Study Of Effects Of MTs On Regulating Heavy Metal Enrichment Mechanism And Its Antiasthma

Objective:Pheretima aspergillum (E. Perrier) is conventionally called "Guang Di Long" and recognized high-quality products in the domestic and foreign medicine markets. However, the problem of exceeding the limit of heavy metal in "Guang Di Long" has restricted its further development. Through experimental research, the team found that exceeding the limit of heavy metal in "Guang Di Long" was:directly related to physical properties heavy metal enrichment and tolerance, and confirmed metal enrichment was (MTs) closely connected with metallothionein. But the problems like deeper explanation that how and why heavy metal enrichment phenomenon in P. aspergillum occur; what is its molecular regulation;how to control the problem of exceeding the limit of heavy metal thoroughly without affecting the existing efficacy have not been able to solve. This research, starting with MTs, attempts to clarify the role of MTs in metal enrichmentin and clear its regulation mechanism of heavy metals, at the same time, to investigate the influence of MTs on airway inflammation and airway hyperresponsiveness through experiment on anima, which can lay a solid foundation forrevealing molecular biology mechanism of heavy metal enrichment characteristics of P. aspergillum, and also provide a scientific basis for further controlling heavy metal enrichment so as to ensure the quality of Chinese traditional medicine.Methods:1. Expressing pET-32a(+)-MT-2 recombinant strains and pET-32a(+) control strains preserved in our laboratory were activated by scraping line method and their expressions were detected and confirmed by SDS-PAGE and western blot after induced by IPTG. 2. The spread plate count and atomic absorption spectroscopy were used to examine the increase of heavy metal tolerance and accumulation characteristics of MT-2 recombinant strains under the stresses at different concentration of Cd.3. After IPTG inducing expression, MT-2 recombinant strain was centrifuged and treated with ultrasonic lysozyme and its expression forms was then analyzed. The model of free radical damage DNA in vitro was established to investigate the protection DNA oxidative damage of MT-2 recombinant protein purified through Ni-chelating byagarose electrophoresis.4. After the design of primers of Act in gene sequences according to related gene sequences in GenBank database, Actin gene of P. aspergillum was amplificated by PCR. PCR products were sequenced at the biotechnology company BGI and were given a similarity search by BLAST to verify the accuracy of sequence. The primers of actin and MT-2 gene were designed and quantitative real-time PCR was used to compare the mRNA expression of MT-2 under the stresses at different concentration levels of Cd.5. Specific primers were designed according to the known MT-2 cDNA sequence and MT-2 gene coding sequence was amplificated, sequenced, compared and analyzed. Base on the sequence,three specific primers were designed. The 5’ promoter sequence was isolated by genome walking technology and Promoter Prediction software was used to analyze its cis-acting elements.6. Airway responsiveness was measured by whole body plethysmographs. The lung sections were stained with either HE or PAS. The total number of cells was counted by trypan blue staining method. Interleukin (IL)-4, IL-5 and IL-13 levels in bronchoalveolar lavage fluid (BALF) and immunoglobulin E (IgE) levels in serum were measured by enzyme-linked immunosorbent assay (ELISA).Results:1. SDS-PAGE gel showed that recombinant MT-2 protein was expressed successfully and Western blot confirmed the validity of the expression product, suggesting that MT-2 recombinant strains can be used in subsequent experiment.2. Heavy metal enrichment experiments showed that expressing MT-2 recombinant proteins’strain ability of resistance to cadmium was obviously higher than that of the control bacteria, and the maximum tolerance dose of cadmium was 250 μM. Heavy metal enrichment experiments showed that compared with blank control bacteria, expressing MT-2 recombinant proteins’strains markedly enhanced its capability of heavy metal enrichment. With the low dose group (10μM and 20μM) of Cd treatments, the adsorption of heavy metal of MT-2 recombinant strains was respectively 1.9 and 8.8 times that of the control group, but with the high dose group of Cd treatments, the adsorption of heavy metal of MT-2 recombinant strains was respectively 4.4 and 8.3 times that of the control group. The results showed that metallothionein played important roles in heavy metal resistance and enrichment.3. SDS-PAGE analysis showed MT-2 recombinant proteins existed in the form of soluble and inclusion body protein and its main form was soluble. The supernatant of lysates were purified with Ni Sefinose Kit, and high purity protein was obtained. In vitro agarose gel electrophoresis indicated that MT-2 recombinant proteins can obviously protect DNA damage caused by free radicals, and the effect was positively correlated with its dose.4. Actin partial cDNA sequence of P. pergilum was acquired by PCR amplification and sequencing, then a similarity search was done by BLAST. The results showed that Actin gene fragments were accurate and reliable and can be used in the research of quantitative PCR. Results from RT-PCR analysis proved that heavy metal cadmium can significantly induce MT-2 mRNA expression with a dose response relationship. There was a significant rise in mRNA expression on the 3rd day after Cd treatment and the maximum level on the 7th day after Cd treatment.5. A 2826 bp MT-2 gene coding sequence was obtained by PCR and sequencing. Compared with the known MT-2 cDNA sequence (accenssion no KC787373.1), it was found that MT-2 gene contained four exons and introns. A 1534 bp promoter region of MT-2 was isolated using the genome gene walking method. The Promoter analysis with online Prediction software revealed that MT-2 promoter contained not only CAAT box, TAAT box and other core promoter elements, but also three specific response heavy metal elements MRE involved in regulating the MT-2 expression.6. Determination of the airway resistance found airway resistance of mice in the asthma model group was significantly higher than that in the control group by administration of saline and different concentrations of acetyl choline(P<0.01), whereas the MTs group had no obvious changes compared with the model group. The differences had no statistical significance (P>0.05). The total number of cells in the BALF of the model group mice was markedly higer than that of the control group (P<0.01), while the MTs group had no obvious decrease, and there was no statistical significance(P>0.05). IL-4, IL-5, and IL-13 in the BALF and IgE in serum increased notably in the mode group compared with the control group (P<0.05), but three cytokines in BALF and ova-specific IgE in serum of MTs group were not significantly different compared with the model group (P>0.05)Conclusion:In the current study, we confirmed the MTs mediated heavy metal enrichment mechanism and its regulation mechanism, and it was a stress product which could protected the body from heavy metal toxicity and palyed no contribution to antiasthma effect in the level of protein, gene and overall level. The result indicated that the MTs could served as a important targets for further removing or blocking the way of enrichment of heavy metal ions the of enrichment of heavy metal ions, which not only strengthened the foundation for further removing or blocking the way of enrichment of heavy metal ions to ensure the quality of the medicinal part by genetic engineering technique, but also opened up a new approach for improving new breeds to ensure the safety and utility in clinical medication from the source.