| Objective:In order to solve the problem of unclear quality control and mechanism of action of the hospital preparation Jiawei-fengfang Pills(JWFFP).Using full-time dual-wavelength fusion traditional TCM fingerprint and stability test,the quality control method of JWFFP was studied and the validity was verified.A rat mammary gland hyperplasia model was established to observe the dynamic pharmacodynamic changes of JWFFP in the treatment of rat mammary gland hyperplasia,and to analyze the mechanism of action by RNA-Seq technology.Method:(1)Fingerprint research methods:GH0525046C18A column(250×4.6 mm,5μm,Beijing Green Baicao Technology Development Co.,Ltd.)was used,acetonitrile(A)-0.01%phosphoric acid water(B)was used as mobile phase gradient elution,detection wavelengths were 248 and 284 nm.The chromatographic data detected at two wavelengths were exported from the Lab Solutions chromatography workstation,and the full-time dual-wavelength fusion was performed.10 batches of self-made JWFFP samples were injected and analyzed,and the data were evaluated by special similarity calculation and evaluation software,and determine the content of 6 index components.(2)Verify validity methods:The JWFFP was placed under the influence factor test,accelerated test and long-term test conditions,respectively.The stability was evaluated by quantitative indexes,which were liquiritin,scutellarin,hesperidin and scutellarein,and the validity was verified by statistical method.(3)Dynamic pharmacodynamics and mechanism research methods:The rat model of mammary gland hyperplasia was established by combining estrogen and progesterone.70 female non-pregnant rats were divided into blank group(CK),model group(MOD),Jiawei Fengfang Pill high-dose(FFW-H),medium-dose(FFW-M)and low-dose(FFW-L)groups,rupixiao group(RPX)and tamoxifen group(TAM)were administered drug intervention.The diameter and height of the second pair of nipples of rats in each group were measured at three time points before modeling,after modeling and after drug administration.On the 0th,10th,20th,and 30th days of administration,the second and third pairs of breast tissues of each group were aseptically harvested,and HE-stained paraffin sections were used to observe the dynamic changes of breast swelling symptoms in rats under different interventions and at different times.On the 30th day of administration,the 3rd and 4th pairs of mammary gland tissues of the rats in each group were harvested,and stored after being snap-frozen in liquid nitrogen.RNA-Seq technology was used to analyze the differential genes of mammary gland hyperplasia in rats treated with JWFFP,and the biological functions and pathways involved in the differential genes were explored by GO and KEGG enrichment analysis.Result:(1)Fingerprint research results:A full-time dual-wavelength HPLC fingerprint of JWFFP was established,19 common peaks were marked and the similarity was above 0.97,and 7 peaks were identified.Among them,6 components have good linearity within the corresponding concentration range,and the average sample recovery rate is 97.31%～101.47%.The determination results of the 10 batches of samples were 0.2418～0.2983mg·g-1 of liquiritin,0.2740～0.2901mg·g-1 of scutellarin,1.2501～1.4391mg·g-1 of hesperidin,0.0545～0.0595mg·g-1 of scutellarein,0.3824～0.4309mg·g-1 of glycyrrhizic acid and 0.0276～0.0298mg·g-1 of wogonin.(2)Verify validity results:The results of the stability test show that the traits and content of JWFFP meet the requirements after being placed for a corresponding time under high temperature,high humidity,strong light,accelerated and long-term test conditions.Under the condition of 25℃,the predicted value of the validity of JWFFP is 23.3207 months.(3)Dynamic pharmacodynamics and transcriptome research results:Before modeling,there was no significant difference in the diameter and height of the second pair of nipples in each group.After modeling,the nipple diameter and height of rats in other groups increased significantly compared with CK group(P<0.05).After 30 days of administration,the diameter and height of each intervention group were significantly lower than those of the MOD group(P<0.05).The dynamic pathological results showed that compared with the CK group,the mammary duct epithelial cells in the MOD group were actively proliferated at each time period,and a small amount of secretions could be seen in the dilated duct lumen.From the 20th day of administration,except for the RPX group,the hyperplasia of mammary gland,the degree of lumen expansion and the hyperplasia of interstitial fibrous connective tissue of the rats in the other administration groups were significantly reduced,and the number of ductal epithelial cells was significantly reduced.The differentially expressed genes between the groups were obtained by RNA-Seq.Compared with the CK group,there were a total of 1113 differentially expressed genes in the rat mammary gland hyperplasia.Compared with MOD,the number of differentially expressed genes in FFW group,RPX group and TAM group were 565,257 and 669,respectively.After comparing the CK group and the MOD group,there were 275 differentially expressed genes that were reversed by JWFFP(compared between the FFW group and the MOD group),including 185 differentially expressed genes that were up-regulated after modeling and down-regulated after JWFFP intervention.These genes are mainly involved in biological functions such as inflammatory response,lipid metabolism,ion transport,and Ras signaling pathway,ErbB signaling pathway,estrogen signaling pathway,etc.There were 90 differentially expressed genes that were down-regulated after modeling and reversed after JWFFP intervention.These genes are mainly involved in functions such as immune response,carbohydrate metabolism,and PPAR signaling pathways.Conclusion:(1)The fingerprint of JWFFP and the content determination method of six components were established by HPLC and full-time dual-wavelength fusion technology.The results showed that the overall quality difference of laboratory-made JWFFP was small,indicating that the quality of the preparation was controllable.The method is simple,accurate and reproducible.Combined with chemometric methods,it can be used as a time-saving and convenient method to control and evaluate the quality of JWFFP preparations.(2)From the stability test results,it can be seen that the stability of JWFFP is good,and the predicted validity is 23 months under the conditions of 25℃ and relative humidity(60±5)%.(3)The rat mammary gland hyperplasia model was successfully established.JWFFP intervention can significantly improve the degree and pathological morphology of nipple hyperplasia in the rat mammary gland hyperplasia model,and the drug’s onset time may start on the 20th day of administration.(4)The transcriptome library of mammary gland tissue of rats with mammary gland hyperplasia treated by JWFFP intervention was successfully constructed.Screened out 275 differentially expressed genes that were up-regulated or down-regulated after JWFFP intervened in mammary gland hyperplasia rats.And comprehensive analysis of GO and KEGG functional enrichment showed that Ras signaling pathway,ErbB signaling pathway,estrogen signaling pathway,PPAR signaling pathway,and arachidonic acid metabolism pathway may be related to the treatment of mammary gland hyperplasia.JWFFP may play a comprehensive role in the treatment of mammary gland hyperplasia in rats by regulating mammary cell proliferation,participating in estrogen and lipid metabolism,and regulating inflammatory response. |
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