Study On The Effect Of Lepisorus Ussuriensis Total Flavonoids Against Rheumatoid Arthritis

Objective To select the anti-rheumatoid arthritis(RA)active components of Lepisorus ussuriensis(Regel et Maack)Ching),and optimize the extraction process of these components,determine its efficacy and mechanism of action on adjuvant arthritis in rats,provide scientific basis for the subsequent development and utilization of Lepisorus ussuriensis.Methods(1)The theoretical active components of Lepisorus ussuriensis anti-RA were selected by network pharmacology.(2)The ultrasonic-assisted extraction method was used,and the single-factor experiment was carried out with ultrasonic power,ultrasonic temperature,ultrasonic time,and liquid-solid ratio as the inspection indicators,and the optimal extraction process of Lepisorus ussuriensis total flavonoids was obtained through response surface process optimization;The total flavonoids of Lepisorus ussuriensis were purified by D101 macroporous resin.(3)A rat adjuvant arthritis model was established by subcutaneous injection of Freund’s complete adjuvant(FCA),and the experimental animals were divided into blank control group(CG),model group(MG),methotrexate group(WPG),tripterygium glycoside group(CPG),Lepisorus ussuriensis total flavonoids high-dose group(LHG)and Lepisorus ussuriensis total flavonoids low-dose group(LLG).Rats were regularly monitored for changes in body weight and toe swelling,the organ index of the rats′ thymus and spleen was calculated,detected the content of TNF-α,IL-1βand IL-6 in serum of each group,HE staining and histomorphology were performed on the rat ankle joints in each group.(4)The content of vitamin C(Vitamin C,VC)in the serum of the rats in each group was detected by high performance liquid chromatography(High Performance Liquid Chromatography,HPLC).(5)Check the 16 S r RNA V3-V4 region in rat feces by Novaseq 6000 PE250 sequencing platform.Results(1)Network pharmacological screening was used to obtain the effective active ingredient of Lepisorus ussuriensis against RA as total flavonoids.(2)The optimal extraction conditions of total flavonoids of Lepisorus ussuriensis were optimized by response surface methodology: ultrasonic power 360 W,ultrasonic temperature 58 ℃,ultrasonic time 50 min,liquid-solid ratio 20:1,the average extraction rate was 17.944%.Ultrasonic extraction of 1.3 kg Lepisorus ussuriensis was carried out according to this method,and D101 macroporous resin was used for purification to obtain 120.05 g Lepisorus ussuriensis total flavonoid extract,with a content of 55.78%.(3)After treatment with Lepisorus ussuriensis total flavonoids,the rats were positive feeding,active status,the body mass has all continued to increase,and their fur is smooth and shiny;compared with the CG group,higher foot swelling in the MG group,with a extremely significant difference(p<0.01),the contents of TNF-α,IL-1β and IL-6 in rats increased significantly(p<0.05);compared with the MG group,splenic index,thymus index,liver index and kidney index were extremely significantly increased in LHG group(p<0.01),and there was no significant difference from the CG group;compared with the CG group,Msynosynoproliferation was hyperplasia in the MG group,and appeared severe cartilage damage and inflammatory cell infiltration,compared with the MG group,In the LHG group,synoviocyte hyperplasia,synovial tissue congestion and chondrocyte destruction were significantly reduced,inflammatory cell infiltration was decreased.(4)Compared with the CG group,the serum vitamin C content of the MG group,CPG group,and LLG group was extremely significantly different(p<0.01),and the serum vitamin C content of the WPG group was significantly different(p<0.05).There was no significant difference between the CG group and LHG group;Compared with the MG group,the serum vitamin C content of all the treatment groups was significantly different(p<0.01).(5)High-dose total flavonoids of Lepisorus ussuriensis can upregulate the abundance of Unclassified,Ralstonia and Oscillospira in the intestinal flora,Faecalibacterium,Bacteroides,Bacteroidaceae,Porphyromonadaceae,Parabacteroides,Dialister,Butyricimonas,Eubacterium,Sarcina,Gemmiger were characteristic bacteria.Conclusions In this study,total flavonoids,the main component of anti-RA,which were screened from Lepisorus ussuriensis,were extracted and purified,and a simple and practical process of extracting and purifying total flavonoids of Lepisorus ussuriensis was obtained;The experimental results show that the total flavonoids of Lepisorus ussuriensis have a significant therapeutic effect on adjuvant arthritis rats,and the mechanism of action may be the upregulation of the abundance of the genera Unclassified,Ralstonia and Oscillospira in the intestinal flora,to increase the vitamin C content in rats with adjuvant arthritis and reduce the content of TNF-α,IL-1β and IL-6 and then play an anti-RA effect.