Anti-inflammatory Analgesic Action Of Panax Notoginseng Saponins And Its Compatibility To The Influence RANKL/OPG Expression Of Synovium The Rats With Collagen Induced Arthritis

Objective:Rheumatoid arthritis is a chronic autoimmune disease, its etiology andpathogenesis is not clear, the basic characteristics of synovitis and progressivebone and cartilage destruction, inhibiting bone destruction is the key to thetreatment of RA. In recent years with the tumor necrosis factor alpha (TNF-α)inhibitors as a representative of biological agents is RA therapeuticbreakthrough, but its effectiveness is limited, the clinical remission rate is notvery ideal, expensive and possible adverse reactions at the same time, make itslimited in clinical application. In recent years, research suggests the naturalplant medicine effective component of RA bone destruction may havetherapeutic effect. This topic proposed three aspects:1.Establish the painmodel in mice, select the optimal dose of Panax Notoginseng Saponinsanalgesia;2.With1%carageen glue as inflammatory, acute inflammation inmice model was established, select Panax Notoginseng Saponins optimumdoses of anti-inflammatory;3.Establish the rat collagen induced arthritis (CIA)model, using ELISA method to detect serum OPG，RANKL，MMP-3contentand the cotent of serum TNF-α, IL-6by protein chip technology; immunehistochemical methods detect the expression of OPG,RANKL and MMP-3inankle joint synovium tissue, investigate the part mechanism of RA bonedestruction, at the same time explore the Tripterygium Glycoside union PanaxNotoginseng Saponins on experimental arthritis inflammatory score and bonedestruction of related factors, provides the experimental basis for the clinicaltreatment of RA. Method:1Establish the mice model of pain and detect pain threshold by grouping1.1Hot plate methodKunming mice, weight18-22g. Put them at constant temperaturetemperature55±0.5℃water bath on the hot plate. Observe and record themice from being placed on the hot plate to lick after sufficient time, as a painresponse latency.Choosing pain reaction incubation period is between10s and30s40female mice, and random divide them into Panax NotoginsengSaponins (50mg/kg,100mg/kg,200mg/kg), iresearch smoothie group (10mg/kg) and normal saline group, there have8mice in each group,(0.3ml/only).After the treatment0.5h,1.0h,2.0h,3.0h mesure the mice pain thresholdchange.1.2Radiant heat stimulation method (the method of flicking tail)Kunming mice, weight18-22g. Put them in the cage, rat tail was loadedon rat tail light pain measurement instrument, direct beam in rat tail1/3(about1-2cm away from the tail, a pen marked with black spots, so that each timethe light point in the same location), measure the time needed for a spin inmice, as pain reaction incubation period.Choose pain response latency for2-6s50male mice,theywere random divided into Panax Notoginseng Saponins(50mg/kg,100mg/kg,200mg/kg), iresearch smoothie group (10mg/kg) andnormal saline group, each group of8, lavage dosage (0.3ml/only). After thetreatment0.5,1.0,2.0h mesure the mice pain threshold change.1.3Acetic acid twisting methodKunming mice, weight18-22g,female.They were randomly divided intoPanax Notoginseng Saponins (50mg/kg,100mg/kg,200mg/kg), iresearchsmoothie group (10mg/kg) and normal saline group, each group of8, eachgroup of continuous irrigation three days,on the3ee day after the treatment of1h,according to0.1mg/kg intraperitoneal injection of0.7%acetic acidsolution, after observing5minutes, then began to record the mice body torsiontimes (with abdominal contraction, body distortion, hind legs stretching, crawl,etc as the judgment standard)15minutes, and calculate the inhibitionrate.Inhibition rate=(the average number of twist body of negative control group-the average number of twist body of treatment group)/the averagenumber of twist body of negative control group*100%.2Establish acute arthritis model of mice by carrageenan and evaluationinflamationChoosing kunming mice,18-22g, male and female half, they wererandomly divided