| In recent years,the rapid development of the agricultural economy has made the widespread use of pesticides a new trend,which has also promoted the development and production of pesticides.However,due to improper use and storage,pesticide poisoning incidents occur frequently,which has become a social public health problem that cannot be ignored.Paraquat(PQ)is a REDOX herbicide that induces oxidative stress by producing reactive oxygen species(ROS).Because of its low production cost,quick effect and strong toxicity,it has been widely used in agricultural production in developing countries.However,the improper use of paraquat makes it one of the major organophosphorus pesticides harmful to public health.After it enters the human body,it destroys the REDOX system and produces too many free radicals,resulting in the gradual loss of functions of the lungs,kidneys,liver and other organs after varying degrees of injury.Studies have shown that liver is one of the main target organs for PQ exposure.Toxins,drugs,environmental pollutants and other foreign substances produce a variety of free radicals during liver transformation and excretion.When the production and consumption of free radicals are out of balance,excessive free radicals will leadto liver damage.At present,there is no specific antidote and effective treatment for paraquat poisoning,and the mortality after poisoning is very high.Therefore,it is urgent to find specific drugs and methods to treat paraquat poisoning.The active ingredient of flavonoids in traditional Chinese medicine has been attracting much attention.Previous studies have proved that flavonoids can remove free radicals,delay senescence,protect cardiovascular and cerebrovascular system,fight diabetes and nephropathy,fight inflammation and fight tumor,and have high development and utilization value.As a medicine,flavonoids have the advantages of low toxicity,small adverse reactions and easy to development.Ophiopogon japonicus(Ophiopogon japonicus)is a commonly used and widely distributed medicinal herb in China.8-Formylophiopogonanone B(8-FOB)is a high isoflavone compound found naturally in radix ophiopogonae.There have been few previous studies on the antagonism of 8-FOB against paraquat induced hepatotoxicity.Therefore,this study aims to explore the protective effect of 8-FOB on PQ-induced hepatotoxicity and its molecular mechanism through in vitro and in vivo.The main research contents and results are as follows:1.Determination of PQ concentration in vitro and the antagonistic effect of 8-FOB on PQ toxicityFirst,normal human hepatocytes(L-02 cells)were used as in vitro cell models.CCK-8 method was used to detect the cell viability of PQ at different concentrations(0 ?M,250 ?M,500 ?M,750 ?M,1000 ?M)after 0,6,12,24,and 48 h.According to the results,PQ with an action time of 24 h and a concentration of 500 M was selected to establish an in vitro hepatocyte injury model.Furthermore,we calculated the IC50 of PQ treated L-02 cells for 24 h,and recorded the cell morphology after PQ treatment at different concentrations for 24 h by optical microscope.The same method was used to evaluate the toxicity of 8-FOB in L-02 cells and the effect of pretreatment for 6 h to inhibitthe toxicity of PQ(500 ?M),then the EC50 of 8-FOB was calculated.The results showed that with the increase of concentration of PQ for 24 h,cells showed different degrees of wrinkling and shrinkage,and the calculated IC50 was 817.3 M.Pretreatment with 8-FOB(10 ?M,40 ?M)significantly increased PQ-induced(500 ?M)reduction in cell activity.The results under the microscope showed that 8-FOB significantly reduced the cell damage induced by PQ,and the semi-maximum effective concentration(EC50)calculated that the 8-FOB in L-02 cells was 8.057 ?M,while the high concentration of 8-FOB(40 ?M)alone did not affect the cell activity,indicating that 8-FOB could effectively antagonise the cytotoxicity of PQ.2.8-FOB improves oxidative stress induced by PQ and its mechanismThen,we further studied the hepatoprotective activity and related mechanism of L-02 hepatocyte model treated with PQ(500 ?M)by 8-FOB pretreatment/non-pretreatment for 6 h.The influence of 8-FOB on total ROS and mitochondrial ROS(mt ROS)content in L-02 cells was detected by the detection kit after PQ treatment for 3 h.Mito-tempo,a mitochondrial targeted antioxidant,and n-acetylcysteine(NAC),a cell ROS scavenger,were used as controls.In PQ applied 24 h after,detection of mitochondrial membrane potential(??m)and ATP levels to assess mitochondrial function,product detection of intracellular lipid peroxide malondialdehyde(MDA)and the changes of antioxidant enzyme superoxide dismutase(SOD)to evaluate the occurrence of oxidative stress.The results showed that PQ caused excessive ROS production in cells and mitochondria,while 8-FOB could reduce the production of excessive intracellular total ROS and mt ROS.Similarly,Mito-tempo and NAC also reduced the levels of intracellular total ROS and mt ROS.Among them,8-FOB of 10 ?M and 40 ?M can effectively inhibit the accumulation of lipid peroxidation product MDA in