Evaluation Of Mitochondrial Protection Of Haematococcus Pluvialis Based On SLC25A46 Gene Knockout Mouse Model

The mitochondria is the"power house"for energy production,having great significance for normal physiological functions in the cell.Haematococcus pluvialis is an ideal source of natural antioxidant astaxanthin and an algae food with important nutrition and research value.In this study,an in vitro yeast model was used to confirm that Haematococcus pluvialis could extend the chronological lifespan?CLS?of yeast cells.A new mitochondria-damaged mouse model induced by the knockout of the SLC25A46 gene?a gene encoding mitochondrial outer membrane protein?was innovatively constructed.The reliability and rationality of the model were fully demonstrated.Finally,the established mitochondria-damaged SLC25A46 gene knockout mouse model was used to evaluate the mechanism of mitochondrial protection function of Haematococcus pluvialis.In the yeast experiment,it was found that Haematococcus pluvialis had no significant effect on the growth of yeast cells at two concentrations?200 and 500?g/m L?.Further research showed that Haematococcus pluvialis could significantly improve the survival rate of yeast cells on the 5th and 7th days of incubation,and greatly extended the chronological lifespan of yeast cells.In order to explore the potential mechanism of the effect of Haematococcus Pluvialis on the chronological lifespan of yeast cells,ROS,MMP and the accumulation of carbohydrate in the yeast cells were determined.The results showed that high?500?g/m L?and low?200?g/m L?doses of Haematococcus pluvialis could significantly reduce the ROS levels in yeast cells by 31.21%?p<0.001?and 19.37%?p<0.001?,showing a concentration dependence.High concentrations of Haematococcus pluvialis could significantly increase the level of MMP by 18.74%?p<0.05?.At a concentration of 500?g/m L?high?and 200?g/m L?low?,Haematococcus pluvialis increade the accumulation of glycogen by 0.60?g/mg?p<0.001?and 0.17?g/mg?p<0.001?.These results indicated that Haematococcus pluvialis could significantly prolong the CLS of yeast cells and possessed potential anti-aging functions.Using CRISPR/CAS9 gene editing technique,mitochondrial damage gene knockout mice were obtained by site-directed knock-out of SLC25A46 gene.Mitochondria-damaged mice(SLC^-/-)with distinct phenotype characteristics were obtained by group propagation and genotype identification.The results of the phenotypic analysis showed that SLC^-/-mice had shortened life span,reduced weight gain,and slow or impaired multiple organs development.In the grip test,the grip strength of 1M and 1.5 M SLC^-/-mice was 0.59 and 0.86 N lower than that of SLC^+/+mice,respectively;In the suspension experiment,the suspension time of 1M and 1.5 M SLC^-/-mice was only 4.00±2.31s and 2.71±1.39 s;Footprint analysis experiments showed that the base width,overlap width,and stride length of SLC^-/-mice were significantly lower than those of the SLC^+/+mice?p<0.05?.These behavioral experiments revealed that SLC^-/-mice had severe muscle weakness,motor disability and severe ataxia.Further investigations of deficiencies in the antioxidant defense system of SLC^-/-mice showed that the lipid peroxide MDA content in the brain tissue of SLC^-/-mice was significantly higher than that of SLC^+/+mice by 0.77 nmol/mg prot?p<0.005?;At the same time,CAT enzyme activity significantly decreased by 0.47 U/mg prot?p<0.05?,indicating a relatively high level of oxidative stress in the SLC^-/-mice.HE staining was used to explore the pathological features of the tissues of the SLC^-/-mice.The staining results showed that SLC^-/-mice liver tissues were loosely arranged with large gapst between the tissues.Microstructure of the mitochondria in SLC^-/-mice was observed under the transmission electron microscopy.The results showed a greatly reduced mitochondrial internal cristae density in the brain and liver of SLC^-/-mice,with obvious vacuolization.Mitochondrial surface area was smaller in the SLC^-/-mice?1.0-3.5 m²?compared to the SLC^+/+mice?3.5-6.0 m²?.The relative abundance of mitochondria in the liver was about 90%with a length-width ratio of about 1.4,characterized by the single morphology and low richness of mitochondria in the liver.Haematococcus pluvialis was administered at a dose of 500 mg/kg/d to evaluate the its mitochondria protection effect in mice.The results revealed that muscle strength of mice significantly increased by 0.53 N?p<0.05?under the intervention of Haematococcus pluvialis.The MDA content decreased by 0.52 nmol/mg prot and 0.67 nmol/mg prot in the brain and liver respectively?p<0.001?.In addition,SOD activity in brain tissue significantly increased by 1.10 U/mg prot?p<0.005?.HE staining showed that the histopathological structure of the liver significantly improved after the intervention of Haematococcus pluvialis.Based on these results,it could be deduced that Haematococcus pluvialis could improve mitochondrial damage in mice,revealing that Haematococcus pluvialis has a protective effect on the mitochondria.In conclusion,Haematococcus pluvialis has been proven to be able to protect and promote mitochondrial function,making it useful as a food,health food or formula food product for special medical purposes with a good prospect for protecting mitochondrial function and reducing oxidative stress.