| Background and purposeNeurodegenerative diseases are generally caused by the chronic and progressive degeneration of the central nervous system. These diseases usually include the Alzheimer’s disease (AD), Amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), Parkinson’s disease (PD), multiple sclerosis (MS) and other related disorders. Up to date, the etiology and pathogenesis of these diseases remain unclear. With the aging population in China, the neurodegenerative diseases such as AD and PD are becoming more and more prevalent. It has been shown that oxidative stress can cause oxidative damage of biological macromolecules, lead to apoptosis, and play an important role in the pathogenesis of AD and PD.Although many drugs have been applied to the treatment of various degenerative diseases of the nervous system,they often can cause serious side effects with a high cost and promote the generation of drug resistance which greatly reduced the effectiveness of long-term medication. Recently, many studies have shown that some traditional Chinese medicines can scavenge free radicals, inhibit oxidative stress and the resultant apoptosis, and thus display significant efficacy in the treatment of AD and PD.The purpose of this study is to analyze the antioxidant capacity of termite fungus garden and compatible medicines, and investigate their effects and possible mechanisms in the treatment of degenerative diseases of the nervous system, which may provide new knowledge for the development of drugs to treat neurological diseases.Methods1.The peritoneal cavities of rats were injected with the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and the PD-model rats were selected by the pole-climbing method. The Pingchan Fang medicines were composed of Astragalus, Tianma, ginkgo biloba, white peony root, Uncaria, forsythia, raw licorice,wild arrowroot,big leave and red Jing tian and berberine.The PD model rats were randomly grouped into treatment group and control group, and then were treated with the Pingchan Fang together with melatonin for21days.The rat behavior was documented. The rat brain sections were prepared and applied for hematoxylin-eosin (HE) staining, Bax/Bcl-2immunohisto chemical staining and TUNEL staining (apoptosis analysis). The content of dopamine (DA) in the brain homogenates was determined, as well as its metabolites homovanillic acid (HVA) and dihydroxy acid (DOPAC).2. The PD models of fruit fly (Drosophila) were prepared by the hybrid of wild-type (UAS-wild-type a-synuclein/+) and UAS-DdC flies. The wild-type and PD flies had been fed with different concentrations of alcohol extract of termites fungus garden for42d, and the life span and the behavior of them were analyzed. The brain tissue biopsy was performed and the sections were treated by HE staining.3. The PD models of mouses were prepared by subcutaneous injection of D-galactose, and were fed with different concentrations of alcohol extract of termites fungus garden, then the Morris water maze place navigation method was used to place mice in the pool from four quadrants. The average latency time was calculated to evaluate the mouse ability to obtain spatial memory. The measurements for SOD, MDA and GSH of these PD model mice were performed to evaluate the establishment of these models and the effects of ethanol extracts of the termite fungus garden. The changes of brain β-amyloid and the histopathological changes in the brains and livers were also examined.4. The oxidative stress model of neuroblastoma SH-SY5Y cells were prepared by H2O2treatment, and the effects of the application of alcohol extract of termites fungus garden are investigated. The MTT assay was used to determine the cell viability at different incubation time points. The SOD, MDA, GSH and other activities were also examined. In addition, the alcohol extract of termites fungus garden was applied both during and after the construction of the models to analyze the prevention and treatment effects of these extracts.5. The gastric cancer cells BGC-823were incubated with the water extract of the termites and fungus garden for24h to observing its effect on the growth of these cells under an inverted microscope. The cell survival was determined by Trypan blue staining method. The cell viability was measured by MTT method. The apoptosis was examined by the flow cytometry. The changes of p53gene expression levels were tedected using quantitative PCR analysis.Results1. The PD animal models were successfully constructed by MPTP method. The significant reductions of the amount of DA, HVA, DOPAC in forebrain tissues were found, accompanied by mental deterioration and movement disorders. The combined use of Pingchan Fang and MT can significantly increase the levels of DA, HVA, DOPAC, increase the expression of Bel-2in rat striatum expressing neurons and glial cells, decrease the expression of Bax, and reduce the apoptosis of DA cells.2. By contrast to wild-type fruit flies (control group), the PD male fruit flies climbing ability is enhanced during maturation, but was decreased significantly during the24to48d after emergence. In the experimental group treated with Termite fungus garden, the climbing ability of female PD flies was gradually decreased with age. The alcohol extract of termites fungus garden can improve the climbing capability of PD flies, and the effects were the most prominent during the18-48d after emergence. The improvement was becoming more prominent as the extract concentration or fly age was increased, and there were no significant difference in the climbing ability between male and female PD flies. The life span of male and female flies can be significantly prolonged by the treatment of the termite fungus garden, with a good dose-dependent manner. The flies with significantly decreased ability of climbing activity (36d) were biopsied to prepare the pathological sections. Compared to control groups, the brain cortex cells of PD flies were thinned and in disorder with increased vacuoles. Application of the fungus garden extracts can increase the brain cell numbers of cells with well arranged array and reduced vacuoles.3. The AD aging mice show great physical and mental decline in the maze test and significantly delayed escape latency. The oxidation and antioxidant indicators were changed significantly. The changes in the brain were consistent with the physiological changes of aging, indicating that AD model mices were successfully established. In the treatment group, the blood SOD activity, GSH and MDA content are significantly improved as compared with the model group. After treatment with different concentrations of the extract of termites and fungus garden, the cell morphology was gradually becoming normal for most of the brain and liver cells of the AD model group, and the amount of brain tissue β-amyloid is also becoming normal.4. The measurement of SOD, MDA and GSH showed that the addition of alcohol extract of termites fungus garden into the culture medium for SH-SY5Y cells can prevent the H2O2-induced cell damage and improve the cell survival, indicating that the extract can fight against the damage caused by H2O2.5. After24h incubation of the water extract of termites and fungus garden, the growth of BGC-823cells was inhibited, accompanied with the cell number and morphological changes. The cell number was reduced and some cells were ruptured. Some cell nucleus was shrunk with less nuclear plasma. Thus, the extract can inhibit the growth of these tumor cells, which is in a dose-dependent manner. This extract can up-regulate the gene expression of p53, which may be the way to affect cell survival.Conclusion1. The combined use of traditional Chinese medicine and Western medicine with an emphasis on the close relationship between the diagnosis and symptom shows great advantages in the treatment of PD and other nervous degenerative diseases.2. The significant improvement of the climbing ability and life span of PD flies suggests the extract of termite fungus garden can improve the movement ability of PD flies, which may be related to its role in the antioxidant reactions.3. The extract of termite fungus garden shows positive effects in the treatment of AD, which is in a concentration-dependent manner.4. The alcohol extract of the termites fungus garden shows protective effects for SH-SY5Y cells against the H2O2induced damage, indicating the presence of antioxidant substances in this extract.5. The extract of termite fungus garden shows pro-apoptotic effects for the gastric cancer cells BGC-823and can up-regulate the p53expression, indicating it is related to the p53-mediated apoptosis. |
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