cells,improve the activity of antioxidant enzyme SOD in cells,and effectively inhibit the oxidative stressinduced by PQ in L-02 cells.Mitochondrial function evaluation results showed that 8-FOB significantly inhibited the PQ induced mitochondrial membrane potential(??m)and ATP levels decrease,suggests that mitochondrial function are protected.In conclusion,these results indicate that PQ disrupts the balance of the REDOX system in L-02 cells,resulting in the overproduction of ROS in mitochondria and the whole cell,further leading to the loss of mitochondrial membrane potential and the decline of mitochondrial productivity,while 8-FOB improves PQ-induced oxidative stress and mitochondrial dysfunction through its antioxidant effect.3.8-FOB antagonized the apoptosis of L-02 hepatocytes induced by PQFurthermore,the apoptosis rate of L-02 cells was increased by flow cytometry after 24 h of PQ treatment,but the apoptosis rate was significantly decreased by 8-FOB pretreatment.We examined the effect of 8-FOB on the activity of Caspase-3,the most important end-shear enzyme in the process of apoptosis.Western blot was used to explore the expression of Caspase-3 from the protein expression level.The results of 8-FOB antagonized the apoptosis of L-02 cells induced by PQ showed that PQ could induce the overexpression of intracellular Caspase-3 enzyme and enhance its activity,thus promoting the apoptosis of L-02 cells.However,8-FOB can inhibit the activity of Caspase-3and increase the protein expression,and reduce the apoptosis rate of cells.However,8-FOB inhibits increased Caspase-3 activity and protein expression levels,reducing the rate of apoptosis.In general,8-FOB exerts its antioxidant effect in vitro,inhibits PQ-induced oxidative stress and apoptosis,plays a protective role in L-02 cells.4.8-FOB reduced oxidative stress and PQ-induced liver injury in miceTo investigate whether 8-FOB has antioxidant and liver protective effects in vivo.In this study,8-week male C57BL/6 mice(20-24 g)were randomly divided into 5 groups: Control group,(0.5% CMC-Na)Solvent group,8-FOBgroup,PQ group,and 8-FOB +PQ group.The 8-FOB was uniformly suspended in 0.5% CMC-Na,and the 8-FOB group and 8-FOB +PQ group were gavage at a dose of 20 mg/kg/day for 3 consecutive days.Mice in the Control group and Solvent group were gavage with saline of equal volume and 0.5% CMC-Na.Mice in the PQ group and the 8-FOB +PQ group were given a single intraperitoneal injection with a single dose of PQ(30 mg/kg)on the fourth day.After PQ 24 h injection,detection of mice serum alanine aminotransferase(ALT)and aspertate aminotransferase(AST)activity of liver tissue with hematoxylin and eosin(H&E)staining to observe histomorphology changes.The activity levels of superoxide dismutase(SOD),glutathione reductase(GSH),catalase(CAT)and MDA in liver tissues were detected,and the changes of liver tissues were detected through histopathology to explore the protective effect of8-FOB on liver injury induced by PQ in mice.After 24 h of intra-abdominal injection of PQ,the activity of ALT and AST in serum was detected,and the histopathological changes of liver were observed with hematoxylin and eosin(H&E)staining.Activity levels of superoxide dismutase(SOD),glutathione reductase(GSH),catalase(CAT)and MDA in liver tissues were detected to explore the protective effect of 8-FOB on liver injury induced by PQ in mice.The results of in vivo experiments showed that 8-FOB could effectively reduce the activity of ALT and AST caused by PQ,significantly improve the pathological damage of liver tissue in mice,and reduce the Suzike injury score.We found that 8-FOB did not cause oxidative stress in the liver,while PQ significantly increased MDA level,decreased GSH and SOD activity in the liver.The 8-FOB pretreatment can obviously inhibit the increase of MDA level,the decrease of GSH and SOD activity induced by PQ.CAT activity in different treatment groups showed no significant change,indicating that CAT activity was not sensitive.These results indicate that 8-FOB effectively reduce liverinjury caused by PQ in function and morphology,inhibit PQ-induced oxidative stress in liver.To sum up,this project studied the antioxidant capacity and liver protection capacity of flavonoids 8-FOB in vitro and in vivo.The study on the antagonistic mechanism of 8-FOB against the toxicity of PQ proved for the first time that8-FOB was an effective antioxidant,which could antagonize the hepatotoxicity induced by PQ at internal and external.The inhibition of oxidative stress in the liver is a key factor for 8-FOB to antagonize the hepatotoxicity induced by PQ,which is characterized by inhibiting the production of MDA and the generation of intracellular and mitochondrial ROS,reducing the levels of GSH and SOD,and reducing the damage of PQ to the liver.The study of this topic shows that8-FOB has the potential for future clinical application because it promotes the understanding of the mechanism of oxidative stress in PQ-induced hepatotoxicity and the development of antagonistic medicines. |
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