| Objective:To establish adjuvant-induced arthritis(AIA)in rats,and study the pharmacodynamics and mechanism of ethanol extract of Tujia ethnomedicine KHS(Xuetong)against rheumatoid arthritis(RA).The research aimed to provide reference for screening new drugs for treatment of RA,and provide scientific basis for further development and utilization of national innovative drugs.Methods:1.HPLC was used to control the quality of alcohol extract from KHS,and the maximum tolerated dose of alcohol extract was determined by MTD.ICR mice were randomly divided into control group(0.3%CMC-Na solution)and KHS alcohol extract group(26 g·kg-1),and were administered for 2 times within 24 h.The mice were observed for 14 d in the diet,drinking water,body weight,mental state and so on.2.To study the inhibitory effect of alcohol extract from the KHS on acute inflammation in mice induced by xylene induced ear edema and carrageenan induced rat paw swelling model.ICR mice were randomly divided into model group,indomethacin group(10 mg·kg-1),KHS of low,medium and high dose group(200?400?800 mg·kg-1),impact assessment of KHS on xylene induced ear swelling model in mice;Wistar rats(male and female)were randomly divided into model group,indomethacin group(100 mg·kg-1),KHS of low,medium and high dose group(200?400?800 mg·kg-1and100?200?400mg·kg-1),impact assessment of KHS alcohol extract carrageenan induced rat paw swelling model.3.To study the analgesia effect of alcohol extract from the KHS by acetic acid induced writhing test and radiation induced rat tail flick test.The ICR mice were randomly divided into model group,Rotundine group(100 mg·kg-1),KHS of low,medium and high dose group(50?100?200 mg·kg-1),observe the effect of KHS on acetic acid induced writhing times of mice;SD rats were randomly divided into model group,Rotundine group(100 mg·kg-1),KHS of low,medium and high dose group(50?100?200 mg·kg-1),impact assessment of KHS on radiation induced rat tail flick latency.4.To study the mechanism of anti-inflammatory effect by establishing model of rat paw swelling induced by carrageenan.Wistar rats were randomly divided into model group,indomethacin group(100 mg·kg-1),KHS at low,medium and high dose group(200?400?800 mg·kg-1).Rats were injected with 0.1ml carrageenan in the right hind foot,anesthetized rats after5 hours of injection,the abdominal aorta was taken out of blood,the serum was separated,and the feet were intercepted.ELISA kits was used to detect the levels of TNF-??IL-1??IL-6 in serum of rats,and the expression levels of COX-1,COX-2 and i NOS were detected by Western Blot.5.AIA was established in male SD rats as described previously,and animals were daily treated by gavage with KHS ethanol extract(1.0 g·kg-1)or vehicle(0.3%CMC-Na)throughout the 30-day experiment.The incidence and severity of arthritis were evaluated using clinical parameters.On day 30,bone destruction of the arthritic joints were assessed by computed tomography(CT)and histopathological analyses.The serum levels of pro-inflammatory cytokines TNF-?,IL-1?,and IL-6 were measured by ELISA.6.AIA was established in male SD rats to systematic study the effect and mechanism of alcohol extract of KHS.60 male SD rats were randomly divided into normal group,model group,indomethacin group(1.0 mg·kg-1),KHS of low,medium and high dose group(200?400?800 mg·kg-1).From the first day of experiment,except normal group,root of rats tail subcutaneous injection of 0.1 ml containing 200?g tested heat inactivated Mtb(H37Ra)of complete Freund's adjuvant(CFA),continuous administration of 30 d.The time of the onset of arthritis was recorded,and the swelling volume of the rats was measured,and the arthritis index was calculated according to the arthritis severity score.The damage degree of the left foot was observed by CT scanning.30d after,the rats were anesthetized,the abdominal aorta was collected,the serum was separated and the hind feet were intercepted.The levels of TNF-??IL-1??IL-6 and IL-17A/F in serum were detected by ELISA kits,and the expression levels of MMP-1,MMP-3 and TIMP-1 were detected by Western Blot.Results:1.From the HPLC chromatogram of 210 nm wavelength under the conditions of the observed KHS alcohol extract containing 5 effective components:Schistandronic