| Objective: To establish the fingerprint of Xiaocuo decoction compound by high performance liquid chromatography(HPLC),and to determine the content of index components,to establish the quality standard of Xiaocuo decoction compound;to predict the effective components,key targets and pathways of Xiaocuo decoction compound in treating acne(Acne)by network pharmacology method,and to verify the prediction results by animal experiments,so as to lay a foundation for further research on Xiaocuo decoction compound.Methods: 1.According to Chinese Pharmacopoeia and literature search,the suitable HPLC chromatographic conditions for Xiaocuo Decoction were explored,and then the fingerprint of Xiaocuo Decoction was established by using this chromatographic condition.After specific investigation,the content of specific indicator components was detected.2.The network pharmacology method is used to predict the effective components,targets and key pathways of Xiaocuo Decoction.3.Establishment of Acne animal model and administration therapy:Propionibacterium acnes suspension and oleic acid were prepared in advance according to the modeling method in the literature,and the model was constructed in the lower part of the ear cortex of the rat right ear.After successful modeling,the experimental animals were divided into blank group,model group,low-dose group of Xiaocuo decoction,medium-dose group of Xiaocuo decoction,high-dose group of Xiaocuo decoction and isotretinoin soft capsule group.According to the equivalent dose conversion method of human and animal,isotretinoin soft capsule group was given a daily dose of 3.125mg/kg,medium-dose groups were given 14.7g/ml,while low-dose,and highdose groups were given 7.3g/kg and 29.4 g/kg,the blank group and the model group were given the same volume of normal saline for 21 days.4.Apparent observation: After gavage,the texture and color of the right ear and ear tissues of rats in each group were observed with naked eyes,as well as the presence or absence of skin lesions such as skin thickening,sebaceous gland detachment,pus and papules.5.Use a vernier caliper to measure the ear thickness of the rat,and then calculate the rate of ear swelling according to the formula.6.Hematoxylin-eosin staining(HE)was used to analyze the pathological results of the right auricle of experimental rats.7.The contents of TNFα and IL 6 in serum of each group of rats were detected by ELISA kit according to its instructions,and the measured results were analyzed.8.Immunohistochemical method was used to detect the expression of TNFα,IL 6 and IL 1β protein in the right ear of rats.9.RT-q PCR expression of m RNA in TNFα,IL 6,MMP3,MMP9 and AKT1 in the right ear of each group of rats was detected by using the technique.10.Western Blot was used to detect the expression of TNFα,IL 6,MMP3,MMP9 and AKT1 proteins in the right ear of rats.Results: 1.Establishment of fingerprint of Xiaocuo Decoction and determination of the content of indicator components: The fingerprint of 15 batches of Xiaocuo Decoction was established,and the similarity was above 0.9.Twenty-five common peaks were identified and eight peaks were identified,namely,weelolide,trichurde,salvianolic acid B,berberine,mangiferin,forsythin,glycyrrhizin and glycyrrhizin.Mangiferin,privet glycoside and glycyrrhizic acid are specific,and their average mass fractions are 1.32 mg/g,1.45 mg/g and 1.07 mg/g respectively.2.Network pharmacology research results: Quercetin,kaempferol,luteolin and other compounds may be the effective components of Xiaocuo Decoction in the treatment of Acne.The process of Xiaocuo Decoction in the treatment of Acne is the result of the synergistic effect of multiple pathways.TNF signaling pathway,HIF-1 signaling pathway,NOD-like receptor and other pathways may be the main pathways involved in the process of Xiaocuo Decoction in its therapeutic effect.Among all signaling pathways,the most advanced one was TNF signaling pathway based on P-value.After network construction of genes in this pathway,it was found that TNFα,IL 6,IL 1β,AKT1,MMP3 and MMP9 were the key genes in this pathway.3.Results of animal experiments:(1)Apparent morphology: The auricles of the blank group were soft and reddish in color,and no skin lesions such as skin thickening,sebaceous gland shedding,pustules and papules appeared.The auricles of the model group were hard and dark red in color,with skin folds,thickening and swelling.Compared with the model group,the high-dose group of Xiaocuo decoction and isotretinoin soft capsule group showed that the ear redness and swelling,skin folds disappeared,papules and pustules disappeared,and capillaries became clear obviously.The low-dose and medium-dose groups of Xiaocuo decoction improved the ear skin to varying degrees.(2)The measurement results of ear thickness and swelling rate in the right ear of rats: After successful modeling,the ear thickness and swelling rate of rats in the model group increased significantly compared with the blank group(P<0.001).Compared with the model group,the ear thickness and swelling rate of rats in the low,medium and high dose groups of Xiaocuo decoction and isotretinoin soft capsule groups decreased significantly(P<0.01,P<0.001).(3)HE staining results: Compared with the blank group,a large number of inflammatory cells appeared in the ear tissue of rats in the model group,as well as inflammatory manifestations such as blurred boundaries of epidermis,dermis and subcutaneous tissues.After drug administration intervention,inflammatory cells were occasionally seen in model rats,and the boundaries of epidermis,dermis and subcutaneous tissue gradually became clear.(4)Changes of TNFα and IL6 in rat serum: After modeling,compared with the blank group,the levels of TNFα and IL 6 in Acne model group were significantly increased,with statistical difference(P<0.001).Compared with Acne model group,the contents of TNF α and IL 6 in serum of rats in the middle and high dose group of Xiaocuo decoction and isotretinoin soft capsule group were significantly decreased(P<0.01,P<0.001).(5)IHC results: Compared with the blank group,the expression of TNFα,IL 6 and IL 1β protein in the ear tissue of rats became darker after the model was established,and the expression of TNFα,IL 6 and IL 1β protein in the ear tissue of rats became lighter after the drug administration.(6)RT-q PCR results: Compared with the blank group,the m RNA levels of AKT1,TNFα,IL 6,MMP3 and MMP9 in the model group were significantly increased(P<0.001).Compared with the model group,the m RNA levels of AKT1,TNFα,IL 6,MMP3 and MMP9 in the high dose group and isotretinoin soft capsule group were significantly decreased(P<0.01,P<0.001).The m RNA levels of AKT1,TNFα,IL 6,MMP3,MMP9 were significantly decreased in the group of Xiaocuo decoction(P<0.01,P<0.001),and the m RNA levels of AKT1,TNFα,IL 6,MMP9 were significantly decreased in the group of Xiaocuo decoction(P<0.05,P<0.01).(7)Western blot results: Compared with the blank group,the relative protein expression levels of AKT1,TNFα,IL 6,MMP3 and MMP9 were significantly increased in the model group(P<0.001).The protein expression levels of AKT1 and IL 6 were down-regulated in the middle and high dose group and isotretinoin soft capsule group,with statistical difference(P<0.05,P<0.01,P<0.001).The low dose group of Xiaocuo decoction had downregulated effect on AKT1 and IL 6,but there was no statistical difference.Conclusion: The fingerprint of Xiaocuo decoction was established by HPLC,the content of three index components was determined,and the quality of Xiaocuo decoction was preliminarily evaluated.In order to explore the effect and mechanism of Xiaocuo decoction,this study first predicted the possible mechanism of Xiaocuo Decoction in the treatment of acne by using the research method of network pharmacology.By consulting the literature at home and abroad,combined with the recent research progress of acne mechanism,and then verified the prediction results of network pharmacology by using animal experiments.It establishes the preliminary quality standard and mechanism research basis for Xiaocuo decoction,so as to facilitate the follow-up research. |
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