae (20mg/kg) and allitridin (75mg/kg) were calculated by certain formula according to the median dose of adult human. Three modes of drug usage were designed. The tested mice were divided four groups, including①infected controls;②treating group: after 30min of viral injection, 100μl of drug were given intraperitoneally with once every day. The longest duration was 7 days;③preventing group: mice was treated with drug for 3 days in advance, then challenged with viruses and didn't treated any more;④preventing plus treating group: mice were given preventing regimen, after viral infection, were treated with drug for 7days of longest duration. 180 mice were randomly divided into four groups of each drug, 18 mice in each subgroup. At 72h after infection, 6 live mice from each subgroup were randomly sacrificed and their blood and liver tissue specimens were obtained for following tests. Another 5 normal mice were served as normal controls for plasma ALT measure.The survival time of tested mice were observed and analyzed by SPSS 12.0 software. Plasma ALT levels of above blood samples were measured by biochemical auto-analyzer. The viral titter of liver tissue specimens were detected by plaque formation assay, and the pathological changes were assessed by Rezkalla analyzing method, in which the ratio of the area of necrosis and inflammatory cell infiltration to the area of whole visual field were used for semi-quantitative analysis.ResultsIt was found that 0.0001 of MOI was the optical viral infection amount for establishing the infected cell models. For shuang-huang-lian, herba houttuyniae and allitridin injections, their MTC were 1000μg/ml, 51.29μg/ml and 7.6μg/ml, their TC50 were 6100μg/ml, 500μg/ml and 75μg/ml, and their IC50 were 304.9μg/ml, 22.06μg/ml and 2.7μg/ml, the treatment index (TI= TC50/ IC50) were 20.07, 22.67 and 27.78, respectively.The results of plaque inhibiting rates of different subgroups of three drugs with different doses showed that for the groups of allitridin with large, medium and small doses plaque inhibiting rates were 69.70%, 57.58% and 45.45% in treating groups, 60.40%, 48.51% and 36.63% in counteracting groups, 56.8%, 39.22% and 25.49% in preventing groups; for the groups of shuang-huang-lian with three doses were 73.77%, 62.50% and 48.08% in treating groups, 69.42%, 57.52% and 42.53% in counteracting groups, 65.87%, 53.15% and 38.79% in preventing groups; for the groups of herba houttuyniae with three doses were 70.53%, 54.55% and 35.79% in treating groups, 65.34%, 51.49% and 32.67% in counteracting groups, 61.76%, 50% and 29.41% in preventing groups, respectively. All three drugs exhibited obvious anti-MHV-3 activities in a dose-dependent way in the infected cell models. By comparison, inhibiting effect was the strongest in treating group, stronger in counteracting group, and less strong in preventing group.All fulminant hepatitis mice died within 5 days after infection. Some model mice treating by three drugs with three regimens survived for 7 days after infection. The results of survival analysis for tested groups of animal models showed that for allitridin drug, survival time of preventing group and preventing plus treating group was significantly longer than those of infected controls and treated group (P<0.05); for shuang-huang-lian, survival time of all three intervention groups were obviously longer than that of infected controls (P<0.05) and that of preventing plus treating group significantly longer than of treating group(P<0.05); for herba houttuyniae, survival time of all three intervention groups were also obviously longer than that of infected controls (P<0.05) and those of preventing group and preventing plus treating group significantly longer than of treating group too.The mean plasma ALT levels are extremely high in infected models with 5573 U/L, and had significantly decreased in all drug intervention groups, ranged from 2716.21 U/L to 4453 U/L (P<0.01). By comparison, ALT levels of all preventing groups and preventing plus treating groups were markedly lower than that of infected controls (P<0.01).Viral titter in liver tissue showed a bit similar changes to plasma ALT, which in all drug intervention groups were significantly lower than of infected models (P<0.05), but only that of preventing plus treating group of shuang-huang-lian injection obviously lower than of corresponding treating group (P<0.05).In infected models includes, the livers were distinctly enlarged with the color of dark reddish brown purple and showed under the light microscope that liver cells died a great lot, the structure of hepatic lobules was severely damaged, there were hemorrhage and thrombosis within hepatic sinuses and inflammatory cell infiltration in the necrotic area. In the mice treated with drugs, there are no obvious differences between treating group and model controls, but in preventing plus treating groups and preventing groups the liver volume was a bit minished with the color of duck red, the liver necrosis and inflammatory cell infiltration were slightly improved and there was no thrombus in hepatic sinuses.ConclusionsA MHV-3infected cell model for screening antiviral agents and studing the pathogenesis of diseases had been successfully established. In cell models in vitro, three traditional Chinese preparations, shuang-huang-lian, herba houttuyniae and allitridin could obviously inhibit the replication of MHV-3 in a dose-dependent way, which might be involved in the process of viral absorption to and entrance into cells and the replication in the cells. By comparison, treatment index of allitridin was the highest; second of herba houttuyniae and third of shuang-huang-lian. In mouse models with fulminant hepatitis caused by MHV-3 virus, all three drugs also showed inhibiting activities against MHV-3 by significantly decreasing the viral load within liver tissues. The anti-MHV-3 effect of shuang-huang-lian was the strongest in vivo. The preventing modes of three drugs could improve the outcome of mice with fulminant hepatitis by obviously prolonging the survival time and alleviating liver disfunction, indicating that preventive effect of these drugs excelled their treating potency, and they might be the candidates of preventive agents for community control during the epidemic period of SARS. |
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