| Purpose:The network pharmacology was used to predict the mechanism of Yingzhongxiao ointment in treating nodular goiter.Based on the analysis results,the mechanism of therapeutic effect of Yingzhongxiao ointment on experimental nodular goiter rats was explored from the perspective of cell proliferation.Material and method:Part I :This part is about the Network Pharmacological Study of Zhizhongxiao in Treating Nodular Goiter.The Chinese medicine system pharmacology technology platform(TCMSP)was used to screen the drug active ingredients and potential targets of the drug with the drug-like properties(DL)?0.18.The Gene Cards and OMIM databases were used to search for nodular goiter disease targets,and Cytoscape software was used to construct a relationship network of "active ingredients-action targets" and "active ingredients-potential therapeutic targets".The STRING database and Cytoscape software were used to construct the target protein interaction network.The biological processes and pathway enrichment analysis of the treatment targets for Yingzhongxiao were performed through DAVID and KEGG databases.Part II:This part is about the effect of Yingzhongxiao on the proliferation of thyroid cells in nodular goiter rats.50 SPF grade 4-week-old Wistar rats,half male and half female.10 rats were selected randomly as the normal group,and the remaining 40 rats were intragastrically administered with a dose of 0.01g/(kg·d)PTU for 14 days to replicate the goiter model.After modeling,40 rats were randomly divided into 4 groups:Model group,Ointment group,Placebo group(only ointment base without drug),LT4 group.From the 15 th day,the rats in the normal group and the model group were reared normally.The rats in the other groups were depilated with 6% sodium sulfide.The rats in the ointment group were given an external application of Yingzhongxiao ointment with a crude drug content of 0.9 g /(kg·d)once a day.The Placebo group was given an external application of the neck once a day without medicinal ingredients.The LT4 group was administrated with 0.2?g /(kg·d)LT4 aqueous solution once a day.Rats in each group were continuously intervened for 28 days.After the intervention,the rats were anesthetized,and serum and thyroid tissue were collected.The contents of TT3,TT4 and TSH in rat serum were detected by ELISA,the morphology of thyroid tissue was observed by HE staining,and the expression of PCNA and VEGF in thyroid tissue of rats was detected by immunohistochemistry and Western blot.Results: 1.(1)In the TCMSP database,with DL?0.18 as the condition,227 active ingredients of 8 flavor drugs of Yingzhongxiao were selected,including 39 types of prunella,63 types of rhubarb,43 types of gardenia,15 types of fritillaria,8 types of curcuma,21 types of indigo naturalis,33 types of mint,and 5 types of borneol.(2)Active ingredients have 2,052 targets,of which prunella has 555 targets,rhubarb has 241 targets,gardenia has 496 targets,fritillaria has 85 targets,curcuma has 55 targets,indigo naturalis has 162 targets,mint has 452 targets and borneol has 6 targets and there are 149 remaining after deleting duplicate targets.(3)A total of 847 targets for nodular goiter disease were found in the Gene Cards and OMIM databases,of which 190 were targets for drug-disease intersection.49 targets belong to prunella,18 targets belong to rhubarb,48 targets belong to gardenia,11 targets belong to fritillaria,2 targets belong to curcuma,14 targets belong to indigo naturalis,45 targets belong to mint,3 targets belong to borneol,and 62 are left after removing duplicate targets.(4)The STRING database was used to construct a therapeutic target protein interaction network map.The PPI network contains 62 target protein nodes,609 interactive connections,the average node degree is 19.6,and the average local clustering coefficient is 0.709.(5)Analyze the biological target of the treatment target,and get 1163 pieces of biological process information.914 are of significant significance(P <0.01).The first 20 significant enrichment pathways mainly involve the response of cells to organics,the regulation of metabolic processes,the regulation of cell proliferation,the regulation of cell death,the regulation of catalytic activity,the regulation of protein metabolic processes,Regulation of stimulus response.(6)KEGG enrichment analysis revealed 128 gene pathways related to bloating and potential treatment targets,of which 112 had significant significance(P <0.01).The first 20 significant enrichment pathways were PI3K-Akt signaling pathway,EB virus infection,and Kaposi.Osteosarcoma-associated herpesvirus infection,MAPK signaling pathway,hepatitis B,prostate cancer,apoptosis,measles,focal spots,TNF signaling pathway,fluid shear stress and atherosclerosis,breast cancer,hepatitis C,P53 Signaling pathway,colorectal cancer,endocrine resistance,AGE-RAGE signaling pathway in complications of diabetes,platinum resistance,bladder cancer,prolactin signaling pathway.2.