Preliminary Study On The Protective Mechanism Of 3,5-Dicafeoylquinic Acid Against Intestinal Injury Induced By Uric Acid Based On Caco-2 Cell Model

Hyperuricemia（HUA）is a metabolic disease caused by long-term purine metabolism disorder and abnormally increased uric acid（UA）production level in the body.The abnormal level of UA in the body is often accompanied by atherosclerosis,coronary heart disease,kidney and intestinal dysfunction.However,the clinical drugs commonly used in the treatment of HUA,such as allopurinol and benzbromarone,have certain damage to the body.How to use functional factors in food to relieve the disease has become a research hotspot in recent years.Based on previous studies in our laboratory,The extract of Artemisia selengensis Turcz has a good alleviating effect on HUA,among which 3,5-dicaffeoylquinic acid（3,5-diCQA）is the main component of dicaffeoylquinic acids in the extract.Most studies have focused on the anti-virus,liver protection and diabetes relief effects of 3,5-diCQA.However,there are few reports on the effect of 3,5-diCQA on hyperuricemia and intestinal absorption characteristics.3,5-diCQA is not as effective as 1,3-diCQA in inhibiting the production of UA by xanthine oxidase in vitro.Therefore,it is speculated that 3,5-diCQA may alleviate HUA by restoring the antioxidant capacity of intestinal cells,maintaining the stability of intestinal barrier,regulating the expression of uric acid transporter,and improving the fluidity of cell membrane.And are these physiological effects related to their intestinal absorption properties?Based on the above contents,this paper has carried out corresponding work,and the specific methods and results are shown as follows:（1）Human colon adenocarcinoma cell（Caco-2）was treated with different doses of 3,5-diCQA and UA for different times,and it was found that UA had obvious damage to Caco-2 cells in a time and dose-dependent manner,while 3,5-diCQA had no obvious damage to Caco-2 cells.500 mg/L UA was used to establish a HUA intestinal injury model in Caco-2 cells.It was found that UA could reduce the viability of Caco-2 cells,significantly increase the level of reactive oxygen species（ROS）,decrease the activity of superoxide dismutase（SOD）,increase the content of malondialdehyde（MDA）,and reduce the integrity of cell membrane.The survival rate of Caco-2 cells was increased when 3,5-diCQA was used for the corresponding time.Medium and high doses（50 mg/L,80 mg/L）of 3,5-diCQA could improve catalase（CAT）activity and SOD activity to a certain extent,and reduce MDA content.Meanwhile,in the HUA cell model treated with 3,5-diCQA for 2 days,medium and high doses of 3,5-diCQA could significantly increase the m RNA expression level of ABCG2,a uric acid efflux protein.It also significantly reduced the m RNA expression level of the uric acid reuptake protein GLUT9,thereby protecting cells from excessive UA damage.Caco-2 cells were used to establish the intestinal single-cell layer model,and it was found that UA had certain damage to the single-cell layer.After the intervention of 3,5-diCQA,the barrier was repaired,and the protein expression levels of tight junction proteins ZO-1 and Occludin were significantly increased.A high dose of 3,5-diCQA could significantly increase the expression level of Claudin-1 protein,thereby maintaining the protective effect of the barrier.In conclusion,3,5-diCQA can relieve intestinal irritation caused by high uric acid by restoring the antioxidant capacity of intestinal cells,maintaining the stability of intestinal barrier,regulating the expression of uric acid transporter,and improving the fluidity of cell membrane,which is a potential anti-HUA food functional factor.（2）Caco-2 cells were seeded in the Transwell chamber.After 21 days of culture,the transepithelial resistance（TEER）value was close to 1000Ω·cm²,and the alkaline phosphatase activity was significantly different between the AP/BL（Apical side/Basolateral side）,indicating that the absorption model was successfully established.The apparent permeability coefficient（Papp）and efflux rate（ER）were calculated by adding different concentrations of 3,5-diCQA to AP/BL for different times.It was found that with the extension of time and the increase of concentration,the apparent Papp changed accordingly,but remained at about 1×10^-6 cm/s or lower than 1×10^-6 cm/s.This indicated that the absorption of 3,5-diCQA was not high,and the ER value was maintained between 1-1.5,indicating that there was a certain efflux mechanism.The addition of verapamil hydrochloride,an inhibitor,reduced ER,indicating that P-gp（P-glycoprotein）may be involved in the efflux of 3,5-diCQA.This absorption property also suggests that 3,5-diCQA may not only play a physiological role in the cell,but also interact with the cell membrane.（3）The interaction between different concentrations of 3,5-diCQA and L-alpha-Dipalmitoyl phosphatidylcholine（DPPC）liposome molecules was characterized by DSC and FTIR.In the range of 0 mol%-5 mol%,3,5-diCQA mainly enters the head region of the DPPC molecule.In the range of 5 mol%to 20 mol%,3,5-diCQA mainly enters the hydrophobic tail chain region of the DPPC molecule.The location of 3,5-diCQA in DPPC liposome molecules changes with the change of molar concentration percentage,which will cause changes in the thermodynamic properties of the complex.At the same time,the synergy of the complex will also change.In the range of 0 mol%-5 mol%,the interaction between 3,5-diCQA and the head of DPPC liposome was strong,and the phase transition temperature of the complex was slightly higher or basically unchanged than that of the blank DPPC liposome.In the range of 5 mol%-20 mol%,the phase transition temperature of the complex was significantly lower than that of the blank DPPC liposome.It is strongly proved that the addition of 3,5-diCQA can improve the fluidity of the membrane,and the good antioxidant physiological activity of 3,5-diCQA May be related to this.（4）Using molecular docking technology,3,5-diCQA was docked with the target uric acid reabsorption proteins GLUT9,OAT4 and OAT10.It was found that the docking binding sites of 3,5-diCQA and UA with the three reabsorption proteins were basically close,and compared with UA and benzbromarone,The binding energies of3,5-diCQA were-9.0,-7.9 and-8.6 for GLUT9,OAT4 and OAT10,respectively,which were preferentially bound to transporters,indicating that 3,5-diCQA could preemptively seize the binding sites of UA to these three proteins.It prevented UA from being reabsorbed into the cell and alleviated HUA.Meanwhile,the binding sites of 3,5-diCQA and Keap1 were SER-602,ARG-415,ASN-382,ASN-387,ARG-380,ARG-382.Among them,AER-602,ARG-415,ASN-382 and ARG-380 were reported to be Keap1-Nrf2 binding sites,indicating that 3,5-diCQA May promote Nrf2 nuclear translocation by preemizing the Nrf2 binding site,thereby activating the expression of antioxidant response elements.