Extraction Process Optimization and Bioactivity Analysis of Andrias davidianus Liver Metallothionein

In this study, the by-products processed from Andrias davidianus were evaluated, and the structure, activity, and functionality of the metallothionein from this amphibian species (AdMT) were investigated. AdMT was extracted from the liver using a centrifugal homogenization method, with the extraction process optimized by means of single-factor and orthogonal experiments. Subsequently, the protein was purified using ion-exchange chromatography. The antioxidative activity of AdMT was assessed by measuring its free radical-scavenging ability, and its cytotoxicity was evaluated using the lactate dehydrogenase release assay. The anti-inflammatory activity of the protein was determined by RAW264.7 cell model of lipopolysaccharide-induced inflammation, and its immunogenicity and effect on cell proliferation were analyzed using the CCK-8 assay. Its effects on the cell cycle and cell migration ability were assessed using flow cytometric and scratch assays, respectively. Its heavy metal detoxification capacity was evaluated using L929 and HaCaT cell models of CdCl₂-induced toxicity. Results showed that, the optimal AdMT extraction conditions were determined to be as follows: Liquid-to-material ratio of 5:1, extraction solution concentration of 0.02 mol/L, extraction temperature of 40 ℃, and extraction time of 1 h, yielding 0.362 mg/g of the protein. AdMT exhibited a 95.01% scavenging rate against ABTS⁺ free radicals and had no significant impact on the lactate dehydrogenase release rates of L929 and HaCaT cells or on the viability of RAW264.7 cells. After 48 h of AdMT treatment, the viability of L929 and HaCaT cells improved to 192.63% and 207.92%, respectively, and their cell proliferation indices increased to 0.56 and 0.53, respectively. Additionally, the migration ability of the L929 and HaCaT cells increased with increasing AdMT concentration, showing scratch healing rates were 63.38% and 76.23%, respectively. In the cell inflammation model, AdMT significantly (P<0.05, P<0.01, P<0.001) reduced the expression levels of inflammatory factors. Furthermore, the survival rate of Cd-exposed cells was restored to that of the control group. These results would provide a reference for the development of AdMT and utilization of by-products processed from Andrias davidianus.