| BackgroundRheumatoid arthritis (RA)is an unexplained chronic multi-system disease, in order to accumulate peripheral joints symmetrically persistent synovitis characteristic features. Synovitis can lead to destruction of cartilage and bone erosion, and ultimately affect the integrity of the joint is a sign of RA. Currently its etiology and pathogenesis is not fully understood, the lack of clear targets and effective clinical treatment, it also gives the treatment of RA bring great difficulties.To further investigate the effect of KDM decoction on bone destruction, this experiment relies on the National Natural Science Foundation of China (No.81173634) and the Guangdong Provincial Natural Science Foundation of China (No.2014A030313403) design.ObjectiveThrough this research, we are hoping to clear off the KDM decoction on bone metabolism in RA, focusing on RANKL/OPG signaling pathway and Wnt/β-catenin signaling pathway in RA bone metabolism and mechanism of action off the KDM decoction mechanism of action, provide a theoretical basis for clinical application.Methods1. The effect of KDM decoction and its components on CIA ratsGrouping:KDM group, KD group, KM group, THH group, MTX group, Model group and Control group. Here, The normal rats as normal control group, MTX as positive control group. Each group has 8 rats.Intervention:To preparation the classic RA model:collagen induced arthritis (CIA). After the secondary immunization in the 14th day of this experiment, treatment measures were taken on the rats. KDM group,KD group, KM group, THH group were given THH dose of 4g/kg/d, MTX group was given MTX dose 0.9mg/kg/w, Model group and Control group were given pure water 4ml/kg/d. All the interventions were given by intragastric administration for 4weeks.Observations indexes:the appearance behavior of rats, the weight, the joint swollen and Arthritis index, the ankle joint pathological changes of synovium and cartilage with HE staining, the serum concentrations of TNF-a with ELISA.Date analysis:Each values is pressed as the mean±standard deviation and was analyzed by SPSS 17.0software. Repeated measures analysis was used for repeated measures data, and ANOVA analysis was used to compare means between three or more groups, and Post Hoc multiple comparisons was used for any two groups. Choose LSD when the variance is homogeneous, and choose Tamhane T2 when the variance is not homogeneous. Statistical significance was determined when P-values were less than 0.05.2. The effect of KDM decoction and its components on Analysis of bone mineral density and trabecular of CIA ratsGrouping:KDM group, KD group, KM group, THH group, MTX group, Model group and Control group. Here, The normal rats as normal control group, MTX as positive control group. Each group has 4 rats.Intervention:To preparation the classic RA model:collagen induced arthritis (CIA). After the secondary immunization in the 14th day of this experiment, treatment measures were taken on the rats. KDM group, KD group, KM group, THH group were given THH dose of 4g/kg/d, MTX group was given MTX dose 0.9mg/kg/w, Model group and Control group were given pure water 4ml/kg/d. All the interventions were given by intragastric administration for 4weeks.Observations indexes:Micro-CT imaging evaluation, bone mineral parameters indicators:bone mineral content (BMC), bone mineral density (BMD); stereological measurement parameters indicators:bone volume fraction (bone volume/total volume, BV /TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), bone surface area/bone volume (BS/BV), trabecular separation (Tb.Sp);structure model index parameter index: trabecular pattern factor (Tb.Pf), structure model index (SMI)Date analysis:Each values is pressed as the mean±standard deviation and was analyzed by SPSS 17.0software. Repeated measures analysis was used for repeated measures data, and ANOVA analysis was used to compare means between three or more groups. Choose LSD when the variance is homogeneous, and choose Tamhane T2 when the variance is not homogeneous. Statistical significance was determined when P-values were less than 0.05.3. The effect of KDM decoction and its components on osteoclasts and osteoblast of CIA ratsGrouping:KDM group, KD group, KM group, THH group, MTX group, Model group and Control group. Here, The normal rats as normal control group, MTX as positive control group. Each group has 8 rats.Intervention:To preparation the classic RA model:collagen induced arthritis (CIA). After the secondary immunization in the 14th day of this experiment, treatment measures were taken on the rats. KDM group, KD group, KM group, THH group were given THH dose of 4g/kg/d, MTX group was given MTX dose 0.9mg/kg/w, Model group and Control group were given pure water 4ml/kg/d. All the interventions were given by intragastric administration for 4weeks.Observations indexes:TRACP&ALP double-staining to observe the number of osteoclasts and osteoblasts ELISA to measure the serum concentration of osteoclasts markers TRACP5b and the osteoblasts markers BALP.Date analysis:Each values is pressed as the mean±standard deviation and was analyzed by SPSS 17.0software. Repeated measures analysis was used for repeated measures data, and ANOVA analysis was used to compare means between three or more groups. Choose LSD when