into Panax Notoginseng Saponins (50mg/kg,100mg/kg,200mg/kg), iresearch smoothie group (10mg/kg) and normal control group,continuous irrigation3d, on the3ee day after the treatment of1h, the right ofthe rat toes intradermal l%carageen glue caused inflammatory, measuredafter inflammatory1h,2h,3h,4h,5h the right rear foot paw volume, calculatethe swelling degree and the inhibition of groups.3Establish collagen induced arthritis model and laboratory testingChoosing Wistar rats weight in180-200g, the number of male and femalewere equal. After measuring the volume of bilateral paw, the bottom of rightpaw was disinfected and established collagen induced arthritis model byinjecting0.2ml collagen from chicken sternal cartilage type II in the tail, righthind limb and back together, and boosted at the14th day to make manufacturethe model of CIA.There are six rats were injected normal saline using thesame method as normal control group.On21st day after modeling, rats thatpresented secondary-response were divided randomly into Model group, LEFgroup, TG group, TG+PNS group, and each group included six samples. Byintragastric administration, they were respectively given LEF (10mg/kg), TG(10mg/kg) and TG (10mg/kg)+PNS (200mg/kg) for42days, and Normalgroup and Model group were given equivalent volume of saline. Aftertreatment6weeks, measuring paw volume, weights and AI, rats weresacrificed and drawn of blood by abdominal aortas under10%chloralic hydrasto get serum. The levels of serum OPG，RANKL and MMP-3were detectedby ELISA, the cotent of serum TNF-a，IL-6were detectde by protein chiptechnology,while the expression of OPG、RANKL and MMP3in synoviumtissues were acquired by immunohistochemical method.Statistical analysis: The results were expressed as x±s. All statistical analyses of data were computed by SPSS13.0. The comparisons of groupswere evaluated by one-way analysis of variance (ANOVA)，P＜0.05wasjudged as a statistical difference.Results：1The effects of Panax Notoginseng Saponins on pain threshold of mice1.1Hot plate methodBefore delivery each pain threshold had no statistical difference (P＞0.05); For0.5h: PNS each dose group and imrecoxib group pain thresholdwere higher than normal control group (P＜0.01), PNS each dose group painthreshold were lower than imrecoxib group (P＜0.01), PNS low-dose painthreshold was lower than the high-dose group (P＜0.01), PNS medium-dosepain threshold is lower than the high dose group, but there was no statisticallysignificant difference (P＞0.05); For1h,2h,3h: PNS each dose group andimrecoxib group pain threshold were higher than physiological saline group (P＜0.01), PNS each dose group pain threshold was lower imrecoxib group (P＜0.01), PNS low, middle dose group pain threshold were lower than the highdose group (P＜0.05).1.2Radiant heat stimulation method (the method of flicking tail)Before delivery each pain threshold had no statistical difference (P＞0.05); For0.5h,1h,2h: PNS each dose group and imrecoxib group painthreshold were higher than in physiological saline group (P＜0.01), PNS eachdose group pain threshold were lower than imrecoxib group (P＜0.01), PNSlow pain threshold, middle dose group were lower than the high dose group (P＜0.05).1.3Acetic acid twisting methodPNS each dose group and imrecoxib group twisting the body startingtime were higher than in normal saline group (P＜0.01), PNS each dose grouptwist body starting time were lower than imrecoxib group (P＜0.01), PNS low,middle dose group twist body starting time were lower than the high dosegroup(P＜0.05); PNS each dose group and imrecoxib group body torsionfrequency were lower than normal saline group (P＜0.05), PNS each dose group body torsion frequency were higher than imrecoxib group (P＜0.01),PNS low, medium dose group body torsion frequency is higher than the highdose group (P＜0.05), the inhibition rate of PNS low, medium and high dosegroup were12.22%,21.75%and26.71%.2Panax Notoginseng Saponins to the inhibition of carrageenan caused footswelling in miceInflammatory1h: PNS each dose group and imrecoxib swelling