The Study On Physiology Response And Change Regularity Of Heavy Metal Cadmium Ion In Pheretima Asperillum

Pheretima aspergillum(E.Perrier) from Megascolecidae, Pheretima Division, is one kind of the original animals-Pheretima received under "The People's Republic of China Pharmacopoeia" (2005 edition).Because of Its main products in both Guangdong and Guangxi, it is called " Lumbricus kwangt ". Because of its curative effect, fine medicine processing,it'squality was recognized as the best in the industry, it is a popular commodity in the domestic medicine sales, but also the designated export varieties.At present, the original animal of China pheretima medicine-pheretima asperillum(PA), still rely on wild resources mainly, with different environmental conditions and harvest processing technologies, ingredient quality is unstable, including heavy metals exceeding which is difficult to control. According to preliminary work:One of the important reasons of heavy metal exceeding of pheretima medicine is the physiological heavy metal enrichment of the original animal-PA. Therefore, wishing to solve this problem from the source, It should be the first step to deeply study the process and mechanism of bioaccumulation of heavy metals, which provides evidence for the next step to discharge heavy metal from PA.With the rapid development of industry and agriculture, The problems of heavy metal soil pollution becomes more and more serious, and the pollution in the soil is hidden, long-term and irreversible. The heavy metal in the soil can be absorbed by organisms so that the metal enrichment concentration of the end of the food chain is very high. The latter characteristic is even more worrying. Because of this feature will not only seriously affect the yield and quality of plants and animals, but also pose a serious threat to the human being and even to the entire biosphere through the food chain or the drugs. Soil animals are important part of the soil ecology system. The toxic heavy metals in soil could be enriched in soil animal bodies by different ways. Much research currently focuses on the effect of different kinds of earthworms on biological characteristics of soil heavy metals from the perspective of ecological toxicology, and on the accumulation of heavy metals in earthworm as signs of environment pollution or as a tool for ecological restoration. less research focuses on the mechanism of heavy metals enrichment in earthworms to improve medical resources in medical earthworm-related research.On the basis of prior research work of our research group, heavy metal-induced experiment was carried out to explore cadmium tolerance of PA, The effect of heavy metal Cadmium ion on the structural of PA gastrointestinal epithelial cells,The effect of heavy metal Cadmium ion on the lysosomal membrane of PA, and immunochemistry to study the metallothionein expression in different tissues of in PA. The corresponding results obtained are summarized as follows respectively:一,Literature ReviewFrom the analysis of existing research literature, at home and abroad on the Study on Characteristics of earthworm enrichment is mainly reflected in the below three aspects:1. The research results indicate that earthworms have a certain heavy metal tolerance and accumulation capacity, and the accumulation and tolerance capacities to different heavy metals were different.2. heavy metal accumulation characteristics of earthworms is mainly used for monitoring and evaluation of current environmental pollution and restoration of the polluted environment.3. It is lacking for The related research to improve medical resources and solve excessive heavy metals in medicinal earthworm.二,Investigation of heavy metal cadmium tolerance of PAIn this study, heavy metal-induced experiment was used to explore the heavy metal cadmium tolerance of PA in two weeks. the results show that PA could tolerate 12 mg/kg cadmium and its growth was hardly affected; when the soil Cd concentration was 18 mg/ kg, the body cadmium concentration was 100 mg/kg, PA mortality rate was 25%, PA growth WAS suppressed significantly, PA body weight sharply decreased to 60%, PA shape and form was changed, indicating PA can't tolerate this concentration of cadmium. When the soil Cd concentration is 24 mg/kg or more, all of the PA died within one week, mortality was 100%, weight, shape, body shape changed greatly, and appears similar to acute poisoning, indicating PA tolerance very poor to this concentration of cadmium. The results also can make the following inferences:PA can tolerate Soil cd maximum concentration of 12 mg/kg in the vicinity. Through Cd concentration and PAs growth analysis found that when the Cd concentration in PA body accumulated to 100 mg/kg, it is already close to cd maximum tolerance concentration, and further indicating that PA Cd tolerance is of in vivo maximum tolerance concentration and in vitro the maximum tolerance concentration. Yu-Fang Song show that Eiseniafoetidasavigny could tolerate up to 290 mg/kg soil cadmium, which is higher 24 times than PA's, suggesting the tolerance characteristics of different types of earthworms significantly different.The distribution of the heavy metal cadmium in different parts of PA showed that the cadmium content of internal organs of well-growthed PA(group 1～4) were 1.8～9.8 times higher than the trunk,. This result shows that PA absorbed Cd mainly through the digestive system, with its tolerance to cadmium may be associated with the body detoxification mechanism. In addition, when PA was put into the soil with high concentration (24 mg/ kg or more) of cadmium, it will quickly die, however, when put into the soil with lower Cd concentration, it will adapt to this environment and survive normally, with its Cd concentration of internal organs and the whole earthworm were as high as 90 mg/kg and 70 mg/kg or more respectly, far more than the concentration of 24 mg/kg. The results suggest that PA was of effective heavy metal detoxification mechanisms with the limit of no more than physiological circumstances, and showed the body's high tolerance to cadmium.The study examined PA's cadmium ions tolerance and in vivo cadmium ions enrichment. The future study should expand the scope of study time to provide a further basis for studying PA metal enrichment mechanism and for the strategy laying down to lift Cd enrichment.