| Objective: Zukamu Granules(ZKMG)is the representative prescription of traditional Uyghur medicine for treating common cold,with the effects of regulating abnormal temperament,clearing heat,sweating and relieving orifice,used for the treatment of cold cough,fever without sweat,sore throat,nasal congestion nose,runny nose,etc.Although the clinical efficacy of ZKMG has been confirmed,its specific mechanism of action is still unclear.In this study,we used the classic anti-inflammatory and analgesic model to investigate its analgesic and anti-inflammatory efficacy.Ccombination with network pharmacology and pharmacology experiments,we focused on the anti-inflammatory mechanism to provide experimental basis for clinical medication.Methods: The method of hot-plate and writhing by acetic acid in mice were made to study the analgesic effect of ZKMG.The inflammation model of LPS-induced acute lung injury in rats and xylene-induced mouse ear swelling were used to investigate the anti-inflammatory effects of ZKMG.HS-SPME-GC-MS technique was used to extract and identify the volatile components of ZKMG,combined with major databases of network pharmacology to predict the anti-inflammatory material basis of ZKMG and its potential mechanism of action.In order to further explore the molecular mechanism of ZKMG inhibiting xylene and LPS-mediated inflammatory reaction,on the basis of the pharmacodynamics of xylene-induced ear swelling model in mice and LPS-induced acute lung injury model in rats,through the method of enzyme linked immunosorbent assay(ELISA)and Western Blot(WB),we conducted in-depth studies on the mechanism of action,such as the effects on mitogen-activated protein kinase(MAPK),Nuclear factor kappa-B(NF-κB)signaling pathway and inflammatory mediators.Results:(1)Analgesic efficacy: ZKMG had significant inhibitory effects on the pain response to hot plate stimulation and abdominal cavity stimulation by acetic acid solution,which had the greatest inhibitory effect at 60 minutes after administration.And the pain threshold increased with the dose,which was significantly different from the control group(P < 0.05).(2)Inflammatory pathological injury: Pathological evaluation showed that ZKMG pretreatment could significantly improve the inflammatory response such as tissue congestion,edema,disordered arrangement of rhabdomyofibers,and a large number of inflammatory cells infiltration in xylene-induced ear swelling model mice,and the inflammatory response such as the alveolar wall significantly thickened,alveolar size uneven and significantly smaller,and massive inflammatory cell infiltration in LPS-induced acute lung injury model rats.In addition,this phenomenon gradually decreased with the increase of ZKMG dosage,among which the high-dose group had the best effect on improving the structural damage of ear and lung tissues and reducing the infiltration of inflammatory cells.(3)Network pharmacology: A total of forty-seven volatile components were identified in ZKMG,and seventeen bioactive components including levomenthol,thymol,eugenol were screened out.Forty-four anti-inflammatory targets were predicted,involving 20 inflammation-related pathways such as MAPK signaling pathway,NF-κB signaling pathway and arachidonic acid metabolism.(4)Content of inflammatory factors: In the xylene-induced ear swelling model of mice,the expression levels of inflammatory factors IL-6,IL-1β and TNF-α in the ear tissue of the model group significantly increased compared with the normal control group(P<0.001).Compared with the model group,the content of inflammatory factors in the dexamethasone group and the various dose groups of ZKMG were significantly reduced(P<0.01)and Zukamu group was dose-dependent inhibition,there was no significant difference between the administration group and the normal control group(P > 0.05).In the LPS-induced acute lung injury model of rats,compared with the normal group,the contents of IL-6,IL-1β and TNF-α in the alveolar lavage fluid of rats in the model group increased sharply(P<0.001).Compared with the model group,the levels of inflammatory factors in the alveolar lavage fluid of rats in the dexamethasone group and various dose groups of ZKMG showed the decline of different degree(P<0.05).(5)Expression of MAPK signaling pathway family proteins: In the xylene-induced ear swelling model of mice,the levels of p-p38,p-ERK,and p-JNK in the model group were significantly increased compared with the normal control group(P<0.001).Compared with the model group,the expression levels of p-p38,p-ERK,and p-JNK were significantly decreased in dexamethasone and various dose groups of ZKMG(P<0.001,P<0.05).In the LPS-induced acute lung injury model,the changes in phosphorylation levels of MAPK family proteins p38,ERK,and JNK in rat lung tissue were basically consistent with those in the xylene-induced mouse ear swelling model.(6)Expression of NF-κB signaling pathway-related proteins: In the model of LPS-induced acute lung injury,compared with the normal control group,LPS can induce the phosphorylation of NF-κB p65 in lung tissue.Compared with the model group,dexamethasone and various dose groups of ZKMG reversed these lung injury manifestations.In addition,the expression of COX-2 and i NOS was significantly enhanced in the model group,while dexamethasone and various dose groups of ZKMG could inhibit its expression to a variable extent.Conclusion:(1)Analgesic effect: compared with the model group,different doses of ZKMG can significantly inhibit the pain response stimulated by hot plate and writhing response induced by acetic acid in mice,indicating that Zukamu particles are effective analgesics for physical and chemical pain.(2)Anti-inflammatory effect: compared with the model group,different doses of ZKMG could significantly improve the inflammatory pathological damage of mouse ear tissue and rat lung tissue,and reduce the level of inflammatory factors in the mouse ear tissue and alveolar lavage fluid of rats,and inhibit the phosphorylation of MAPK signaling pathway family proteins p38,ERK,and JNK in ear tissue and lung tissue,as well as the phosphorylation of NF-κB signaling pathway-related protein NF-κB p65 and the expression of COX-2and i NOS in rat lung tissues.The high dose ZKMG had the best improvement effect,indicating that ZKMG has certain anti-inflammatory activity,which may be through inhibiting the phosphorylation or expression of related proteins to prevent the activation of MAPK and NF-kappa B signaling pathways to regulate the inflammatory response,thereby exerting therapeutic effects on xylene-induced ear swelling in mice and LPS-induced acute lung injury in rats.In addition,the compounds obtained by HS-SPME-GC-MS were screened and summarized through multiple databases,and the synergistic effect of ZKMG on multi-component,multi-target and multi-pathway was studied by online network construction method,so as to predict the material basis and mechanism of ZKMG on anti-inflammation.At the same time,combined with the pharmacological experimental,it provides a reliable method to reveal the complex mechanism of action of ZKMG.In conclusion,ZKMG is an effective therapeutic agent against acute upper respiratory tract infection,with analgesic and anti-inflammatory effects to alleviate headache,sore throat,nasal congestion and runny nose,and the mechanism of analgesic and anti-inflammatory effects is clear,in order to provide better clinical medical services. |
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