the variance is homogeneous, and choose Tamhane T2 when the variance is not homogeneous. Statistical significance was determined when P-values were less than 0.05.4. The regulation of KDM decoction and its components on RANKL/OPG signaling pathway of CIA ratsGrouping:KDM group, KD group, KM group, THH group, MTX group, Model group and Control group. Here, The normal rats as normal control group, MTX as positive control group. Each group has 8 rats.Intervention:To preparation the classic RA model:collagen induced arthritis (CIA). After the secondary immunization in the 14th day of this experiment, treatment measures were taken on the rats. KDM group, KD group, KM group, THH group were given THH dose of 4g/kg/d, MTX group was given MTX dose 0.9mg/kg/w, Model group and Control group were given pure water 4ml/kg/d. All the interventions were given by intragastric administration for 4weeks.Observations indexes:Immunohistochemistry to measure the protein expression of RANKL and OPG, ELISA to measure the serum concentration of RANKL and OPG.Date analysis:Each values is pressed as the mean±standard deviation and was analyzed by SPSS 17.0software. Repeated measures analysis was used for repeated measures data, and ANOVA analysis was used to compare means between three or more groups. Choose LSD when the variance is homogeneous, and choose Tamhane T2 when the variance is not homogeneous. Statistical significance was determined when P-values were less than 0.05.5. The regulation of KDM decoction and its components on Wnt/β-catenin signaling pathway of CIA ratsGrouping:KDM group, KD group, KM group, THH group, MTX group, Model group and Control group. Here, The normal rats as normal control group, MTX as positive control group. Each group has 4 rats.Intervention:To preparation the classic RA model:collagen induced arthritis (CIA). After the secondary immunization in the 14th day of this experiment, treatment measures were taken on the rats. KDM group, KD group, KM group, THH group were given THH dose of 4g/kg/d, MTX group was given MTX dose 0.9mg/kg/w, Model group and Control group were given pure water 4ml/kg/d. All the interventions were given by intragastric administration for 4weeks.Observations indexes:QPCR was used to detect the mRNA expression of Wnt3a, β-catenin and Dickkopf-1.Date analysis:Each values is pressed as the mean±standard deviation and was analyzed by SPSS 17.0software. Repeated measures analysis was used for repeated measures data, and ANOVA analysis was used to compare means between three or more groups. Choose LSD when the variance is homogeneous, and choose Tamhane T2 when the variance is not homogeneous. Statistical significance was determined when P-values were less than 0.05.Results1. The effect of KDM decoction and its components on CIA ratsRats general, body weight changes:compared with the Control group, Model group, the intervention group were mental fatigue, limited mobility, less shiny gray coat color; slow weight gain was statistically significant (P<0.05); and Model group, KDM group, KD group, KM group improved weight gain was statistically significant (P<0.05); compared with the MTX group, KDM group, KM group improved weight gain was statistically significant (P<0.05).Rat paw swelling:Compared with Model group, KDM group, KD Group, KM group AI score improvement was statistically significant (P<0.05); compared with the MTX group, the Medicines AI score improvement was not statistically significant (P>0.05). Compared with Model group, the intervention group left, right foot volume improvement was statistically significant (P<0.05); compared with the MTX group, KDM group, KD Group, KM group left, right foot volume improvement was statistically significant (P <0.05).Rat ankle pathology Rating:Compared with Model group, KDM group, KM group synovial pathology score decrease was statistically significant (P<0.05); compared with the MTX group, the Medicines synovial pathological scores showed no significant difference (P>0.05). Compared with Model group, KDM group, KD Group rickets reduce processing rates was statistically significant (P<0.05); compared with the MTX group, the Medicines rickets treatment score difference was not statistically significant (P>0.05).Serum TNF-a concentrations:Compared with Model group, MTX group, KDM group, KD Group, KM serum TNF-a concentration reduction was statistically significant (P<0.05); compared with the MTX group, KDM group, KM Group reduce serum TNF-a concentration was statistically significant (P<0.05).2. The effect of KDM decoction and its components on Analysis of bone mineral density and trabecular of CIA ratsBone mineral parameters indicators:Compared with Model group, MTX group, KDM group, KD Group, KM group ankle bone mineral content (BMC) increase was statistically significant (P<0.05); and MTX group comparison of various groups on the right ankle BMC medicine was not statistically significant (P>0.05). Compared with Model group, KDM group, KD Group ankle bone mineral density (BMD) was significantly increased (P<0.05);compared with the MTX group, the Medicines ankle BMD was not statistically significant (P>0.05).Stereological measurement parameters indicators:Compared with Model group, KDM group, KD Group, KM group ankle bone volume fraction (bone volume/total volume, BV/TV) was