The Effect Of Homocysteine On The Mitochondrial Function Of Caco-2 Cells And Its Mechanism

BACKGROUND: Mitochondria can not only provide energy for cells,but also contribute to many other physiological activities.many studies have shown that persistent mitochondrial dysfunction can lead to chronic inflammation,which in turn leads to a series of chronic diseases,including rheumatism,cardiovascular disease,neurological disease or metabolic diseases.Inflammatory bowel disease is a chronic non-specific intestinal inflammatory disease caused by a variety of complex factors.Intestinal barrier dysfunction of IBD can lead to diarrhea through the ion leakage mechanism,and can cause secondary intestinal exogenous antigen uptake of mucosal inflammation.intestinal epithelial cells are subjected to long-term cell updated processes that require a high energy base.IBD has been shown to be associated with intestinal epithelial cells' mitochondrial abnormalities.Homocysteine(Hcy)is an amino acid derived from methionine,which includes an sulfhydryl in the middle.Recent studies have shown that Hcy participates in a variety of chronic diseases including neuromuscular lesions,diabetes,obesity,cardiovascular disease pathophysiological processes.Homocysteine can increase the sensitivity of cells to oxidative stress by inhibiting the expression of large amounts of antioxidant enzymes.Recent studies have shown that the expression and activity of heme oxidase-1 and glutathione peroxidase are impaired in vascular endothelial cells treated with high concentrations of homocysteine.Similar with these results,IBD patients also have a decreasing in antioxidant capacity.Intravenous methionine diet and hyperhomocysteinemia are associated with endothelial and epithelial cells dysfunction and have been supported by the three antioxidant effects in the mouse colitis model [19]. Homocysteine can affect the permeability of vascular endothelial cell barrier by promoting matrix metalloproteinase active.Previous studies have found that the level of Hcy in plasma and colonic mucosa of IBD patients is significantly higher than that of normal people.In the experimental colitis rat model,subcutaneous injection of Hcy can promote the release of inflammatory factors,which lead to the colonic mucosa oxidative damage and increase the severity of inflammation.Objective: Intestinal epithelial cells provide an important barrier function between the internal environment and the external environment of the body and play an important role in the development of intestinal inflammation.Caco-2 cells were treated with different concentrations of Hcy for different times to observe the changes of mitochondrial function in Caco-2 cells,and understand more about the effect of Hcy on mitochondrial function of intestinal epithelial cells.Method:1.the effect of homocysteine(Hcy)on the activity of Caco-2 cells with different concentrations and in different timesThe experiment was divided into two gruops which respectively were normal group and Hcy group,Caco-2 cells growed 24 hours then were treated with Hcy.The cells were treated respectively with 10?mol/L,25?mol/L,50?mol/L,100?mol/L,500?mol/L and 1000 ?mol/L Hcy.The normal group was treated with the same amount of cell culture medium.The effects of Hcy on the growth of Caco-2 cells were observed by MTT and LDH at different time(3,6,12,24 h).2.the effect of different concentrations of homocysteine(Hcy)on oxidative damage of Caco-2 cells in different timesThe cells were treated with 10?mol/L,50?mol/L and 100?mol/L respectively for 24 hour.The normal group was treated with the same amount of cell culture medium in same times.The levels of MDA,SOD,GSH-Px were measured.3.the effect of different concentrations of homocysteine(Hcy)on mitochondrial function of Caco-2 cells in different timesThe cells were treated with 10?mol/L,50?mol/L and 100?mol/L respectively for 24 hour.The normal group was treated with the same amount of cell culture medium in same times.The following indicators were tested for each group: SDH and ATPase levels.The cells were treated with 50?mol/L,100?mol/L,500?mol/L and 1000?mol/L Hcy for 3 hour and 6 hour.The normal cells were treated with the same amount of cell culture medium in the same time.The effects of Hcy on the the mitochondrial swelling degree of Caco-2 cells were observed.The cells were treated with 10 ?mol/L,50 ? mol/L and 100 ? mol/L respectively for 1 hour.The normal group was treated with the same amount of cell culture medium in same times.The effects of Hcy on the the mitochondrial membrane potential of Caco-2 cells were observed.Result:1.Effects of different concentrations of Hcy on the growth of Caco-2 cells in different times:Compared with the control group,the MTT absorbance decreased significantly(P<0.05)with the increase of Hcy concentration,and the LDH value was significantly higher than that of the control group(P <0.05).With the same concentration of Hcy,the absorbance of MTT decreased significantly with the prolongation of the time while LDH value increased significantly.Hcy had the ability to inhibit the growth of Caco-2cells to a certain extent and with the increase of concentration and the prolongation of time,the ability of inhibited effect was stronger.2.Effects of Hcy on oxidative damage in Caco-2 cells:Compared with the control group,the level of ATPase decreased in Hcy treated group and the amplitude of ATPase decreased much more huge with the increase of the Hcy concentration.The level of SDH increased in Hcy treated group and the amplitude of SDH increased much more huge with the increase of the Hcy concentration.statistical analysis showed p>0.05,it had not a significant difference.Therefore with the increase of concentration,Hcy had the effect of oxidative damage to Caco-2 cells..3.Effects of Hcy on mitochondrial function in Caco-2 cells:The level of SDH in the Hcy treated group(10?mol / L,50?mol / L,100?mol / L)was lower than that in the control group(P <0.05).Hcy inhibits ATPase in a concentration-dependent manner,which may have the ability to inhibit the SDH level.The absorbance of mitochondria at527 nm decreased slowly in the control group,suggesting that mitochondria were swollen due to abnormal osmotic pressure.The decrease of the absorbance of mitochondria in absorbance in Hcy treated group for 3 h and 6 h was lower than the control group,suggesting that the function of mitochondrial had damaged and was not sensitive for the changes in environmental p H.After adding 0.3 mmol / L Ca Cl2 reaction buffer,the mitochondrial absorbance of the control group decreased significantly,which indicated that the mitochondria were obviously swollen.Hcy treated group for 3h was lower than that of normal group,and reached a balance at 4min.Hcy treated group for 6h reached a balance at 2 min.The effect of Hcy on mitochondrial function interfered the sensitivity of mitochondria to high concentrations of calcium in he environment.The red fluorescence intensity of the control group was94.8%,and the green fluorescence intensity was 4.73%.With the increase of Hcy concentration(50?mol / L,100?mol / L,500?mol / L,1000?mol / L),the red fluorescence intensity decreased gradually,67.7%,62.9%,60.8% and 59.4%respectively,and the green fluorescence intensity was 28.4%,31%,34.3% and 36.3%respectively.The red fluorescence intensity is weakened and the green fluorescence intensity is enhanced,suggesting that the mitochondrial membrane potential decreases with the increase of Hcy concentration.With the increase of Hcy concentration,the green fluorescence intensity gradually increased and the red fluorescence intensity decreased gradually,suggesting that the mitochondrial membrane potential decreased nwith Hcy in the proportional relationship,with the increase of Hcy concentration,the mitochondrial membrane potential decreased more significantly,the more severe mitochondrial damaged.Conclusion:1.Hcy has the ability to inhibit the growth of Caco-2 cells;2.Hcy promotes lipid peroxidation and inhibits antioxidant enzyme levels with oxidative damage on Caco-2 cells;3.Hcy reduces the levels of ATPase and SDH in Caco-2 cells and damaged mitochondrial energy metabolism.4.Hcy influences mitochondrial swelling and membrane potential in Caco-2 cells,suggesting that Hcy may influence intestinal mucosal barrier function by damaging the mitochondrial function of intestinal epithelial cells.