degreesbelow the normal saline group (P＜0.01), PNS low, middle swelling degreeswere higher than imrecoxib group (P＜0.01), PNS high dose group swellingdegrees was higher than imrecoxib group, but there was no statisticallysignificant difference (P＞0.05), PNS low and middle dose swelling degreeswere higher than the high dose group (P＜0.05), PNS low, middle and highgroup inhibition rate were25%,60%,77.1%; Inflammatory2h: PNS mediumand high dose group and imrecoxib swelling degrees below the normal salinegroup (P＜0.01), PNS low-dose swelling degrees below the normal salinegroup, but there was no statistically significant difference (P＞0.05), PNSeach dose group swelling degrees were higher than imrecoxib group (P＜0.05),PNS low-dose swelling degrees was higher than the high dose group (P＜0.01), PNS middle dose group swelling degrees was higher than the high dosegroup, but there was no statistically significant difference (P＞0.05), theinhibition rate of PNS low, medium and high dose group were0.21%,40.3%,57.6%; Inflammatory3h: PNS medium and high dose group and imrecoxibgroup swelling degrees below the normal saline group (P＜0.01), PNSlow-dose swelling degrees below the normal saline group, but there was nostatistically significant difference (P＞0.05), PNS each dose group swellingdegrees higher than imrecoxib group (P＜0.05), PNS low and middle dosegroup swelling degrees higher than the high dose group (P＜0.05), theinhibition rate of low, medium and high dose group were0.63%,34.7%,54.5%; Inflammatory4h: PNS medium and high dose group and imrecoxibswelling degrees below the normal saline group (P＜0.01), PNS low-doseswelling degrees below the normal saline group, but there was no statistically significant difference (P＞0.05), PNS low and middle dose group swellingdegrees higher than imrecoxib group (P＜0.01), PNS high dose groupswelling degrees higher than imrecoxib group, but there was no statisticallysignificant difference (P＞0.05), PNS low-dose swelling degrees higher thanthe high dose group (P＜0.01), the PNS middle dose group swelling degreeshigher than the high dose group, but there was no statistically significantdifference (P＞0.05), the inhibition rate of PNS low, medium and high dosegroup were20.0%,52.5%,60.3%; Inflammatory5h: PNS each dose groupand imrecoxib group swelling degrees below the normal saline group (P＜0.05), PNS low and middle dose group swelling degrees higher than imrecoxibgroup (P＜0.01), PNS high swelling degrees higher than imrecoxib group, butthere was no statistically significant difference (P＞0.05), PNS low-doseswelling degrees higher than the high dose group (P＜0.01), the PNS middledose swelling degrees higher than the high dose group (P＞0.05), theinhibition rate of PNS low, medium and high dose group were32.7%,73.4%and88.2%.3TG, PNS and its compatibility to the effect of rat weight changeBefore modeling, there was no statistical differences among the weight ofeach group (P＞0.05). On21st day after modeling, the weight-gain value ofmodel group was12.69±1.16, TG group11.80±1.58, LEF group12.02±1.76,TG+PNS group12.32±1.73were lower normal control group24.42±3.09(P＜0.01). But there was no statistical differences among them (P＞0.05). Aftertreatment6weeks the weight-gain value of TG group was11.30±1.53, LEFgroup15.22±2.44, TG+PNS group16.95±4.40, and model group10.54±1.91.Compared with model group, there were no statistical differences among TGgroup(P＞0.05), but there were statistical differences among LEF group（P＜0.05) and TG+PNS group(P＜0.01), there was statistical differences betweenTG group and it (P＜0.01).4TG, PNS and its compatibility to the effect of rat AI changeOn21st day after modeling, the AI value of Model group was11.50±1.87,TG group11.65±1.05, LEF group11.17±0.98, TG+PNS group10.97±1.75. But they were no statistical differences among them (P＞0.05). Aftertreatment6weeks the AI change value of TG group6.33±1.21, LEF group5.83±0.75, TG+PNS group5.66±1.37were lower model group was11.67±1.03(P＜0.01), but there was no statistical differences among eachtreatment group (P＞0.05).5TG, PNS and its compatibility to the effect of rat paw swelling-inhibitionratio changeAfter treatment4weeks, the both paws swelling degree LEF group(83.92±8.35,73.48±9.53), TG group (87.02±21.63,79.62±14.15), TG+PNSgroup (75.28±14.77,74.91±11.94) were lower model group (120.16±11.41,108.65±9.21)(P＜0.01), Compared with TG group, there were no statisticaldifferences among LEF group, TG+PNS group and it (P＞0.05), Theinhibition ratio of LEF group, TG group and TG+PNS group were respectively31.26%,27.15%,34.20%; After treatment6weeks, the both paws swellingdegree LEF group (64.15±3.48,61.34±8.34), TG group (70.16±10.99,64.73±8.15), TG+PNS group (56.65±6.34,53.11±2.99) were lower modelgroup (120.24±16.73,109.74±16.57)(P＜0.01); Compared with TG group,there was no statistical differences between LEF group and it (P＞0.05), butthere was statistical differences between TG+PNS group and it (P＜0.05). Theinhibition ratio LEF group, TG group and TG+PNS group were respectively45.38%,41.33%,52.24%.6TG, PNS and its compatibility to the effect of rat serum level of OPG,RANKL, MMP-3, TNF-α, IL-6change6.1Comparison of serum OPG level (pg/ml)The level of serum OPG in Model group was (2685.87±169.74), TGgroup (2371.38±150.74), LEF group (2071.35±114.98), TG+PNS group(1853.16±128.89), and normal control group (1611.39±105.82). Comparedwith normal control group, there were statistical differences among modelgroup, LEF group, TG group (P＜0.01), there was statistical differencesamong TG+PNS group (P＜0.01); Compared with Model group, there werestatistical differences among LEF group, TG group and TG+PNS group (P＜ with TG group there was statistical differences (P＜0.01).6.2Comparison of serum RANKL level (pg/ml)The level of serum RANKL in Model group was30.97±1.78, TG group26.76±1.33, LEF group24.55±1.16, TG+PNS group22.32±1.18and normalcontrol group20.01±1.91. Compared with normal control group, there werestatistical differences among model group and TG group (P＜0.01), there werestatistical differences among LEF group and TG+PNS group (P＜0.05)；Compared with Model group, there were statistical differences among the LEFgroup and TG+PNS group (P＜0.01), there was statistical differences amongthe TG group(P＜0.05), the level of serum RANKL TG+PNS group were thelowest, compared with TG group there was statistical differences (P＜0.05).6.3Comparison the proportionality of serum OPG/RANKLThe proportionality of serum OPG/RANKL in model group was96.12±7.39, TG group91.44±2.47, LEF group88.31±8.73, TG+PNS group79.68±8.42and normal control group77.52±4.50. Compared with normalcontrol group, there was statistical differences among LEF group and TGgroup(P＜0.05), there was no statistical differences among TG+PNS group（P＞0.05）; Compared with model group, there was no statistical differencesamong TG group (P＞0.05), there was statistical differences among LEFgroup and TG+PNS group (P＜0.01), the TG+PNS group were the lowest,compared with TG group there was no statistical differences (P＞0.05).6.4Comparison of serum MMP-3(ng/ml)The level of serum MMP-3in model group was21.46±1.71, TG group18.78±1.15, LEF group16.36±0.93, TG+PNS group14.72±0.59and normalcontrol group12.75±1.10. Compared with normal control group, there werestatistical differences among model group, LEF group and TG group (P＜0.01), there was statistical differences among TG+PNS group (P＜0.05);Compared with model group, there were statistical differences among LEFgroup, TG group, TG+PNS group (P＜0.01), and the TG+PNS group was thelowest, compared with TG group there was statistical differences (P＜0.01). 