(1)There was no statistical difference in the body weight of rats in each group(P > 0.05).Compared with the normal group,the thyroid weight and relative weight of the rats in each group increased significantly(P <0.01).Compared with the model group,the thyroid weight and relative weight of the placebo group were not statistically different(P > 0.05).The thyroid weight and relative weight of the LT4 group decreased significantly(P <0.01);compared with the LT4 group,the thyroid weight and relative weight of the placebo group decreased significantly(P <0.01).(2)In the normal group,the size of follicles in thyroid tissue was uniform,the epithelial cells were arranged regularly,the gelatinous in the follicular cavity was uniform,and a small number of capillaries were visible.The thyroid tissue follicles in the model group and the placebo group were irregular in size and shape,with large differences.The epithelial cells were significantly proliferated,the arrangement was disordered,the glial distribution was uneven,and the surrounding capillaries were abundant.Compared with the model group,the size and arrangement of thyroid tissue follicles in the ointment group were relatively regular,the proliferation of epithelial cells improved,the distribution of colloids was more uniform,and the capillaries around the follicles decreased.In the LT4 group,the thyroid follicles recovered to a medium size,and the arrangement was relatively regular.The colloids in the follicular cavity were relatively uniform,and the capillary distribution around them was reduced.(3)There was no significant difference in serum TT3 levels among the groups(P>0.05).Compared with the normal group,the serum TT4 of each group was significantly reduced(P<0.01);Compared with the model group,the serum TT4 of the LT4 group was elevated(P<0.05),and there was no statistical difference in the other groups(P>0.05);Compared with the LT4 group,the TT4 level in the placebo group was lower(P<0.05).Compared with the normal group,the serum TSH in each group was significantly increased(P<0.01);Compared with the model group,the serum TSH in the LT4 group was significantly reduced(P<0.01),and there was no statistical difference in the other groups(P>0.05);compared with the LT4 group,there was a significant difference between the ointment group and the placebo group(P<0.01).(4)The average optical density of PCNA positive expression in thyroid tissue of each group of rats was calculated: compared with the normal group,the PCNA positive expression in each group was significantly enhanced(P<0.01);compared with the model group,the PCNA expression in the placebo group was not Statistical differences(P>0.05),PCNA expression in ointment group and LT4 group were significantly reduced(P<0.01);compared with LT4 group,PCNA expression in ointment group was significantly reduced(P<0.01).(5)Compared with the normal group,the expression of PCNA protein in each group was significantly enhanced(P <0.01);compared with the model group,the expression of PCNA protein in the placebo group was not statistically different(P> 0.05),and the expression of PCNA protein in the oinment group and the LT4 group significantly reduced(P <0.01);compared with the LT4 group,PCNA protein expression in the ointment group was significantly reduced(P <0.05).(6)The average optical density value of VEGF positive expression in thyroid tissue of each group of rats was calculated: compared with the normal group,the VEGF expression in each group was significantly enhanced(P<0.01);compared with the model group,there was no statistics in the placebo group(P>0.05),and the expression of VEGF in ointment group was significantly reduced(P<0.01,P<0.05).Compared with LT4 group,the expression of VEGF in ointment group was significantly reduced(P<0.01).(7)Compared with the normal group,the expression of VEGF protein in each group was significantly enhanced(P <0.01);compared with the model group,there was no statistical difference in the expression of VEGF protein in the placebo group(P> 0.05),and the expression of VEGF protein in the ointment group and the LT4 group was significantly reduced(P <0.01);compared with the LT4 group,the ointment VEGF protein expression was significantly reduced in the group(P <0.01).Conclusion:1.Yingzhongxiao ointment may treat nodular goiter by participating in cell proliferation,apoptosis and other processes.2.Yingzhongxiao ointment may treat nodular goiter by reducing PCNA and VEGF expression and inhibiting thyroid cell proliferation. |
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