Effect Of Cyanate On Oxidative Stress In Liver And Its Mechanism

Background The incidence of chronic kidney disease(CKD)at home and abroad is increasing year by year and has become one of the public health problems that endanger human health.In recent years,a large number of clinical data have confirmed that the CKD population with hyperlipidemia is significantly higher than the general population,and is characterized by elevated plasma triglycerides and low-density lipoprotein levels.Dyslipidemia not only runs through the course of the disease as a common complication of CKD patients but also promotes the occurrence of cardiovascular diseases(CVD)and causes the death of CKD patients.At the same time,there are many reports of the deterioration of renal function caused by the disorder of lipid metabolism.Clinical and laboratory studies have found that hyperlipidemia can promote macrophage infiltration,foam cell formation,and glomerular sclerosis to exacerbate kidney damage,so hypercholesterolemia and hypertriglyceridemia have been shown to be the progression of chronic kidney disease Independent risk factors.It can be seen that the disorder of lipid metabolism and the impairment of renal function have a causal relationship with each other.The pathological factors and mechanisms involved are extremely complex.The current research has not yet fully elucidated the relationship and related mechanisms of the two.It is currently believed that the production of large amounts of uremic toxins in patients with CKD can cause damage to cells,tissues,and organs or trigger systemic reactions such as immune disorders,inflammatory injuries,and metabolic disorders,causing clinical changes in various organs.Cyanate(cyanate)is a uremic toxin that has attracted researchers' attention in recent years.It can be produced by urea through a nonenzymatic reaction in the body,or by myeloperoxidase(MPO)in smoking or inflammation.Catalytic oxidation of hydrogen peroxide(H2O2)and thiocyanate.CKD patients have high urea content due to the retention of metabolic waste,and the body itself is in a state of low inflammation,so the body has a higher concentration of cyanate.The cyanate salt mainly modifies a variety of proteins with important physiological functions in the body through carbamylation to play a pathological role.A large number of reports in the literature that cyanate carbamylated modified albumin,hemoglobin,low-density lipoprotein,high-density lipoprotein,etc.,are important pathological mechanisms leading to vascular endothelial dysfunction,atherosclerosis,and CVD in CKD patients.In recent years,there have also been reports that cyanate can induce vascular endothelial cell dysfunction by inducing the production of reactive oxygen species(ROS).This direct injury has attracted the attention of researchers.Under normal circumstances,ROS is a natural product of the body's metabolism and plays an important role in cell signaling and metabolic balance in the body.However,under pathological conditions(such as high fat,high glucose,hypoxia,etc.),ROS levels increase sharply,leading to an imbalance of the body's oxidative and antioxidant systems,and severe damage to cell structure and function cause oxidative stress damage.Previous studies have focused on changes in the oxidative system,and the Nrf2 pathway in the antioxidant system has been a hotspot in recent years in oxidative stress research.Under oxidative stress,Nrf2 is uncoupled from Keap1,and Nrf2 enters the nucleus from the cytoplasm and combines with the antioxidant element(ARE)to induce the expression of downstream heme oxygenase-1(HO-1)and other antioxidant enzymes,Through antiinflammatory,anti-oxidation,and regulation of cell apoptosis and other mechanisms to play its protective role.Recently,it has been reported in the literature that Nrf2 can also regulate the m TOR pathway by regulating the phosphorylation of AMPK,thereby regulating the lipid synthesis in vivo.The current research shows that the antioxidant defense system of CKD patients has defects,and the ROS level in patients is significantly higher than that of the normal population,and the levels of several plasma oxidation markers have increased,but is this related to dyslipidemia in CKD patients? No relevant reports have been seen.The liver is the center of lipid metabolism regulation,and also the site where the urea cycle produces urea.Therefore,we hypothesize that the mechanism of dyslipidemia in patients with CKD,in addition to the mechanism of cyanate modification of carbamylation of low-density lipoprotein and high-density lipoprotein,cyanate directly acts on liver cells to induce oxidative stress Injuries and disorders of lipid metabolism are also a possibility.Objective In the first part of this study,male C57/BL6 mice purchased at the Animal Experimental Center of Chongqing Medical University were used as research objects.Modeling was performed using cyanate feeding method to simulate the cyanate concentration in CKD patients.Effects of rat liver metabolism and oxidative stress;the second part of the study used human normal liver cells HL-7702 as the research