| Objective: Gout is a metabolic disease, high levels uric acid in the circulation leads to the deposition of monosodium urate crystals in the organization. Acute gouty arthritis(AGA) is caused by monosodium urate(MSU) crystals deposited in the joints and surrounding tissue, an acute rheumatic diseases characterized by inflammatory cells infiltration, joint swelling and severe pain. MSU crystals activate the NLRP3 inflammasome, leading to the secretion of IL-1β, and then stimulate neutrophil cell activation, TNF-α, IL-6 and other pro-inflammatory cytokines and chemokines release, inflammatory cell recruitment in inflammatory sites, then trigger the cascade inflammation reaction. TGF-β1 plays an important role in AGA spontaneous remission. Western medicine treatment of acute gout currently rely on colchicine, non-steroidal anti-inflammatory drugs, glucocorticoid and biological agents. However, the application of these drugs or are associated with serious side effects, or costly. Understanding of gout Traditional Chinese Medicine(TCM) has a long history. This disease belongs to the rheumatism category of TCM. Clinical treatment of acute gout used by traditional Syndrome Differentiation have been achieved certain effect, and the security is good, however there are less effective, slower onset and other issues. Effective components in chinese medicine is the extract of traditional Chinese medicine, obtain the good curative effect in clinical inflammatory disease application. In the context of modern integrated Traditional Chinese and Western Medicine, this research aims to find the optimal combination compound in the anti-inflammatory effective components aimed at AGA rat from Pre screening of plant effective components of Traditional Chinese Medicine by orthogonal design method. Further validated therapeutic effect on AGA of compound made by effective components, and explore its related antiinflammatory mechanism, thus provide the basis for development of modern medicine compound preparation of effective component used for fast and powerful treat gout.Methods: 1 Screening the best compatibility of effective components of Traditional Chinese Medicine by Orthogonal t Value Design.1.1 Optimal drug components Compound Screening.SD rats were randomly divided into 12 groups according to the ma i n effect screening orthogonal t value table arrangements, intragastric ad ministration by orthogonal t value schedule of compound drug, establishe d AGA model in rats 5 days later. Set another blank control group, nor mal saline intragastric administration, injected PBS as a negative control and calculate the degree of swelling.1.2 Analysis of the interaction of auxiliary medicine from the compound.SD rats were randomly divided into 4 groups according to adjuvant interaction orthogonal t value table, gave the corresponding compound drugs. At the same time, set model group and blank control group, esta blished AGA model 5 days later and calculate the degree of swelling. 1.3 Optimal doses of compound drug screening.SD rats were randomly divided into 9 groups according to dosage screening orthogonal t value table. Meanwhile set model group and blan k control group, established AGA model 5 days later and calculate the degree of swelling.2 Anti inflammatory effect of compound of effective components in AGA rats.SD rats were randomly divided into Effective Components of the o ptimal Compound(ECC) group, Colchicine group, Model group and Nor mal control(Control) group. Model group and the normal control group were given an equal volume of normal saline, treatment group received two corresponding drugs intragastric administration for 5 days. Except t he blank control group were prepared by AGA model, the control group was Injected with PBS at 6th day. Observing the rats swelling degree, swelling rate and gait. Each group were randomly selected 8 rats collect ed serum and ankle 24 hours after modeling. The remainder of each gro up continue to worked as before,rats serum and ankle joint specimens were collected 120 hours after modeling.ELISA was used to detect serum IL-1β, IL-6, TNF-α, MIF, TGF-β1 levels. Ankle joints specimens was prepared slices after decalcified, observed synovial pathological changes with HE staining, while the expression of NLRP3, IL-1β and TGF-β1 in synovium tissues were acquired by immunohistochemical method.3 Statistical analysis:All statistical analyses of data were computed using Statistical Package for Social Sciences(version 13.0). The comparisons of groups were evaluated by repetitive measurement or one-way analysis of variance(ANOVA). Grade data using non parametric test. The differences were consi dered statistically significant at P<0.05.Results:1 Screening the best compatibility of effective components of Traditional Chinese Medicine by Orthogonal t Value Design.Through the screening of Orthogonal t value method based on swelling degree, found that SN high dosage, TGP dosage, PNS low dosage and TG dosage can play the best anti-inflammatory efficacy.2 Anti inflammatory effect of compound of effective components in AGA rats2.1 Anti inflammatory effect of ECC in the advanced stage of inflammation.2.1.1 Effects of ECC on ankle swelling degree in each group.There was no statistical differences among ECC group and Colchicine group(P>0.05), ankle swelling degree in ECC group, Colchicine group and Model group reach the peak 8 hours after modeling, were significantly higher than those in normal persons(P<0.01), ECC group and Colchicine group remarkably lower than the model group(P<0.01), while ECC group and Colchicine grouphad no significant difference.