Anti-inflammatory And Analgesic Effect And Related Mechanisms Of Ginsenoside Metabolite Compound K

Ginsenoside metabolite compound K(20-O-?-D-glucopyranosyl-20(S)-ginophenol,CK)belongs to the group of protopanaxadiol-type saponins.It was isolated by Japanese researchers in 1972 and produced from ginsenosides(such as Rb1,Rb2 and Rc)though various transformation methods.Studies have shown that the pharmacological activity of ginsenosides was mainly mediated through its metabolic component CK,and CK has a variety of pharmacological activities such as anti-inflammatory,anti-tumor,anti-allergy,anti-diabetes,liver protection,neuroprotection,and cardiovascular protection effects.Our previous study found that CK exerted immunomodulatory and anti-arthritic effects in animal models of adjuvant-induced arthritis(AA)and collagen-induced arthritis(CIA).We found that CK significantly reduced the global assessment and arthritis index in CIA mice and AA rats;reduced the number of joint swelling and improve joint histopathology,inhibited synovial cell proliferation and angiogenesis,reduced inflammatory cell infiltration in synovium and bone destruction.CK reduced the spleen index,reduced the histopathological changes in spleen,including the thickness of the periarterial lymphoid sheath and marginal zone,lymph node proliferation and germinal center formation.In addition,CK exerted an immunomodulatory effect on AA rats and CIA mice by reducing abnormal activation of immune cells(dendritic cells,T cells and B cells)and restoring cytokine balance.These results suggest that the immunomodulatory activity of CK may be one of the important mechanisms of its anti-inflammatory effect.However,whether CK has an anti-inflammatory and analgesic effect on acute inflammation remains unclear.In this study,based on the previous studies,the anti-inflammatory activity of CK was evaluated in xylene-induced ear swelling model and carrageenan-induced inflammation model.The hot plate test,acetate-induced writhing test and carrageenan induction inflammatory pain model were established to evaluate the analgesic effect of CK.The possible mechanism was explored by detecting the levels of PGE2,COX-1 and COX-2.Objective:To evaluate the anti-inflammatory and analgesic activity of CK and the possible mechanism.Methods:1.Xylene-induced ear swelling model and carrageenan-induced rat paw swelling were used to evaluate the anti-inflammatory effect of CK on acute inflammation.Kunming mice were treated with CK(7,14,28,56,112,224 mg/kg)and positive control drug celecoxib(28 mg/kg)for 5 days after continuous intragastric administration.One hour after the last administration,the inhibition rate of drugs on the xylene-induced ear was calculated.SD rats were treated with CK(5,10,20,40,80,160 mg/kg)and positive control drug celecoxib(20 mg/kg)continuously for 5 days.One hour after the last administration,0.1 ml of 1% carrageenan was injected intracutaneously to induce inflammation.The paw volume was measured at intervals of 1 h after inflammation,and the difference between the paw volume before and after inflammation was calculated as the degree of swelling and the inhibition rate of swelling of each group was calculated.2.The analgesic effect of CK was evaluated using hot-plate tests,acetic acid-induced writhing and carrageenan-induced inflammatory pain models.Kunming mice were continuously administered intragastrically with CK(7,14,28,56,112,224 mg/kg)for5 days,and the positive control drug sufentanil(40 ?g/kg)was subcutaneously injected.One hour after the last administration,the latency of the hot plate reaction was measured to evaluate the analgesic effect of CK.Kunming mice were administered with CK(7,14,28,56,112,224 mg/kg)and positive control drug celecoxib(28 mg/kg)continuously for 5 days.After the last administration for 1 hour,0.4 ml of 0.6% acetic acid solution were intraperitoneally injected.Five minutes later,the number of writhings was observed and recorded within 5 minutes.The inhibition rate of the test drug on the writhing of the mice was calculated to evaluate the analgesic effect of CK.SD rats were treated with CK(5,10,20,40,80,160 mg/kg)and positive control drug celecoxib(20 mg/kg)for 5 days after continuous gavage and One hour after the last administration,the right foot was injected intradermally with1% carrageenan 0.1ml to induce inflammation.Pain thresholds were measured by using mechanical nociceptive stimuli after injected carrageenan 1,2,3,4,5,and 6h,respectively.The measured value(g)of the paw flexion response before and after administration of the test animal was used as a pain threshold.3.The levels of prostaglandin E2(PGE2),cyclooxygenase-1 and cyclooxygenase-2(COX-1)in carrageenan-induced rat paw swelling and gastric mucosal were detected by