| Objective: Chickpea is a kind of plant grown in Xinjiang，it is Uighur residents’favorite food. Uighur residents has used chickpeas as drug to treat with disease. Muleicount is a main chickpea-producing area, because of its unique original naturalenvironment, the local production of chickpeas has good quality and is thought as naturalgreen food. In order to enhance the added value of chickpeas and develop functional foodand drug resource, we detected nutrients and bioactive substances in chickpea andextracted metallothionein from chickpea. On the basis of previous research, we usemetallothionein extract from chickpea to prevent and treat plumbum toxicity in vivo andin vitro conditions. We hope to find a new approach for the prevention of plumbumpoisoning in children. Methods:1) Atomic absorption spectrometer (AAS) was used todetect metal elements and metallothionein in chickpea. Amino acid analyzer was used todetect content of amino acids and protein in chickpea. Centrifugation and cadmium-hemoglobin method was used to extract metallothionein from chickpea. Highperformance liquid chromatography (HPLC) was used to purify protein. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-Page) and MALDI-TOF-MS was usedto analyse amino acid sequence of the metallothionein from chickpea.2) Kunming micewas treated as animal model of plumbum poisoning was established, metallothioneinextracted from chickpea was used to treat mice against plumbum poisoning. Pathologicalchange of liver and kidney tissues was observed under a litht microscope, transmissionelectron microscopy was used to observe ultrastructure changes of kidney tissue. Contentchange of the SOD, GSH-Px and MDA in liver, kidney and blood serum was detected.Plumbum ion content changes in tissues of liver, kidney, brain and bone was detected byAAS method. ELISA was used to detected content of the MT in liver, kidney, brain andbone tissues. RT-PCR was used to detected expression of the MT1-E mRNA and MT2A mRNA in liver and kidney tissues. Single cell gel electrophoresis (SCGE) was used todetected lymphocytic DNA damage.3) Lymphocyte, hippocampal nerve cell and bonemarrow mesenchymal cell were cultured in vitro as cell model of plumbum poisoningwas established. According to the half inhibitory concentration (IC50) in pre-experimentto determine plumbum concentrations. Metallothionein extracted from chickpea was usedto treat against the toxicity of plumbum in different cell models. ELISA was used todetected content of the LDH in three cell models. The AAS method was used to detectedthe content of the Ca2+and K+and Pb2+in lymphocyte, hippocampal nerve cells and bonemarrow mesenchymal cells at24h,48h and72h time points. Total protein content in threecell models was detected by BCA method. Flow cytometry was used to observe theapoptosis of lymphatic cells, hippocampus nerve cells and bone marrow stromal cells.Hoechest33258fluorescence was used to detected the lymphocyte apoptosis.Transmission electron microscopy was used to observe ultrastructure changes oflymphatic cells. Immune fluorescence assay was used to detected the expression of Bcl-2and Bax in hippocampus nerve cells and bone marrow stromal cells. Results:1) Thechickpeas grow in Mulei count contains17kinds of amino acids which included all thehuman body essential amino acids and total content of amino acid reached18.8%. Thechickpeas contains many kinds of minerals that human body needs, such as potassium,sodium, calcium, iron, zinc, copper, magnesium, chromium, etc. Among these minerals,the content of Kaliun and magnesium and calcium is higher and reach to9256mg/kg and1289mg/kg and1074mg/kg respectively. Plumbum has not been detected, Cadmiumcontent is5.9μg/kg, total mercury content is2.58μg/kg, total arsenic content is4.1μg/kg,metallothionein content in chickpea is0.16mg/g; the molecular weight of the chickpea-metallothionein is about10Da. The results of MALDI-TOF-MS show that there were96protein matched from NCBI BLAST. Based on isoelectric point and molecular weightrange, two qualified protein (gi|330253730and gi|355496648) were selected anddisplayed17protein peptides matched from NCBI BLAST.2）As animal experimentalresults display, the level of eating, drinking and activity has decreased significantly intreated mice. Under optical microscope,we observed tissue edema and disorderedarrangement cells of liver and kidney tissue stained by HE in mice. Exogenousmetallothionein from chickpeas was given to mice to treat tissue damage caused byplumbum, poisoning symptoms were improved significantly after the intervention. Undertransmission electron microscopy, we observed that contraction of renal tubular epithelialcells, a large number of vacuoles in mitochondria, disordered arrangement of microvilli and mitochondrial membrane swelling in positive control group mice. Cell damageconditions was reduced in negative control group mice when metallothionein was givento prevent plumbum toxicity. In positive control mice group, the content of GSH-Px andSOD in liver, kidney and serum decreased while content of MDA increased. Comparedwith positive group, content of GSH-Px and SOD increased while content of MDAdecreased in intervention group, the difference was statistically significant (P＜0.05); thecontent of sulfydryl in high dose of intervention group increased while the content ofplumbum in liver, kidney, brain, bone and whole blood decreased in high and middledose groups after the intervention, the difference was statistically significant (P＜0.05);Compared with negative group, the content of metallothionein increased in interventiongroup, the difference is statistically significant (P＜0.05); As shown in single cell gelelectrophoresis result, metallothionein treating caused lymphocytic DNA damagerepairing, DNA damage degree reducing and comet cell number decreasing; As RT-PCRresult display that expression of MT1-E mRNA and MT2A mRNA increased in livertissue of positive group mice while the expression decreased in intervention groups (P＜0.05); In kidney tissue, the expression of MT1-E mRNA and MT2A mRNA increased inpositive group mice while the expression decreased in intervention groups (P＜0.05)；3）As shown in cells experimental results, proliferation rate of lymphocytes, hippocampalneuronal cells and bone marrow stromal cells reduced when given the plumbum,proliferation rate of cells elevated after given the chickpea–metallothionein. Comparedwith positive control group, the content of Pb2+and LDH in three cell models reducedsignificantly in high and middle dose group at24h,48h and72h time point, the differencewas statistically significant (P＜0.05); the content of K+reduced significantly in highdose group at24h,48h and72h while the content of Ca2+reduced significantly in highand middle dose group at48h and72h, the difference was statistically significant (P＜0.05); As shown in Hoechest33258fluorescent dye result, lymphocytes apoptosis ratedecreased in intenvention group which given chickpea–metallothionein treatmentcompared with positive control group; Under transmission electron microscopy, theapoptotic body were observed and cell surface cilia and intercellular contact disappearedwhile those cell damage symptoms reduced in intervention groups. Compared withpositive control group, the expression of Bax decreased while the expression of Bcl-2increased in intervention groups; the ratio of bcl-2/bax increased in high doseintervention group (P＜0.05). Flow cytometry results showed that the apoptosis rate ofcells in positive group increased, then the apoptosis rate of cells increased when chickpea-metallothionein was used to prevent plumbum toxicity. Conclusion: Chickpeawhich grow in Mulei count in Xinjiang contains essential amino acids and minerals, doesnot contain plumbum metals, it’s total mercury content is much lower than the standardsstipulated by the chinese state in food (0.02mg/kg), cadmium content is much lower thanthe standards stipulated by the Japanese state in brown rice (1.0mg/kg) and the Germanstate in white wine (0.1mg/kg), total arsenic content is much lower than the standardsstipulated by the chinese state in grain (0.7mg/kg); In this experiment, metallothioneinextracted from chickpeas was used to prevent and control the toxic effects of plumbum,results show that chickpeas-metallothionein has antagonism to plumbum toxicity eitherin vivo or in vitro state. The study results provide the basis for health food and functionfood development in the future; at the same time, it provides a new method for preven-tion and treatment of children’s plumbum poisoning. |
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