Therapeutic Effect Of Eupatilin On Hyperuricemia Combined With Acute Gout Arthritis Model Rats

Objective:Gout is an inflammatory joint disease associated with high levels of serum uric acid.Its pathological features are the deposition of monosodium urate crystals in the joints and surrounding tissues,leading to strong inflammatory response and pain.The main treatment methods are lowering serum uric acid level and anti-inflammatory therapy.This study aims to study the pharmacological effects of Eupatilin on xanthine oxidase activity in vitro,uric acid lowering and anti-arthritis in hyperuricemia combined with gout arthritis model rats.Methods:1.Uv spectrophotometric microplate method was used to measure the absorbance at 295 nm to determine the inhibitory effect and mechanism of Eupatilin on XOD activity in vitro,and the Ki was calculated at the same time.2.Hyperuricemia rat model was induced by gavage of potassium oxazinate + hypoxanthine once a day for 14 days,that are both 300mg/kg.Blood canthal was taken to detect uric acid level on the 7th and 14 th days.After successful modeling,except blank group and model group,Eupatilin were given low,medium and high dose treatment for 1 week.Correspondingly,allopurinol was used as a positive control One week later,in addition to the blank group(normal saline),20 m L(20 mg/m L)MSU crystal solution was injected into the ankle cavity of the right hind limb of rats to establish the model of hyperuricemia complicated with gout arthritis.Inflammation index and obstacle index of rats were observed 6 h,12 h,24 h and 48 h after injection of MSU crystal.Meanwhile,ankle swelling volume was measured by toe volume meter and swelling degree was calculated.At the end of the 23 rd day,serum was collected to detect biochemical indexes(SUA,Cr,BUN,GOT,GPT,ADA and XOD)and oxidative stress indexes(MDA,SOD,GSH and CAT).Serum LEVELS of IL-1β,IL-6 and TNF-αwere determined by ELISA.HE staining was used to observe the injury of ankle joint and kidney.Western blot and RT-PCR were used to detect the protein and m RNA expression levels of GLUT9 and URAT1 in kidney,respectively.The protein expression levels of GLUT9,URAT1 and ABCG2 in ileum were detected by Western blot.2.Hyperuricemia rat model was induced by gavage of potassium oxazinate(300 mg/kg)+ hypoxanthine(300 mg/kg)once a day for 14 days.Blood canthal was taken to detect uric acid level on the 7th and 14 th days.After successful modeling,except blank group and model group,positive drug(allopurinol)and isoseranthin low-dose,medium-dose and high-dose treatment were given for 1 week after 1 hour of gavage of modeling drug.One week later,in addition to the blank group(0.9% Na Cl),20 m L(20mg/m L)MSU crystal solution was injected into the ankle cavity of the right hind limb of rats to establish the model of hyperuricemia complicated with gout arthritis.Inflammation index and obstacle index of rats were observed 6h,12 h,24 h and 48 h after injection of MSU crystal.Meanwhile,ankle swelling volume was measured by toe volume meter and swelling degree was calculated.At the end of the 23 rd day,serum was collected to detect biochemical indexes(SUA,Cr,BUN,GOT,GPT,ADA and XOD)and oxidative stress indexes(MDA,SOD,GSH and CAT).Serum LEVELS of IL-1β,IL-6 and TNF-α were determined by ELISA.HE staining was used to observe the injury of ankle joint and kidney.Western blot and RT-PCR were used to detect the protein and m RNA expression levels of GLUT9 and URAT1 in kidney,respectively.The protein expression levels of GLUT9,URAT1 and ABCG2 in ileum were detected by Western blot.Results:1.In vitro experiments showed that Eupatilin had a certain inhibitory effect on XOD activity,which increased with the increase of concentration;The inhibition mechanism of XOD was mixed reversible inhibition,and the inhibition constant Ki of isseraflavin on XOD was calculated by Dixon plot as 136.57.2.The model of high uric acid combined with gout arthritis was established successfully.In the experimental study of lowering uric acid of Eupatilin,it was found that compared with the MC group,isseranthin significantly reduced the serum levels of UA,Cr,BUN and GPT,indicating that isseranthin has a certain liver and kidney protection effect.In addition,isseranthin reduced the oxidative stress damage and inflammatory symptoms of the kidney in rats compared with the MC group.Eupatilin decreased serum uric acid levels by inhibiting the activities of ADA and XOD in serum and liver and regulating the expression levels of GLUT9,URAT1 and ABCG2 in kidney and ileum.3.In the inflammation inhibition experiment of Eupatilin on gout arthritis,after injection of MSU crystal solution into the ankle cavity of rats for 48 h,Eupatilin significantly reduced the inflammation index and ankle swelling VS model group.In addition,compared with the MC group,Eupatilin reduced serum IL-1β level,inhibited the phosphorylation of IκBαand P65 and the protein expression of NLRP3,and alleviated ankle inflammation.Conclusion:Eupatilin could inhibit XOD activity in vitro,thus inhibiting the production of uric acid.Eupatilin alleviated renal oxidative stress injury and inflammatory symptoms caused by high serum uric acid,and also reduced serum uric acid level and inhibited joint inflammation in hyperuricemia rats with gout arthritis.This may be related to the regulation of GLUT9,URAT1 and ABCG2 protein expression in kidney and ileum,inhibition of ADA and XOD activity,inhibition of PHOSPHORylation of IκBα and P65,and NLRP3 protein expression.