acid,D-epigalbacin,Heteroclitalactone A,Schisanlactone E,Nigranoic acid of the of extract samples,determination of the content of showed the highest content is Schisanlactone E,the lowest is D-epigalbacin.2.MTD method was used to measure the maximum tolerance dose of mice to the alcohol extract of KHS,which was 26g·kg-1(equivalent to 253 times the daily dosage of adults).Compared with the control group,no mice had death in the control group,the health status was normal,the body weight kept increasing,and the drinking water was normal.3.Compared with the model group,KHS of low,medium and high dose group(100?200?400 mg·kg-1)could significantly inhibit the ear edema induced by xylene in mice(P<0.05 or P<0.01);KHS of low,medium and high dose group(200?400?800 mg·kg-1)could significantly inhibit the carrageenin induced swelling degree in female rats(P<0.05 or P<0.01),KHS of low,medium and high dose group(100?200?400 mg·kg-1)could significantly inhibit the carrageenan induced male rats paws swelling(P<0.05 or P<0.01).4.Compared with the model group,KHS of low,medium and high dose group(50?100?200 mg·kg-1)can significantly reduce the number of writhing(P<0.05 or P<0.01).KHS of low medium and high dose group(50?100?200 mg·kg-1)could significantly prolong the latent period of radiation induced rat tail flick(P<0.05 or P<0.01).5.Compared with model group,KHS of low,medium and high dose group(200,400,800 mg·kg-1)can significantly reduce the serum of rats induced by carrageenan TNF-?,IL-1?,IL-6 expression levels of inflammatory cytokines(P<0.05 or P<0.01).Western Blot protein detection the results show that compared with the model group,the KHS can significantly reduce the expression level of COX-1,COX-2 and i NOS protein in rats with paw tissue(P<0.05 or P<0.01).6.Treatment with 1.0 g·kg-1KHS significantly inhibited the onset and progression of AIA.The vehicle-treated rats all developed severe arthritis,while the incidence of AIA in the KHS-treated rats was as low as 55%(P=0.035).The KHS-treated rats exhibited 1.8-to 2.3-fold reduction of paw swelling,and gained 10 to 20%more body weight than the vehicle-treated AIA rats throughout the experiment.CT and histopathological examinations revealed that KHS markedly protected AIA rats from cartilage and bone destruction of joints.The serum levels of TNF-?,IL-1?,and IL-6were significantly decreased in the KHS-treated rats than the vehicle-treated AIA rats(P<0.05 or P<0.01).7.Compared with the normal group,rat paw swelled,arthritis index increased significantly,infiltration of inflammatory cells in toe tissueincreased,pannus formation,small animal CT findings in rats after foot joint bone destruction,TNF-alpha,IL-1 beta,IL-6,IL-17A,IL-17F in serum by sub expression increased.Toe tissue MMP-1,MMP-3,TIMP-1 protein expression level increased significantly(P<0.05,P<0.01 or P<0.001).The incidence rate of arthritis model rats was 100%,compared with the model group,KHS at low,medium and high dose group(200?400?800 mg·kg-1)arthritis incidence rate were 90%,60%,30%,KHS can significantly inhibit the rat paw swelling,reduce arthritis index(P<0.05,P<0.01 or P<0.001).Histopathological observation of the alcohol extract of KHS can significantly reduce the inflammatory cell infiltration,synovial proliferation and reduce the formation of pannus(P<0.05 or P<0.01)in the paws of AIA rats.CT examinations showed that the KHS can significantly inhibit the destruction of articular cartilage and bone erosion(P<0.05 or P<0.01).The molecular mechanism study found that KHS can significantly inhibit TNF-?,IL-1?,IL-6,IL-17A,IL-17F etc.KHS can also reduce the MMP-1,MMP-3 protein expression and increased TIMP-1 protein expression.Conclusion:Tujia ethnomedicine KHS(Xuetong)has high safety and good analgesic and anti-inflammatory effects;KHS plays a role in anti-inflammatory effect by reducing expression level of COX-1,COX-2 and i NOS protein and decreasing content of TNF-?,IL-1?,IL-6;KHS treatment of RA effect is obvious,the relative mechanism may be through suppression of TNF-?,IL-1?,IL-6,IL-17A,IL-17F,reduce the expression level of MMP-1 and MMP-3 protein,increase the expression levels of TIMP-1 protein play a protective role in the joint. |
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