Research Of The Regulation Of Selenoprotein K On Apoptosis And Necroptosis Of Chicken Liver

Selenium（Se）,as an important member of the trace element family,plays a pivotal role in regulating body metabolism,protecting health system and maintaining normal animal production.Se has antioxidant,anti-inflammatory and homeostasis functions in the body.However,animals cannot directly ingestion selenium,so they must use the food chain to convert Se from plants or feed into selenium protein which is needed by the animal body.As the main form of Se in animal biological function,the expression of selenoprotein is closely related to the growth of animal and the quality of poultry products.Selenoprotein K（SELENOK）is a member of selenoprotein family,which is highly expressed in many tissues such as heart,ovary,lung and liver.SELENOK is located in the endoplasmic reticulum and plays an important role in the regulation of protein processing and synthesis.However,as the main organ of protein synthesis in the body,the function of liver and the health state of liver cells are not supported by detailed reports on the regulation of SELENOK and related regulatory mechanisms.In this experiment,120 one-day-old broilers were randomly divided into two groups.One group was the control group,which was fed selenium enriched diet（selenium content was 0.278mg/kg）,and the other group was the low selenium experimental group.The bulk diet was low selenium diet（selenium content was 0.039mg/kg）purchased from Longjiang County.Twenty broilers were randomly selected from each group to be euthanized from the 15th,25th and35th days of feeding,respectively,and were recorded as 15+Se group,15-Se group,25+Se group,25-Se group,35+Se group and 35-Se group,respectively.Liver tissues were collected and stored according to detection requirements.Chicken hepatocellular carcinoma cells（LMH）were divided into 6 groups based on the establishment of SELENOK knockdown model,which were NC+Se group,NC-Se group,Vehicle+Se group,Vehicle-Se group,si RNA+Se group and si RNA-Se group.TUNEL staining,liver function detection,ROS staining,AO/EB staining,Fluo-5N staining,flow cytometry,real-time quantitative PCR,Western-blot,transmission electron microscopy and other techniques were used.SELENOK content,oxidative stress index and ROS content,PTEN/PI3K/AKT pathway related gene expression,Ca²⁺related gene expression,endoplasmic reticulum stress related gene expression,and apoptosis and programmed necrosis related gene expression were detected in chicken liver tissue and LMH cells.In order to explore and elucidate the mechanism of SELENOK on apoptosis and necroptosis of liver cells in Se-deficiency chickens.The results are as follows:（1）In order to test whether the model of Se-deficiency in chicken liver was established successfully,the expression of SELENOK in chicken liver was detected,and the results showed that the expression of SELENOK decreased in the low selenium group.The clinical manifestations of oozing diathesis were observed.si RNA was transfected into LMH cells,and the m RNA expression of SELENOK in LMH cells was detected to confirm the successful construction of the SELENOK knockdown model.The contents of ALT,ALP,CHE and AST in serum were detected,indicating that the liver function was impaired when the expression of SELENOK was down-regulated（p<0.05）.（2）The microscopic structure of LMH cells was observed by transmission electron microscope.The transmission scanning of chicken liver tissue showed that the 35+Se group presented smooth round nuclei with normal chromatin distribution,intact inner and outer nuclear membranes,and intact nuclear structure and morphology.The 35-Se group showed obvious apoptotic features,including nucleus shrinkage,nucleolar shrinkage,chromatin condensation,marginal aggregation,and a large number of vacuoles in the cell.Microscopically,fracture and swelling of the endoplasmic reticulum were observed in the 35-Se group,and ribosomes detached from the endoplasmic reticulum were detected.TUNEL staining showed that apoptosis was more obvious in-Se group.Apoptosis was detected in SELENOK knockdown LMH cells by AO/EB staining,which was verified by flow cytometry.The m RNA and protein levels of apoptosis-related genes Bax,Bcl-2,Caspase-3,Caspase-12 and Cyt-c in chicken liver and LMH cells showed that apoptosis occurred in the Se deficiency group/SELENOK knockout group（p<0.05）.