三,The effect of heavy metal Cadmium ion on the structural of PA gastrointestinal epithelial cells1. The effect of cadmium on the skin of PAIn animal classification, PA is belong to Oligochaeta huge Lumbricidae Earthworm. due to the long life in the soil, the skin is the first barrier to resist external soil pollution and make an early, direct response to the toxicity of the soil. Our results show that the structure of the skin of the PA contaminated with 3-6mg/kg cadmium chliride has undergone a significant change although its growth performance in no obvious change, with stratum corneum thickening. the skin defense system of the PA contaminated with 3-6mg/kg cadmium chliride was damaged and the stratum corneum was thinned. Normal Stratum corneum is non-cell layer, colorless and transparent, multi-layer collagen fibers, allowing air through, and to protect the loss of PA body water, at the same time, it can resist the erosion of exogenous chemicals. Thickening of the stratum corneum is a compensatory response to exogenous chemical substances to avoid the toxic effects of exogenous substances. Previous results show that the main component of the cuticle is acidic polysaccharides, which is required for the protection of animals body against the invasion of exogenous substances. The thinning of the stratum corneum Notes the destruction of its structure, the body's defense function declines. In this study, cadmium chloride also affected the secretion of PA reticular epidermal mucous cell. under normal circumstances, the body can rule out a large of nitrogen to maintain the body's metabolic balance and to provide a protective barrier against the external environmental stimulation. When PA was exposed to different doses of cadmium chloride, the structure of the stratum corneum and epidermal cells changed significantly, reflecting the early defense response of the PA to heavy metals.2. The effect of heavy metal on the body wall of PABy comparing the HE staining of the body wall of PA, we found that the damage degree of different muscle layers is different. The fracture of the outermost ring muscle layer occurred only somewhere, the longitudinal muscle in the middle space is different, some longitudinal muscle showed large crack chamber, and some arranged very compact, no fracture. But the inside drill muscle underwent a more serious injury, some formed large fractures, and some completely fragmented, it is clear to seethat the inside drill muscle injury is significantly seriouser than the outer ring muscles layer of the body wall. This shows that the damage of the body wall is from his body interior, showing that the active substances derived from intestinal positive absorption, rather than passive diffusion through the skin absorption.3. The effect of heavy metal on PA gastrointestinal mucosal epithelial cellsGastrointestinal tract is the main digestive organs, animals eat contaminated soil, the poison in the soil directly stimulate the gastrointestinal tract, likely to cause the lesion of gastrointestinal epithelial cells. This study found that in the cadmium chloride 3mg/kg group and 6mg/kg group, a large number of lysosomal hyperplasia could be seen in the PA intestinal epithelial cells, microvilli, cilia arranged in irregular, disordered. However, with increasing exposure dose, damage increased, mainly mitochondrial swelling, endometrial disappear, mitochondria showed vacuolization. This is mainly because mitochondria is cell's energy center, power station, and one of the most sensitive cell organelles, when it is impacted by environmental adverse factors, different morphological changes occur. While in the group contaminated with the high concentration cadmium chloride, such as 30mg/kg, the microvilli of the PA intestinal epithelial cells contracted, necrotic ulcer lesions occurred in the ciliated cells.四,The effect of heavy metal Cadmium ion on the lysosomal membrane of PANeutral red is a weak basic dye, it can go across the cell membrane into cell lysosomes by non-ionic passive diffusion. as environmental stresses, cell lysosomal membrane permeability changes and loses its stability, enzyme membrane becomes easy to break, the dye gradually leakes to the cytoplasm, the cells will be stained red. Neutral red staining as the current already established technique for the evaluation of the toxicity of compounds on cells, has been widely used in chemicals, pharmaceuticals, surfactants, food additives and pesticides for mammalian animal, human and fish cell toxicity test. In this experiment, cadmium induced PA coelomocytes lysosomal damage is obvious, neutral red retention time of lysosomes in the cells showed an increase with the cadmium concentration gradually decreased, which is consistent with reports of foreign-related, indicating lysosomal neutral red retention time in the cells can be used for evaluation the toxic effects of heavy metal Cd in soil on PA.Compared Group 2 (cadmium chloride concentration of 3mg/kg) with Group 3 (cadmium chloride concentration 6mg/kg) (Cd exposure 7 d and 15 d), lysosomal neutral red retention time is of no significant difference (p> 0.05), shows PA has some tolerance and accumulation at low concentrations of heavy metal ions Cd, indicating that PA can lift heavy metal Cd through their circulatory system. But PA's heavy metal tolerance and capacity is limited, with the heavy metal concentration and exposure time longer, heavy metals cytotoxicity on PA is of obvious differences, such as the group 5 (cadmium chloride concentration of 18mg/kg) (Cd exposure 7 d and 15d), lysosomal neutral red retention time is of significant difference (p<0.05). When the heavy metals uptake of PA is more than their own tolerance range, high heavy metals in soil will be directly toxic to earthworm activity and impact on their lives, so that PA growth is inhibited, such as the experimental group 7 and Group 6, because the concentration of cadmium ions is too high, all of the PAs died when raised to 7 days and 9 days.五,The expression of Metallothionein in different tissues of PAIn this study, immunohistochemistry was used to explore the expression of Metallothionein in different tissues of PA. immunohistochemistry clearly shows that PA metallothionein was mainly expressed in intestine, although there was positive result in the epidermis similar to their epidermal MT expression, among the control group and HE staining there was similar positive results, indicating that it is not the expression of MT, but formed by PA pigment cells, which is consistent to A,J.Morgan and S.R.StUrzenbaum's research results. Although PA epidermis cuticle is of high permeability, there may be a small amount of heavy metals by passive diffusion into the surface, MT expression were not found in its muscle layer and the epidermis. However, there were visible brown spots between the intestinal epithelial cells and basement membrane, and with the increase of Cd concentration, MT expression was growing, indicating heavy metal accumulation of PA is by intestinal epithelial cells. This study provides a basis for our further study about the MT transcriptional regulation of PA and the lifting of heavy metal.