significantly increased (P<0.05); and MTX group, KDM group, KD group ankle BV/TV statistically significant increase (P<0.05). Compared with Model group, KDM group, KD Group, KM group ankle trabecular number (Tb.N) increase was statistically significant (P<0.05); compared with the MTX group, KDM group, KD Group ankle joint Tb.N increase was statistically significant (P<0.05). Compared with Model group, KDM group ankle trabecular thickness (Tb.Th) increase was statistically significant (P<0.05); compared with the MTX group, KDM group increased statistically ankle Tb.Th significance (P<0.05).Compared with Model group, KDM group ankle bone surface/volume (BS/BV) decrease was statistically significant (P<0.05); compared with the MTX group, the Medicines BS/BV no statistical change significance (P> 0.05). Compared with Model group, the intervention group ankle trabecular separation (Tb.Sp) decrease was statistically significant (P<0.05); compared with the MTX group, KDM group, KD Group reduced ankle Tb.Sp there was statistically significant (P<0.05).Structure model index parameter index:Compared with Model group, the intervention group ankle bone trabecular pattern factor (Tb.Pf) decrease was statistically significant (P<0.05); compared with the MTX group, KDM group, KD group ankle Tb.Pf decrease was statistically significant (P<0.05). Compared with Model group, the intervention group ankle structure model index (SMI) decrease was statistically significant (P<0.05); compared with the MTX group, KDM group, KD Group, KM group ankle SMI lowered Statistics significance (P<0.05).3. The effect of KDM decoction and its components on osteoclasts and osteoblast of CIA ratsOsteoclast cell number and function:Compared with Model group, the intervention group ankle joint OC cells reduce the number of statistically significant (P<0.05); compared with the MTX group, KDM group, KD Group, KM group ankle joint OC cells reduce the number of there was statistically significant (P<0.05). Compared with Model group, MTX group, KDM group, KD Group, KM serum TRACP 5b concentration reduction was statistically significant (P<0.05); compared with the MTX group, KDM group, KD serum TRACP 5b concentration decreased statistically significant (P<0.05).Osteoblast number and function:Compared with Model group, the intervention group ankle OB cells increased the number of statistically significant (P<0.05); compared with the MTX group, KDM group, KD Group, KM group ankle joint OB number of cells increased there was statistically significant (P<0.05). Compared with Model group, KDM group, KD BALP serum concentration was statistically significant (P<0.05); compared with the MTX group, KDM group, KD BALP serum concentration was statistically significant (P<0.05).4. The regulation of KDM decoction and its components on RANKL/OPG signaling pathway of CIA ratsRANKL protein:Compared with Model group, the intervention group ankle joint RANKL protein expression decrease was statistically significant (P<0.05); KDM group ankle joint RANKL protein expression compared with the MTX group was significantly reduced (P<0.05). Compared with Model group, MTX group, KDM group, KD Group, KM serum RANKL concentration reduction was statistically significant (P<0.05); compared with the MTX group, KDM group, KD serum RANKL concentration reduction was statistically significant (P<0.05).OPG protein:Compared with Model group, the joint expression of OPG increased ankle intervention groups was statistically significant (P<0.05); compared with the MTX group, KDM group, KD Group ankle elevated expression of OPG was statistically significant (P<0.05). Compared with Model group, the intervention group serum OPG concentration was statistically significant (P<0.05); compared with the MTX group, KDM group, KD serum OPG concentration was statistically significant (P<0.05).5. The regulation of KDM decoction and its components on Wnt/β-catenin signaling pathway of CIA ratsWnt3a mRNA expression:Comparison and Modem group, MTX group, KDM group, KD Group, KM group ankle Wnt3a mRNA expression was significantly increased (P <0.05); compared with the MTX group, KDM group, KD Group, mRNA expression of KM group ankle Wnt3a increased statistically significant (P<0.05).β-catenin mRNA expression:Compared with Model group, THH group, KDM group, KD group, mRNA expression of KM group ankle elevated β-catenin was statistically significant (P<0.05); compared with the MTX group, KDM group, KD group, mRNA expression of KM group ankle elevated P-catenin was statistically significant (P<0.05).Dickkopf-1 mRNA expression:Compared with Model group, the intervention group ankle joint Dickkopf-1 mRNA expression was significantly reduced (P<0.05); KDM group ankle joint Dickkopf-1 mRNA expression compared with the MTX group, lower there was statistically significant (P<0.05).ConelutionThe KDM decoction and its components can reduce the expression of RANKL of CIA rats, increase the expression of OPG, and inhibit RANKL/OPG signaling pathway to activate osteoclasts, inhibiting bone resorption; while increasing Wnt3a,β-catenin mRNA expression, reduce Dickkopf-1 mRNA expression, activation of Wnt/β-catenin signaling pathway to osteoblasts,promote bone formation. Such imbalances resorption and formation relative to restore balance. |
PDF Full Text Request