6.5Comparison of serum TNF-α level (pg/ml)The level of serum TNF-a in model group was166.99±3.44, TG group153.69±5.35, LEF group144.60±4.08, TG+PNS group136.18±3.13andnormal control group126.24±4.00. Compared with normal control group,there were statistical differences among them (P＜0.01); Compared withModel group, there were statistical differences among them (P＜0.01), and theTG+PNS group was the lowest, compared with TG group there was statisticaldifferences (P＜0.01).6.6Comparison of serum IL-6level (pg/ml)The level of serum IL-6in Model group was104.90±5.38, TG group92.18±4.86, LEF group83.95±5.78, TG+PNS group74.07±4.88and normalcontrol group66.96±4.74. Compared with normal control group, there werestatistical differences among model group, LEF group and TG group (P＜0.01), there was statistical differences among TG+PNS group (P＜0.05);Compared with model group, there were statistical differences among them (P＜0.01), and the TG+PNS group was the lowest, compared with TG groupthere was statistical differences (P＜0.01).7TG, PNS and its compatibility to the effect of pathological change of ratanklesThere were no pathological changes of ankles in normal control group. Inmodel group, there were synovial cells proliferating, a great quantity ofinflammatory cells infiltrating in synovium, synovium liners graduallythickening, synovial cells proliferating in the join region of synovium andcartilage, cartilages damaged, bone destruction in subchondral and trabecularbone, and even the integrity structures of cartilage and trabecular bone werenot observed. The pathological changes of ankles were all alleviated in eachtreatment group. The pathological score in model group was2.33±0.51, TGgroup1.83±0.41, LEF group1.50±0.55, TG+PNS group1.33±0.52. Comparedwith model group, there were statistical difference among LEF group，TG+PNS group and it (P＜0.01), but there was no statistical differencesbetween TG group and it (P＞0.05), the pathological score in TG+PNS group was the lowest, compared with TG group there was statistical differences（P＞0.05）.8TG, PNS and compatibility to the effect of OPG, RANKL and MMP-3expression of synovium tissue in rat8.1Comparison of OPG expression in each ankle synovial tissuesThe expression of OPG in synovium tissue of ankles in model group was64.50±3.08, TG group75.00±3.63, LEF group84.17±3.19, TG+PNS group93.83±4.83and normal control group102.00±2.61. Compared with normalcontrol group, there were statistical differences among them (P＜0.01);Compared with model group, there was statistical differences among them (P＜0.01), the TG+PNS group was least, compared with TG group there wasstatistical differences (P＜0.01).8.2Comparison of RANKL expression in each ankle synovial tissuesThe expression of RANKL in synovium tissue of ankles in model groupwas70.00±3.35, TG group76.00±4.20, LEF group85.83±3.50, TG+PNSgroup94.50±4.23and normal control group104.50±4.59. Compared withnormal control group, there were statistical differences among them (P＜0.01);Compared with model group, there was statistical differences between TG andit (P＜0.05), there were statistical differences among LEF and TG+PNS and it(P＜0.01), the TG+PNS group was least, compared with TG group there wasstatistical differences (P＜0.01).8.3Comparison of MMP-3expression in each ankle bone tissuesThe expression of MMP-3in bone tissue of ankles in Model group was82.83±4.07, TG group92.83±4.62, LEF group98.83±5.53, TG+PNS group106.00±4.73and normal control group116.33±5.43. Compared with normalcontrol group, there were statistical differences among them (P＜0.01);Compared with model group,there were statistical differences among them (P＜0.01), the TG+PNS group was least, compared with TG group there wasstatistical differences (P＜0.01).Conclusions：1Various pain model and arthritis model research confirmed that Panax Notoginseng Saponins has obvious analgesic and anti-inflammatory effects,and its therapeutic effects existed dose-effect relationship.2Tripterygium glycoside had significant inhibition effect to the jointswelling, arthritis index and serum inflammatory cytokine TNF-a, IL-6ofCIA rats, Panax Notoginseng Saponins combined with Tripterygium glycosidewere shown in the anti-inflammatory enhancers effect.3Tripterygium glycoside combined with Panax Notoginseng Saponinscan obviously lowered RANKL, OPG and MMP-3expression of serum andsynovial in the CIA rats, its effect is superior to used Tripterygium glycoside,it prompted compatibility application had a synergistic effect on inhibition ofbone destruction.