| Objective: Diandao Mujin San from Yizongjinjian（Golden Mirror of Orthodox medicine）,containing Chinese herbs including Radix Aucklandiae and Radix Curcumae,has pain relieving and Qi and blood circulation promoting effects.It is commonly used in the treatment of Qi stagnation and blood stasis syndrome.In this experiment,the mouse models with Qi stagnation and blood stasis syndrome and acute inflammation were established to observe and evaluate the anti-inflammatory and analgesic effects of Diandao Mujin San,and to explore its possible mechanism preliminarily.Methods: Experiment 1: The experiment was divided into 6 groups: normal group（group A）,model group（group B）,celecoxib positive group（group C）,Diandao Mujin San（1:1）treatment group（group D1）,Diandao Mujin San（1:2）treatment group（group D2）,Diandao Mujin San（2:1）treatment group D3.Group A and group B were given the same dose of normal saline,group C and group D were given celecoxib and Diandao Mujin San mixture by gavage pretreatment.One week later,0.1% adrenaline was injected subcutaneously to replicate the syndrome of Qi stagnation and blood stasis.The analgesic effect of the drug was evaluated by hot plate test and acetic acid writhing test,and the whole blood viscosity and plasma viscosity of mice were measured at the same time.Experiment 2: Firstly,the anti-inflammatory effective compounds,targets and pathways of Diandao Mujin San were predicted by web pharmacology and online data platform,and then acute inflammation model was constructed using xylene and lipopolysaccharide（LPS）to verify the anti-inflammatory mechanism pathways of Diandao Mujin San.Firstly,the anti-inflammatory effective compounds,targets and pathways of Diandao Mujin San were predicted through the network pharmacology and online data platform,and then the acute inflammation model was constructed with xylene and lipopolysaccharide（LPS）to verify the anti-inflammatory mechanism pathway of Diandao Mujin San.Experiment 2: First,the anti-inflammatory active compounds,targets and pathways of Diandao Mujin San were predicted by network pharmacology and online data platform.Then,the acute inflammation model was constructed using xylene and lipopolysaccharide（LPS）to verify the anti-inflammatory mechanism pathway of Diandao Mujin San.（1）Grouping and drug administration: as in Experiment 1,xylene was applied to the right ear of mice one week later to calculate the degree of swelling and swelling inhibition rate of ear tissue in each group and compare the differences among groups.HE staining was used for pathological observation to observe the protective effect of Diandao Mujin San on xylene-induced ear swelling in mice,and Western blot was used to detect the expression levels of NOS3,HIF-1α and VEGF proteins in the ear tissue.（2）Grouping and drug administration: as in Experiment 1,after one week,the mice in the five groups except the normal group were given subcutaneous injection of LPS to induce the acute inflammation model,and the normal group was given injection of normal saline.Serum TNF-α and IL-6 levels in each group were detected by ELISA,and the spleen index of each group was calculated.The expression levels of NOS3,HIF-1α and VEGF proteins in the spleen of the mice were detected by Western blot.Results: In experiment 1,hot plate test,the latencies of mouse hot plate thresholds in group B were significantly lower than those in group A（P < 0.05）and those in groups C and D were significantly higher than those in group B（P < 0.05）30s and 60 s after modeling.Compared with the group D,the latencies of mouse hot plate thresholds in group D were significantly increased（P < 0.05）.However,the latencies of mouse hot plate thresholds in the groups with different proportions of Diandao Mujin San had no statistical difference,and there was no significant difference in the latencies of mouse hot plate thresholds in each group within 120 s after modeling.In the acetic acid writhing test,the body writhing times of mice in group B were significantly increased as compared with those in group A（P < 0.05）,the body writhing times of mice in groups C and D were significantly reduced as compared with those in group B（P < 0.05）,and the body writhing times of mice in group D were significantly reduced as compared with those in group C（P < 0.05）.The body writhing times of mice in the group with different proportions of had no statistical difference.In the test of whole blood viscosity and plasma viscosity of mice,the whole blood viscosity and plasma viscosity of mice in group B were significantly increased than those in group A（P < 0.05）,while the whole blood viscosity and plasma viscosity of mice in group C and group D were significantly decreased as compared with those in group B（P < 0.05）.The whole blood viscosity and plasma viscosity of mice in group D were significantly decreased as compared with those in group C（P < 0.05）.There was no statistical difference in whole blood viscosity and plasma viscosity in the groups with different proportions of Diandao Mujin San.In Experiment 2,network pharmacology predicted that 20 effective compounds of Diandao Mujin San were obtained,including sitosterol,stigmasterol and artichoxin,and 40 anti-inflammatory targets of Diandao Mujin San were obtained,which were mainly enriched in HIF-1 signaling pathway,IL-17 signaling pathway,TNF signaling pathway and VEGF signaling pathway.