object,cyanate and TBHQ(Nrf2 agonist)as processing factors,and detected HL-7702 cells' oxidative stress Levels and expression levels of Nrf2-related pathways,and studying the possible mechanism of cyanate action on the liver,providing theoretical and experimental evidence for the clinical application of TBHQ for the treatment of hyperlipidemia in CKD patients.Methods Part I: 70 C57 BL / 6 wild-type(WT)mice purchased and raised from the Experimental Animal Center of Chongqing Medical University were randomly divided into the normal group(30)and cyanate group(40).Mice in the cyanate group were fed a cyanate solution at a concentration of 1% every day,weighed every three days,and plotted the bodyweight curve of the mice.At the end of the experiment,all animals were sacrificed by dislocation after anesthesia,and then mouse blood was collected for determination of liver function,bilirubin,and cholesterol;liver tissue was collected,and sections were stained to observe the pathological damage of mouse liver tissue;oxidative stress kits were used to detect SOD,CAT,MDA,NO related indicators;Western Blot was used to detect the protein expression of Nrf2-related pathways in the liver.Part II: Using human normal liver cells HL-7702 as the experimental object,the CCK-8 method was used to detect the effect of cyanate on the viability of HL-7702 cells;the DCFCH method was used to detect the ROS content in the cells;the oxidative stress kit was used to detect HL-7702.SOD,CAT activity and MDA,NO content in cells;Western Blot to detect the protein expression of Nrf2-related pathways in HL-7702 cells;immunofluorescence method to detect the localization and expression of Nrf2,HO-1 in cells;electron microscope observation Ultrastructure and lipid deposition of 7702 cells.Results Part I: 1.The body weight curve shows that the weight of the mice in the cyanate group is significantly reduced compared with the control group; the blood biochemical results show that the ALT,AST,and ALP levels in the cyanate group are increased compared with the control group,LDH,CHE,HDL levels were significantly reduced,while TC and LDL levels were significantly increased,and statistically significant(P <0.05).2.The results of the enzyme chemistry test showed that compared with the control group,the activity of SOD and CAT in the liver tissue of the cyanate group mice decreased significantly,the content of MDA and NO increased significantly,and there was a difference(P <0.05).3.The results of the tissue section show that compared with the control group,the liver tissue of mice in the cyanate group is disorderly arranged,there is more lipid deposition,and the glycogen metabolism ability is reduced.4.Western Blot results showed that compared with the control group,the expression levels of Nrf2,HO-1,and p-AMPK in the liver tissue of the cyanate group mice were significantly reduced,p-m TOR,p-S6 K and p-S6 The expression level of was significantly increased and had statistical significance(P <0.05).Part II: 1.The results of the CCK-8 method showed that after cyanate treatment of HL-7702 cells for 24 h,as the concentration of cyanate increased,the cell viability significantly decreased.Compared with the control group,it was significantly reduced(P <0.05).2.The results of DCFCH detection of ROS showed that compared with the control group,after 24 h of cyanate-treated cells,the intracellular ROS level was significantly increased and had differences(P <0.05);while TBHQ pretreated cyanic acid There was no significant change in the cellular ROS level in the salt-loaded group.3.The results of enzyme chemistry test showed that compared with the control group,the activity of SOD and CAT in cells of the cyanate group decreased significantly,and the contents of MDA and NO increased significantly,with statistical significance(P <0.05);The activity of SOD and CAT and the content of MDA and NO in the cells of the pretreated cyanate loading group returned to normal levels.4.Western Blot results showed that compared with the control group,the expression levels of Nrf2,HO-1 and p-AMPK in the cyanate group cells were significantly reduced,and the expression levels of p-m TOR,pS6 K and p-S6 were obvious Elevated and had statistical significance(P <0.05);the protein expression level in cells of cyanate group pretreated with TBHQ had no significant difference compared with the control group.5.Immunofluorescence results showed that compared with the control group,Nrf2 entry into the cyanate group cells was significantly reduced,and the HO-1 expression level was significantly reduced;The nuclear situation and the expression level of HO-1 returned to normal levels.Conclusions 1.Cyanate can cause weight loss,liver function decline,liver oxidative stress injury,and abnormal lipid metabolism in mice.2.Cyanate may inhibit the activity of Nrf2 / HO-1,and then activate the expression of m TOR pathway,which may cause liver oxidative stress injury and abnormal lipid metabolism.3.TBHQ can relieve the oxidative stress damage of cyanate to the liver and may become a clinical drug for the targeted treatment of hyperlipidemia in CKD patients.