enzyme-linked immunosorbent assay(ELISA).The expression of COX-1 and COX-2 in carrageenan-induced rat paw swelling and gastric mucosal were detected by western blot.In vitro,the effect of CK(10-9,10-8,10-7,10-6,10-5 mol/L)on COX-1 and COX-2 activity by measuring the production of 6-keto-PGF1? and PGE2 in rat peritoneal macrophages,respectively.Results:1.CK has a direct anti-inflammatory effect,inhibited xylene-induced ear edema and carrageenan-induced paw swelling Compared with the normal group,xylene induced redness in the left ear of mice.Compared with the model group,CK(7,14,28,56,112,224 mg / kg)and celecoxib(28mg / kg)significantly reduced xylene-induced ear swelling in mice.With increasing the dose of CK,its inhibitory effect on ear swelling gradually increased,and CK(224mg/kg)has been achieved maximum inhibition efficiency almostly(the inhibition rate was 93.9%).Carrageenan-induced paw edema in rats was used to evaluate the direct anti-inflammatory activity of CK and celecoxib was used as a positive control drug.Compared with the normal group,the rats paw edema appeared after intradermal injection carrageenan 1h,reached peak after 4h and then remission.Compared with the normal group,there was no significant difference in the degree of paw edema in each group after inflammation 1h.CK(80,160 mg / kg)and celecoxib(20mg / kg)at2h,3h,4h,5h and 6h can reduce the swelling of the paw,CK(40,80,160 mg / kg)at4h,5h,6h can reduce the paw edema volume.2.CK has a peripheral analgesic effect,which can reduce the number of writhing caused by acetic acid and increase the pain threshold of carrageenan-induced inflammatory pain.Evaluation of central analgesic activity of CK by hot plate method.Compared with the control group,CK(7,14,28,56,112,224 mg / kg)had no effect on the pain threshold of hot plate-induced whereas the pain threshold of sufentanil(40 ?g / kg)group was significantly increased,indicating that CK had no analgesic effect on hot plate-induced pain.In this study,acetic acid-induced writhing was used to evaluate the peripheral analgesia of CK.After intraperitoneal injection of 0.6% acetic acid for5 minutes,the mice showed a significant writhing.Compared with the model group,CK(224 mg / kg)and celecoxib(28 mg / kg)significantly reduced acetic acid-induced writhing in mice(P <0.01).Carrageenan-induced inflammatory pain is used to evaluate the analgesic effect of CK on pain induced by mechanical stimulate.Compared with the normal group,the rats pain threshold decreased after intradermal injection carrageenan 2h,reached peak after 4h and then pain threshold increased.Compared with the model group,CK(40,80,160 mg / kg)and celecoxib(20 mg / kg) significantly increased the pain threshold of rats at 3h,4h,5h and 6h after inflammation.3.CK exert anti-inflammatory and analgesic effect via reduce COX-2 expression and down-regulated the level of PGE2 In carrageenan-induced rat paw edema model,compared with the normal group,the level of PGE2 increased in the swollen paw,and CK(10,20,40,80,160 mg/kg)decreased the level of PGE2 in swollen paw.The level of PGE2 in the gastric mucosa of the model group did not change,and CK had no effect on it.Compared with the normal group,the expression of COX-2 in the swollen paw and gastric mucosa increased of the model group,and CK(20,40,80,160 mg / kg)reduced the expression of COX-2.Compared with the normal group,the expression of COX-1 had no change in the swollen paw and gastric mucosa of the model group.CK had no effect on the expression of COX-1.After normal rat peritoneal macrophages were treated with CK(10-9,10-8,10-7,10-6,10-5mol/L),there was no change in the production of 6-keto-PGF1?,suggesting that CK had no effect on the activity of COX-1.After blockade of constitutive expression of COX-1,the expression of PGE2 increased in rat peritoneal macrophages after LPS(1 ?g / ml)stimulated for 20 h.CK(10-9,10-8,10-7,10-6,10-5mol/L)had no effect on the production of PGE2 in rat peritoneal macrophages,suggesting that CK had no effect on the activity of COX-2.Conclusions:1.CK reduced xylene-induced mouse ear oedema and carrageenan-induced rat paw swelling,which suggest that CK has an anti-inflammatory effect on acute inflammation.2.CK reduced the number of writhing induced by acetic acid in mice,significantly increased the pain threshold of carrageenan-induced inflammatory pain in rats,but has no analgesic effect on hot plate-induced pain.These results suggested that CK has a peripheral analgesic effect.3.CK reduced the level of PGE2 and the expression of COX-2 in carrageenan-induced paw edema,but had no effect on the expression of COX-1.These results suggest that the anti-inflammatory and analgesic effects of CK were related to decrease the expression of COX-2 and reduced the level of PGE2.