Study On The Efficacy Of ?-linolenic Acid Attenuating Aluminum-induced Cellular Inflammatory Response

Aluminum is an inorganic pollutant,which is mainly accumulated in the immune system to produce toxicity,thereby adversely affecting human and animal health.Alpha-linolenic acid(ALA),as an important dietary ingredient,has excellent anti-inflammatory activity,which can reduce various cellular inflammatory factors activated by external stimuli,thereby resisting inflammation.However,at present,the immunotoxicity mechanism of aluminum and the anti-aluminum toxicity ability of ALA are still poorly understood,and further investigation is needed.This study will be divided into four groups:control group,injury group,protection group and ALA control group.AlCl₃-induced mouse RAW264.7 macrophages are selected as the inflammation model,and they are cultured with ALA for 24 hours to detect the level of apoptosis,The expression of inflammatory factors and key proteins in related inflammatory signaling pathways clarifies the anti-inflammatory effect and mechanism of ALA induced by aluminum,and provides a new perspective for the development of?-linolenic acid nutrition and health products.The main research results are as follows:1)CCK-8 method was used to detect cell viability and construct AlCl₃-induced inflammation damage model.The results showed that after stimulating RAW264.7cells with 2,8,10 and 15mM AlCl₃ for 24h,the cell viability was inhibited and decreased in a dose-dependent manner.Considering comprehensively,the AlCl₃concentration for establishing the inflammation model was selected to be 10 mM,and the time was 24 h.2)CCK-8 method also used to screen protective oil lipids and their concentration.The results showed that ALA at concentrations of 10,20,50,and 75?M and DHA at concentrations of 10,20,and 50?M were non-toxic to cells after treatment for 24hours,respectively.The comparison found that when 10 mM AlCl₃ and 50?M ALA were co-cultured,the cell viability was increased by 23.4%,about 77%,and reached the peak value compared with aluminum alone.Compared with ALA,the protective effect of DHA is not as significant as the former.Therefore,ALA(50?M)was selected for follow-up experimental research.3)DAPI staining and flow cytometry were used to observe the morphological changes of RAW264.7 cells under different treatments.AlCl₃ induced apoptosis and increased fluorescence,while the administration of ALA significantly reduced the increase in apoptosis and decreased fluorescence significantly.Flow cytometry quantitative detection further verified the above results.4)Based on the detection of DCFH-DA,it was found that compared to the control group,Alexposure caused a more than two-fold enhancement of the intracellular ROS content.After the addition of ALA,the reactive oxygen content was significantly reduced,which was only 37%of the AlCl₃ group(p<0.001).5)Use nitric oxide colorimetry and ELISA to detect the effect of ALA on the levels of inflammatory factors NO,TNF-?,IL-1?and IL-6 in RAW264.7 cells induced by AlCl₃.The results showed that 50?mol/L of ALA significantly reduced the secretion of the above-mentioned inflammatory factors,which can indicate that ALA has a certain anti-inflammatory effect.6)Western blot analysis confirmed that ALA significantly inhibited the protein levels of NF-?B p-p65,p-JNK,p-p38 and p-ERK1/2 in AlCl₃-induced RAW264.7cells.(38.5%,19.7%,46.7%and 8.3%,p<0.01).We also tested whether ALA also inhibited the activation of JAK2/STAT3 and initiated the activation of the pathway negative regulatory protein SOCS-3.As expected,ALA inhibited Al-induced phosphorylation of JAK2 and STAT3(82.5%and 18.8%,p<0.01)and increased the expression of SOCS-3(24.0%,p<0.01).Western blot analysis confirmed that the anti-aluminum immunotoxicity of ALA is related to NF-?B-mediated JAK2/STAT3/SOCS3 pathway and HO-1 activation.Conclusion:This study revealed for the first time that ALA can alleviate the immune toxicity induced by aluminum through MAPK and NF-?B-mediated JAK2/STAT3/SOCS3 pathways and activation of HO-1.These new perspectives to clarify the molecular mechanism of aluminum toxicity and the potential of ALA to resist aluminum toxicity,unearth the new function of ALA to reduce aluminum-induced cell apoptosis,and provide a research basis for the development of new anti-inflammatory drugs.