（3）Through the detection of oxidative stress indexes in chicken liver tissues,the results showed that compared with+Se group,the content of antioxidant enzyme T-SOD,CAT activity and T-AOC content in chicken liver tissues in-Se group were significantly decreased,while the levels of H₂O₂and MDA were significantly increased（p<0.05）.These results indicate that Se-deficiency/SELENOK knockout can cause oxidative stress in chicken liver tissue,and the occurrence of oxidative stress can be alleviated by adding trace element Se in diet/adding sodium selenite in medium to increase SELENOK content.（4）Through the detection of PTEN/PI3K/AKT pathway genes,the results showed that the expression of PTEN was increased in the Se-deficiency group/SELENOK knockout group,and the m RNA and protein expressions of PI3K inhibited by PTEN were significantly down-regulated;meanwhile,the m RNA and protein expression levels of AKT,the downstream gene of the pathway,were significantly reduced（p<0.05）.These results suggest that the decreased expression of SELENOK activates the PTEN/PI3K/AKT pathway through oxidative stress in chicken liver cells.（5）Fluo-5N staining was used to stain LMH intercellular free Ca²⁺,and it was found that a large amount of Ca²⁺was abnormally released in SELENOK knockdown group.The m RNA and protein levels of calcium homeostasis related genes SERCA and CAMK-IV in chicken liver and LMH cells were detected,and their expressions were significantly increased.These results indicated that the down-regulation of SELENOK expression could lead to the destruction of calcium ion homeostasis and calcium overload in chicken liver cells.（6）The m RNA and protein expressions of ER stress-related genes ATF4,ATF6,el F-2α,GRP78,GRP94,IRE and XBP1 were significantly increased in the Se-deficiency group/SELENOK knockout group（p<0.05）.These results suggest that the down-regulation of SELENOK can aggravate the occurrence of endoplasmic reticulum stress in chicken liver.（7）m RNA and protein expressions of HSP60,HSP70 and HSP90 of HSP family were detected,and the results showed that the above gene expressions were significantly increased in Se-deficiency/SELENOK knockout group（p<0.05）.These results indicate that chicken liver adapts to such stress under the condition of Se-deficiency and SELENOK knockout.Meanwhile,the elevated expression of HSP family also helps chicken liver cells to restore physiological protein conformation during and after oxidative stress,and is also a sign of the initiation of stress response mediating cell protection and tissue damage.In addition,we found that the m RNA and protein expressions of HSP60,HSP70 and HSP90 increased in a time-dependent manner,suggesting that HSP response was gradually enhanced with the extension of Se-deficiency treatment time.（8）The m RNA and protein expressions of Cyclin D,CDK4,Cyclin E and E2F1 in chicken liver tissue and LMH cells were detected,and the results showed that the above genes were significantly increased（p<0.05）.Meanwhile,m RNA and protein expressions of Cyclin A,Cyclin B and CDK1/2were significantly down-regulated（p<0.05）.The results of flow cytometry showed that G1/S phase cell arrest occurred in chicken liver cells with Se-deficiency/SELENOK knockout,which aggravated the damage of chicken liver with decreased SELENOK expression.In conclusion,dietary Se-deficiency can lead to decreased expression of SELENOK in chicken liver,decreased antioxidant capacity of chicken liver cells,abnormal accumulation of ROS,activation of PTEN/PI3K/AKT pathway,abnormal release and imbalance of Ca²⁺,and endoplasmic reticulum stress of chicken liver cells.At the same time,the expression of HSP family was increased to cope with abnormal physiological conditions,and finally induced apoptosis and programmed necrosis of chicken liver cells.Meanwhile,the decreased expression of SELENOK can induce G1/S phase arrest of chicken liver cells through the PTEN/PI3K/AKT pathway,increase the expression of p53,and aggravate the occurrence of chicken liver cell apoptosis.This study filled in the blank of the mechanism of SELENOK regulating apoptosis in chicken liver,clarified the important significance of SELENOK on the physiological activity of chicken liver,revealed the mechanism of SELENOK in the occurrence of Se-deficiency disease,and provided theoretical basis and ideas.