| Purpose:1 To clarify the efficacy of Youguiwan and Shaoyao Gancao Decoction on knee pain in KOA rats by means of pain behavioral measurement,histomorphological observation and inflammatory mediators detection.2 To explore the mechanism of Youguiwan and Shaoyao Gancao Decoction on peripheral sensitization of KOA rats based on TRPV4/p38 MAPK/CREB signaling pathway.3 To investigate the mechanism of Youguiwan and Shaoyao Gancao Decoction on central sensitization of KOA rats based on regulation of bradykinin receptor on p38 MAPK signaling pathway.Material and method:1 Effects of Youguiwan and Shaoyao Gancao Decoction on pain behavior,tissue morphology and inflammatory mediators in KOA ratsSixty-four healthy adult SD rats were selected for the experiment,the rats were provided by Liaoning Changsheng Biotechnology Co.LTD,and were raised in the Animal Experiment Center of Liaoning University of Traditional Chinese Medicine.The experiment began after one week of adaptive feeding.Before modeling,rats were divided into sham operation group(n=14)and modeling group(n=50)by random number.Modified Hulth method was used to replicate KOA rat model.HE staining was used to evaluate the models of two rats in the sham operation group and two rats in the modeling group.Random number table method was used to divide the rats in the modeling group into sham operation group,model group,Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group,with12 rats in each group,6 males and 6 females.After successful modeling,Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were dissolved in equal volume of 0.9% normal saline according to their crude drugs 361.8mg/kg,145.8mg/kg and 1.08mg/kg respectively,and gavage orally once a day.Rats in sham operation group and model group were given equal volume 0.9% normal saline intragastric administration.The period of intragastric administration was 4 weeks.The mechanical withdrawal threshold of rats was measured by stimulating the middle part of the left hind foot with Von Frey filaments at day 0,7,14,21 and 28.The general state of the rats was observed after intragastric administration.The serum and synovial tissues of rats were collected,and HE staining method was used to observe the histopathological changes of synovial tissues of KOA rats;ELISA method was used to detect the expression levels of IL-1β,TNF-α,SP,PGE2 and BK in serum of KOA rats;ELISA method was used to detect the expression levels of IL-1β,TNF-α,SP,PGE2 and BK in synovial tissues of KOA rats.2 The mechanism of Youguiwan and Shaoyao Gancao Decoction on peripheral sensitization of KOA rats based on TRPV4/p38 MAPK/CREB signaling pathwayModel grouping,model replication and model intervention are the same as before.Six rats were randomly selected from each group,and L3-L5 dorsal root ganglion was sampled.Western Blot method was used to detect the expression levels of TRPV4,p-TRPV4,p38 MAPK,p-p38 MAPK,CREB and p-CREB in dorsal root ganglion of KOA rats;immunohistochemical method was used to detect the expression levels of p-TRPV4,p-p38 MAPK and p-CREB in rat dorsal root ganglion of KOA rats.3 The mechanism of Youguiwan and Shaoyao Gancao Decoction on central sensitization of KOA rats based on the regulation of bradykinin receptor on p38 MAPK signaling pathwayModel grouping,model replication and model intervention are the same as before.The remaining 6 rats in each group were selected,and the thalamic tissues of rats were collected.ELISA method was used to detect the expression levels of IL-1β,TNF-α and BK in brain tissues of KOA rats;immunofluorescence method was used to detect the expression levels of B1 R,B2R and their co-expression in brain tissue of KOA rats;Western Blot method was used to detect the expression levels of p38 MAPK,p-p38 MAPK,B1 R and B2 R in brain tissue of KOA rats;immunohistochemistry was used to detect the expression levels of p-p38 MAPK in brain tissue of KOA rats.Results:1 Effects of Youguiwan and Shaoyao Gancao Decoction on pain behavior,tissue morphology and inflammatory mediators in KOA rats1.1 The general state of rats was observed: There was no death in each group during the experiment.Rats in the sham operation group were in good condition during the whole experiment,with normal drinking water and bowel movements,white and shiny hair,significant increase in body weight and normal knee movements.Rats in model group were in depressed state,irritable,withered and yellow hair,claudication,and severe swelling and deformation of the left knee joint.Compared with model group,the above performance of rats in Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group was improved to varying degrees.1.2 MWT changes of rats in each group were detected by Von Frey filaments: Within 28 days,MWT of the sham operation group did not change significantly,but the model group showed a downward trend,and the Youguiwan and Shaoyao Gancao Decoction group increased compared with the Youguiwan group and Celecoxib capsule group;the increasing speed of Youguiwan and Shaoyao Gancao Decoction group and Celecoxib capsule group was similar during the period of 1-7 days,and the increasing speed was higher than Youguiwan group;the improvement rate of Youguiwan group was the fastest in the period of 8-14 days.The Youguiwan group and Celecoxib capsule group increased the most rapidly during the 22-28 days.There was no significant difference in MWT among all groups before intragastric administration in the first day(P>0.05).On day 28 of administration,MWT of Youguiwan and