（1）In the xylene-induced mouse ear swelling experiment,the auricle swelling degree of the group B was significantly increased and the swelling inhibition rate was significantly reduced as compared with that of the group A（P < 0.05）.The auricle swelling degrees of the groups C and D were significantly decreased and the swelling inhibition rate was significantly increased as compared with that of the group B（P < 0.05）.The auricle swelling degree of the group D was significantly reduced and the swelling inhibition rate was significantly increased as compared with that of the group C（P < 0.05）.There was no statistical difference in the auricle swelling degree and the swelling inhibition rate of the Diandao Mujin San in different proportions groups.HE staining showed that the pathological structure of the ear tissue in group B was significantly congested and edematous.However,in the pretreatment group D,the tissue edema and congestion were significantly improved,the arrangement of striated muscle fibers was restored,and inflammatory cell infiltration was significantly reduced.In the group C,only a small amount of edema and inflammatory cell infiltration were observed.Western blot results showed that the expression levels of NOS3,HIF-1α and VEGF proteins in the ear tissues of group B were significantly higher than those of group A（P < 0.01）.The protein expression levels of NOS3,HIF-1α and VEGF in the groups C and D were significantly lower than those in the group B（P < 0.05）,there was no statistical difference between the groups C and D,and no statistical difference between the groups with different proportions of Diandao Mujin San.（2）In the LPS-induced acute inflammation experiment,the spleen index of mice in group B was significantly increased as compared with that in group A（P < 0.01）,and the spleen indexes of mice in groups C and D were significantly decreased as compared with that in group B（P < 0.05）.There was no significant difference between groups C and D,or between the groups that were treated with different proportions of Diandao Mujin San.The levels of serum TNF-α and IL-6 in the group B were significantly higher than those in the group A（P<0.05）.The levels of serum TNF-α and IL-6 in the groups C and D were significantly lower than those in the group B（P<0.05）.The serum TNF-α and IL-6 levels in the group D were significantly lower than those in the group C（P<0.05）.There was no statistical difference in the serum TNF-α and IL-6 levels in the groups with different proportions of Diandao Mujin San.Western blot results showed that the expression levels of NOS3,HIF-1α and VEGF proteins in the spleen of group B were significantly higher than those of group A（P < 0.01）.The expression levels of NOS3,HIF-1α and VEGF proteins in groups C and D were significantly lower than those of group B（P < 0.05）.There was no significant difference in histone expression between group C and D.There was no significant difference between the groups with different proportions of Diandao Mujin San.Conclusion: Diandao Mujin San has obvious analgesic effect on mice of Qi stagnation and blood stasis syndrome and can effectively improve blood viscosity of that syndrome.Diandao Mujin San has obvious anti-inflammatory effect.It can significantly reduce the ear swelling and spleen index of mice with acute inflammation,and reduce the expression levels of inflammatory cytokines TNF-α and IL-6.The anti-inflammatory mechanism may be through the reduction of the expression levels of HIF-1α,NOS3,HIF-1α and VEGF protein in the VEGF signaling pathway.The anti-inflammatory and analgesic effects of Diandao Mujin San with different compatibility proportion were same,indicating that the effects of Qi and blood circulation promoting and pain relieving with different compatibility proportion of Radix Aucklandiae and Radix Curcumae were same.However,it is necessary to choose different combinations according to the dialectical evidence for clinical application. |
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