(P>0.05).2.1.2 Effects of ECC on ankle swelling rate in each group.ECC group and Colchicine group had no significant difference(P>0.05), trend of swelling rate in ECC group, Colchicine group and Model group were significantly higher than those in normal persons(P<0.01), ECC group and Colchicine group remarkably lower than the model group(P<0.01).2.1.3 Gait grade at post-model 8 hours in each group.Compared with normal control group, other three groups activities are reduced and shows different degree modeling lower limb claudication(P<0.01); compared with Model group, ECC group obviously improved(P<0.01), Colchicine group also improved(P<0.05), ECC group and Colchicine group had no significant difference(P>0.05).2.1.4 Comparison of serum cytokine levels in each group.IL-1β levels of ECC group lower than Colchicine group(P<0.05),(P<0.05), Compared with normal Control group, IL-1β level of Model group significant rise(P<0.01); IL-6 levels in each group had no significant difference(P>0.05); The levels of TNF-α in ECC group significantly lower than Model group( P<0.01), and Compared with control group Colchicine group increase slightly(P<0.05), Model group were the highest, compared with Control group had significant difference( P<0.01); compared with normal Control group, levels of TGF-β1 in other three groups significantly rise(P<0.01), Compared with Colchicine group ECC group significantly increased( P<0.01), and greatly more than Model group(P<0.01); levels of MIF in ECC group significantly lower than Model group( P<0.01), Compared with Model group Colchicine group had decreased(P<0.05), whereas Model group were higher than Control group(P<0.05).2.1.5 Pathological change of rat ankles in each group.HE staining section showed that in model group, a large number of in flammatory cell infiltration, synovial thickening and edema. Compare d with Model group, ECC group and Colchicine group were improved. There were no pathological changes of ankles in normal control group.2.1.6 Expression of NLRP3, IL-1β, TGF-β1 expression in ankle synovial t i s sues in each group.Compared with normal Control group, The expression of NLRP3 in ECC group, Colchicine group and Model group marked increased, there were statistical differences among them(P<0.01), Compared with Model group, ECC group and Colchicine group expression of NLRP3 were obviously weaker(P<0.01), ECC group significantly lower than Colchicine group(P<0.01); ECC group and Colchicine group IL-1β expression in synovium were decreased significantly compared to Model group(P<0.01), while Compared with Control group, IL-1β expression in Model group obviously enhanced(P<0.01); The expression of TGF-β1 increased significantly in other group excepted Control group(P<0.01), whereas compared with Model group, ECC group and Colchicine group the expression of TGF-β1 were significantly decreased(P<0.01), and ECC group obviously enhanced compared to Colchicine group(P<0.01).2.2 Anti inflammatory effect of ECC in the remission stage of inflammation.2.2.1 Effects of ECC on ankle swelling degree in each group.Ankle swelling changes were significant differences between the groups(P<0.01),The changes of swelling degree in ECC group, colchicine group and model group were significantly reduced at 36 hours after modeling, and the normal control group was relatively stable. swelling degree changes in ECC group and Colchicine group compared to Control group had no significant difference at 120 hours after modeling(P>0.05).2.2.2 Effects of ECC on ankle swelling rate in each group.The changes of swelling rate significant differences in all groups(P<0.01), From the swelling rate change in trend, ECC treatment group and Colchicine group significantly lower than Model group(P<0.01).2.2.3 Comparison of serum cytokine levels in each group.The levels of IL-1β in ECC group and Colchicine group were lower than Model group(P<0.05); The levels of ECC IL-6, TNF-α, MIF in each group there were no statistical significance(P>0.05); TGF-β1 levels in ECC group lower than Model group(P<0.05), compared with Model group Colchicine group decreased significantly(P<0.01), Model group was significantly higher than control group(P<0.01).2.2.4 Pathological change of rat ankles in each group.There were still inflammatory cells infiltration in Model group, syno vial tissue edema reduced, pathological manifestations of ECC group an d Colchicine group both improved, swelling relieve and Control group ha d no abnormalities.2.2.5 Expression of NLRP3, IL-1β, TGF-β1 expression in ankle synovial tissues in each group.The expression of NLRP3 in ECC group significantly lower than C olchicine group(P<0.01), ECC group and Colchicine group significantl y expression decreased(P<0.01), the expression of NLRP3 in ECC tre atment group, Colchicine group and Model group were obviously higher than Control group(P<0.01); the expression of IL-1β in ECC group and Colchicine group obvious lower than Model group(P<0.01), wher eas Model group significantly increased compared to Control group(P<0.01); the expression of TGF-β1 in ECC group obviously higher than C olchicine group and Control group(P<0.01), compared with Model gr oup, ECC group were increased notablely(P<0.01), whereas the expres sion of TGF-β1 in Colchicine group significantly lower than Model grou p(P<0.01), the expression of TGF-β1 in Model groupsignificantly enh anced compared to Control group(P<0.01).Conclusions : 1 The optimal anti-inflammatory Compound of Effective Components from TCM is SN high dosage, TGP dosage, PNS low dosage and TG dosage by the screening of Orthogonal t value method.2 The optimal anti-inflammatory Compound of Effective Components from TCM can significantly inhibit AGA, and can improved joint pathological changes.3 The optimal anti-inflammatory Compound of Effective Components from TCM may inhibit the NLRP3 inflammasome, then downregulation of IL-1β, TNF-α and MIF, as well as upregulate TGF-β1 to play antiinflammatory ef f ect of AGA. |
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