Shaoyao Gancao Decoction group was significantly higher than that of sham operation group(P<0.01),and the difference was statistically significant;there was no significant difference between model group and sham group(P>0.05);compared with the model group,the MWT of Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group was significantly higher(P<0.05 or P<0.01),the differences were statistically significant;MWT of Youguiwan and Shaoyao Gancao Decoction group was higher than that of Youguiwan group(P<0.05),and the difference was statistically significant;there was no statistical significance between Youguiwan and Shaoyao Gancao Decoction group and Celecoxib capsule group(P>0.05).1.3 HE staining results of synovial tissues of knee joints of rats in each group showed: The synovial tissues of rats in the sham operation group were normal,and the cells were flat and neatly arranged,without hyperemia edema,inflammatory cell infiltration and neovascularization.Synovial tissue of model group was infiltrated by inflammatory cells and neovascularization,and synovial cells were hyperplasia and disordered.There were a few inflammatory cell infiltration and neovasculogenesis,mild proliferation and disorder of synovial cells in the Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group,and Youguiwan and Shaoyao Gancao Decoction group was superior to the Youguiwan group and Celecoxib capsule group.1.4 The contents of IL-1β,TNF-α,SP,PGE2 and BK in serum of each group were detected by ELISA: Compared with the sham operation group,the contents of IL-1β,TNF-α,SP,PGE2,BK in model group,Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly increased(P<0.05 or P<0.01),the difference was statistically significant;compared with model group,the contents of IL-1β,TNF-α,SP,PGE2 and BK in Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly decreased(P<0.01),and the differences were statistically significant;the contents of IL-1β,TNF-α,SP,PGE2 and BK in Youguiwan and Shaoyao Gancao Decoction group were significantly lower than those in Youguiwan group and Celecoxib capsule group(P<0.01),the differences were statistically significant.1.5 The contents of IL-1β,TNF-α,SP,PGE2 and BK in synovial tissues of each group were detected by ELISA: Compared with the sham operation group,the contents of IL-1β,TNF-α,SP,PGE2,BK in model group,Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly increased(P<0.05 or P<0.01),the difference was statistically significant;compared with model group,the contents of IL-1β,TNF-α,SP,PGE2 and BK in Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly decreased(P<0.01),and the differences were statistically significant;the contents of IL-1β,TNF-α,SP,PGE2 and BK in Youguiwan and Shaoyao Gancao Decoction group were significantly lower than those in Youguiwan group and Celecoxib capsule group(P<0.01),the differences were statistically significant.2 The mechanism of Youguiwan and Shaoyao Gancao Decoction on peripheral sensitization of KOA rats based on TRPV4/p38 MAPK/CREB signaling pathway2.1 The expression results of TRPV4,p-TRPV4,p38 MAPK,p-P38 MAPK,CREB and p-CREB in dorsal root ganglion of rats in each group showed: There were no significant differences in TRPV4,p38 MAPK and CREB in dorsal root ganglion among all groups(P>0.05);compared with the sham operation group,the expression levels of p-TRPV4,p-p38 MAPK and p-CREB in the model group,Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly increased(P<0.01),and the differences were statistically significant;ccompared with model group,the expression levels of p-TRPV4,p-p P38 MAPK and p-CREB in Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly decreased(P<0.01),and the differences were statistically significant;compared with Youguiwan group,the expression levels of p-TRPV4,p-p38 MAPK and p-CREB were significantly decreased(P<0.01),and the differences were statistically significant;compared with Celecoxib capsule group,the expression levels of p-TRPV4 and p-p38 MAPK in Youguiwan and Shaoyao Gancao Decoction group were significantly decreased(P<0.01),but the expression levels of p-CREB were not significantly different(P>0.05);Meanwhile,the comparison results of p-TRPV4/TRPV4,p-p38 MAPK/p38 MAPK and p-CREB/CREB among all groups were identical with the comparison results of all phosphorylated protein groups mentioned above.2.2 Immunohistochemical method was used to detect the protein expression levels of p-TRPV4,p-p38 MAPK and p-CREB in the dorsal root ganglion of rats in each group:Compared with sham operation group,the protein expression levels of p-TRPV4,p-p38 MAPK and p-CREB in other groups were significantly increased(P<0.01),and the differences were statistically significant;compared with model group,the protein expression levels of p-TRPV4,p-p38 MAPK and p-CREB in Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly decreased(P<0.01),and the differences were statistically significant;compared with Youguiwan group,p-TRPV4,p-p38 MAPK and p-CREB in Youguiwan and Shaoyao Gancao Decoction group were significantly decreased(P<0.05 or P<0.01),the difference was statistically significant;compared with Celecoxib capsule group,p-p38 MAPK and p-CREB in Youguiwan and Shaoyao Gancao Decoction group were significantly decreased(P<0.01),while p-TRPV4 was not significantly different(P>0.05),and the difference was not statistically significant.3 The mechanism of Youguiwan and Shaoyao Gancao Decoction on central sensitization of KOA rats based on the regulation of bradykinin receptor on p38 MAPK