| Fructus Schisandrae, Fruit of Schisandra chinensis (Turcz.) Baill, was regarded as a top grade in traditional Chinese herbal medicine and officially recorded in the Chinese Pharmacopoeia. In traditional Chinese medicine, it is a common ingredient in prescriptions which has the effects of quenching thirst, invigorating kidney and stabling mind. The main active ingredients Schisandrin compounds. In this experiment, restraint stress loading was used as the experimental model. The influences of Schisandra Lignans Extract (SLE), the standards extract of schisandra, on the stress induced liver injury and liver cancer metastasis were studied and the anti-stress effect and possible mechanism of SLE were also investigated.This study was mainly focus on the research of Schisandra lignans, the major component of schisandra, through different extraction methods and determination of total lignans content. Results will be used to determine the optimal extraction procedures and total content determination method. At the same time, SLE fingerprints will be established by HPLC. Comparing the total content of lignans obtained by UV spectrophotometry and chromotropic acid method, UV spectrophotometry is more accurate and stable. Therefore UV spectrophotometry was used. Through the study of using different extraction method, the optimal extraction method was using macroporous resin with 90% ethanol as eluant where total lignan content was the highest (51%). Initial establishment of SLE fingerprints was completed by HPLC to identify the major peaks, which were Schizandrin, Gominsin A, Gominsin C, Deoxyschizandrin,γ-Schizandrin and Schi- zandrin C.Seven week old male KM mice was used in stress-induced liver injury experiment, using restraint stress animal model to evaluate the anti-stress effect SLE. Stress-induced liver injury was induced in mice after 18 h restraint stress load. Plasma alanine aminotransferase (ALT) and Cortico-sterone (Corticosterone) activity were determined by Reitman-Frankel assay. Liver Malondialdehyde (MDA) levels and antioxidant capacity (ORAC) levels were tested using thiobarbituric acid reactive substances assay and fluorescence microplate photometer respectively. The key free radical scavenging enzymes SOD and GPX activity were determined by colorimetry. RT-PCR was used to detect changes in liver SOD, GPX and glucocorticoid receptor (GR) mRNA expression. After 18 h restraint stress loading, ALT values in control group was 17.53±4.67 IU/L, while stress model group was 96.70±6.27 IU/L. Serum ALT levels in mice loaded with restraint stress was significantly increased (P<0.01), which suggested that restraint stress can cause serious liver damage. Mice Corticosterone levels in plasma were significantly increased, which showed that restraint stress-induced liver injury is related to changes of stress hormone levels. Administration of Schisandra standard extract can significantly reduce the stress-induced elevation of serum ALT activity in mice. ALT values in SLE low dose group was 34.76±2.26 IU/L while high dose group was 29.70±5.04 IU/L, showing that Schisandra standard extract has certain protective effect towards stress-induced liver damage. Levels of lipid peroxide (MDA content) was significantly higher in liver tissue of restraint-stressed mice. Besides, antioxidant capacity index (ORAC value) were significantly reduced. These phenomenon were indicating that antioxidative ability of liver tissue were significantly lowered, and the liver was under lipid peroxidation status. This also showed that stress-induced liver damage may be related to the increase of free radicals and endogenous antioxidant consumption in liver tissue. SLE high and low dose could ease the situation of lipid peroxidation in liver to a different extent. At the same time, SLE could increase ORAC levels, which illustrated that the protective effect of SLE on stress-induced liver damage may be associated with the remission of liver oxidative damage. Further mechanism studies showed that mice of stress model group, compared with control group, had a remarkably lowered SOD and GPX level in liver tissues. Mean while, RT-PCR results showed that mRNA expression of SOD1 and GPX in restraint stressed mice liver were significantly lowered (P<0.01). This result showed that restraint stress led to reduced antioxidant capacity. When comparing with restraint stress group, liver tissue SOD1 and GPX mRNA expression in SLE high dose group of were significantly increased (P<0.05). Experimental results showed that SLE could alleviate oxidative damage in mice liver, which might because of the active ingredients in SLE can increase both the enzyme activity and mRNA expression of free radical scavenger SOD and GPX. Therefore, Schisandra standard extract could lower plasma Corticosterone content in mice, reduce excessive MDA level in liver tissue and increase ORAC level, improve the activity and mRNA expression of scavenging key enzymes SOD and GPX. These are all indicating that the protective effect of Schisandra standard extract on stress-induced liver injury is related to the alleviation of lipid peroxidation of liver cell caused by stress.DAB/2 mice were tail vein injected with P815 tumor cells,3 days after 20 h of restraint stress load, to establish stress-induced cancer metastasis animal model. Changes in number of liver metastasis clones and spleen index were tested. Flow cytometry was used to determine changes in T lymphocyte subsets and CTL killing activity and RT-PCR was used to detect mRNA expression changes of liver Fas and Fas-L. Results showed that 12 days after intravenous injection of P815 tumor cells on tail in restraint stressed mice, the number of liver metastasis clones in control group was 10.80±3.59. Liver metastasis clones was increased to 31.40±4.90 in stress model group, demonstrating that restraint stress-induced liver cancer metastasis rate was significantly increased (P<0.01). Compared with the stress model group, the number of liver metastasis clones in SLE low and high dose were 26.40±4.81 and 18.40±3.72 respectively (P< 0.05). This result showed that SLE has significant inhibition on stress-induced carcinoma metastasis of P815 cell. Immune results showed that restraint stress lowered spleen index, while significantly reducing the total number of spleen cells and decreased Th and Tc cell ratio in spleen cells. SLE (100 and 200 mg/kg) administration can improve these indicators in varying degrees. Results of CTL activity tests, which is closely related to cancer combating, showed that restraint stress could significantly reduce CTL activity of spleen (LU10/spleen) when compared to control group (P<0.01). While Comparing with the stress model group, oral administration of 100 and 200 mg/kg SLE in mice could elevate spleen CTL activity (LU10/spleen) to different levels (P<0.05). In addition, research found that liver Fas/Fas-L mRNA expression ratio of stress model group decreased significantly compared with the control group (P<0.01). Compared with the stress model group, liver Fas/Fas-L mRAN expression was significantly increased in both SLE 100 and 200 mg/kg group. Results indicated that the anti-metastasis mechanism of SLE might because of the increase of Fas/Fas-L mRNA expression.In summary, SLE can be improved liver damage and cancer metastasis caused by restraint stress load. Its mechanism may be related to the elevated immune functions and alleviation of oxidative damage within cells. Therefore, the reveal of liver protective effects of SLE from anti-stress perspective using modern scientific methods are urgent and bound to form a good social benefits. |
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