signaling pathway3.1 The contents of IL-1β,TNF-α and BK in each group were detected by ELISA: Compared with sham operation group,the contents of IL-1β,TNF-α and BK in model group,Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly increased(P<0.01),and the differences were statistically significant;compared with model group,the contents of IL-1β,TNF-α and BK in Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly decreased(P<0.01),and the differences were statistically significant;compared with Youguiwan group and Celecoxib capsule group,the contents of IL-1β,TNF-α and BK in Youguiwan and Shaoyao Gancao Decoction group were significantly decreased(P<0.01),and the differences were statistically significant.3.2 Immunofluorescence method was used to detect the levels of B1 R,B2R and their co-expression in the brain tissues of rats in each group,and the results showed: Compared with sham operation group,the number of B1 R,B2R and B1 R and B2 R co-expressed positive cells in model group,Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly increased(P<0.01),and the differences were statistically significant;compared with model group,the number of B1 R,B2R and B1 R and B2 R co-expressed positive cells in Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly decreased(P<0.01),and the difference was statistically significant;Compared with Youguiwan group and Celecoxib group,the number of B1 R,B2R and B1 R and B2 R co-expression positive cells were significantly decreased(P<0.05 or P<0.01),the difference was statistically significant.3.3 Expression levels of p38 MAPK,p-p38 MAPK,B1 R and B2 R in KOA rat brain tissue3.3.1 Western Blot method was used to detect the protein expression levels of p38 MAPK and p-p38 MAPK in the brain tissues of each group,and the results showed: There was no significant difference in p38 MAPK in brain tissues among all groups(P>0.05);compared with sham operation group,the expression level of p-p38 MAPK in model group,Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group was significantly increased(P<0.01),and the difference was statistically significant;Compared with model group,the expression level of p-p38 MAPK in Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group was significantly decreased(P<0.01),and the difference was statistically significant;Compared with Youguiwan group and Celecoxib capsule group,the expression level of p-p38 MAPK in Youguiwan and Shaoyao Gancao group was significantly decreased(P<0.01),and the difference was statistically significant;The comparison of p-p38 MAPK/p38 MAPK among groups was consistent with that of p-p38 MAPK.3.3.2 Western Blot method was used to detect the protein expression levels of B1 R and B2 R in the brain tissues of each group,and the results showed: Compared with sham operation group,the expression levels of B1 R and B2 R in model group,Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly increased(P<0.01),and the differences were statistically significant;compared with model group,the expression levels of B1 R and B2 R in Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group were significantly decreased(P<0.01),and the differences were statistically significant;compared with Youguiwan group and Celecoxib capsule group,the expression level of B1 R and B2 R in Youguiwan and Shaoyao Gancao Decoction group were significantly decreased(P<0.05 or P<0.01),the difference was statistically significant.3.3.3 Immunohistochemical method was used to detect the expression level of p-p38 MAPK protein in the brain tissues of each group,and the results showed: Compared with sham operation group,the expression level of p-p38 MAPK in model group,Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group was significantly increased(P<0.01),and the difference was statistically significant;compared with model group,the expression level of p-p38 MAPK in Youguiwan and Shaoyao Gancao Decoction group,Youguiwan group and Celecoxib capsule group was significantly decreased(P<0.01),and the difference was statistically significant;compared with the Youguiwan group,the expression level of p-p38 MAPK in Youguiwan and Shaoyao Gancao Decoction group and Celecoxib capsule group were significantly decreased(P<0.05 or P<0.01),the difference was statistically significant.Conclusion:1 Youguiwan and Shaoyao Gancao Decoction can improve the general state of KOA rats,reduce the mechanical withdrawal threshold of KOA rats,and improve the synovial histopathological morphology of KOA rats.The expressions of IL-1β,TNF-α,SP,PGE2 and BK in serum and synovial tissue of KOA rats were inhibited,and the expressions of IL-1β,TNF-α and BK in brain tissue were alleviated,and knee pain symptoms of KOA rats were improved.Youguiwan and Shaoyao Gancao Decoction is effective in treating knee pain in KOA rats.2 Youguiwan and Shaoyao Gancao Decoction may regulate the peripheral sensitization of KOA by inhibiting the over-activation of TRPV4/p38 MAPK/CREB signaling pathway in rat dorsal root ganglion.3 Youguiwan and Shaoyao Gancao Decoction may regulate the central sensitization of KOA by down-regulating the expression of B1 R and B2 R in rat brain tissue and inhibiting the over-activation of p38 MAPK signaling pathway.4 The p38 MAPK signaling pathway is involved in pain sensitization in KOA rats,and Youguiwan and Shaoyao Gancao Decoction may regulate the process of pain sensitization by inhibiting the over-activation of p38 MAPK signaling pathway,thus alleviating the pain symptoms of KOA. |
PDF Full Text Request