| Diabetes Mellitus(DM)is a public health issue that is of increasing concern to countries around the world.Diabetic Retinopathy(DR)is the most important,most serious and most difficult treatment for blindness in DM patients.It is also one of the four most common blind diseases in the world.Bushen Huoxue prescription(composed of Rehmanniae radix,Salviae miltiorrhizae radix et rhizoma,Puerariae lobatae radix,and Ginseng radix et rhizoma)was developed by the members of this research group and obtained invention patents.The previous clinical and pharmacological experiments showed that Bushen Huoxue prescription was used to prevent and treat DR,and the effect was good.In the early stage of the research group,the effective substances of Ginseng radix et rhizoma and Salviae miltiorrhizae radix et rhizoma in the Bushen Huoxue prescription were studied.On the basis of previous research,this paper is based on the multi-dimensional spectrum-effect relationship to study the effective substances of Bushen Huoxue prescription in preventing and treating DR,and lays a foundation for the development of new DR Chinese medicine with independent intellectual property rights.12 batches of Bushen Huoxue prescription samples were obtained by uniform design method.12 batches of sample fingerprints were established by HPLC-UV-ELSD and the differences of 12 batches of samples were evaluated by cluster analysis.The results showed that 23 common peaks were identified in the HPLC-UV fingerprint,among which the peaks 1-15,17 and 21 were from Puerariae lobatae radix,the peaks 18-20,22 and 23 were from Salviae miltiorrhizae radix et rhizoma,and the 16 th peak was Salviae miltiorrhizae radix et rhizoma.Shared with Puerariae lobatae radix.Fourteen common peaks were identified in the HPLC-ELSD fingerprint,wherein the 1’-7’ peak was derived from Puerariae lobatae radix,the 8’peak was derived from Salviae miltiorrhizae radix et rhizoma,and the 9’~14’ peak was derived from Ginseng radix et rhizoma.By comparing with the reference substance,seven components in the compound were identified,respectively,the peak of 6/4’ was puerarin,the peak of 22/8’ was salvianolic acid B,the peak of 10’ was ginsenosideRg1,the peak of 11’ was ginsenoside Rb1,the peak of 12’ was ginsenoside Rc,the peak of 13’ was ginsenoside Rb2,and the peak of 14’ was ginsenoside Rd.In the HPLC-UV fingerprint of Bushen Huoxue prescription,12 batches of samples were divided into 5 categories by cluster analysis,in the HPLC-ELSD fingerprint,12 batches of samples were divided into 3 class.The rat model of DR was induced by high fat diet + STZ.The changes of retinal structure were detected by pathological tissue.In the DR retina,immunohistochemistry was used to detect the protein expression level of vascular endothelial growth factor(VEGF)and hypoxia-inducible factor-1α(HIF-1α).The Elisa method was used to detect the content of advanced glycation end products(AGEs)in rat plasma.The results of HE staining showed that compared with the normal control group,the retinal tissue of the experimental model group showed obvious increase of new capillary in the outer and inner layers,capillary microthrombus formation,disorder of perivascular cells,and ganglion cell degeneration or necrosis,obvious thickening of the optic nerve fiber layer,neurofibrillary edema,degeneration and other pathological damage,suggesting that the rat DR model was successfully constructed.Compared with the model group,the degree of lesions in each drug-administered group was alleviated,suggesting that each drug-administered group has a certain therapeutic effect.The results of retinal digestion showed that compared with the normal control group,the ratio of endothelial cell number to pericyte cell number in the model group was significantly increased,which was significantly different(P<0.01).Compared with the model group,the ratio of endothelial cell number to pericyte cell number in the each drug groups were significantly lower,with a very significant difference(P<0.01).The results of immunohistochemistry showed that the optical density values of retina tissues with VEGF and HIF-1α protein expression were remarkably increased in the model group when compared with the normal control group(P<0.01).Compared with the model group,the accumulation of both in the retina of each administration group decreased to some extent,indicating that 12 batches of Bushen Huoxue prescription have certain therapeutic effects on DR.The results of ELISA showed that the plasmaconcentration of AGEs in the model group was notably increased when compared with that of the normal control group(P<0.05).Compared with the model group,the concentration of AGEs in the plasma of the rats in each administration group was reduced to some extent.The TOPSIS analysis method was used to comprehensively analyze and evaluate the efficacy of 12 batches of Bushen Huoxue prescription.The results showed that the original group and the drug group 2 had the best effect in preventing and treating DR.Partial least squares regression(PLSR)was used to study the correlation between the common peak area of 12 batches of Bushen Huoxue prescription and VEGF,HIF-1α,AGEs in DR rat,and to find out the medicine effective component that contributed to “efficacy”,establish a model of the multi-dimensional spectrum-effect relationship of Bushen Huoxue prescription.The results showed that puerarin,salvianolic acid B,ginsenoside Rd and one components with undetermined structure(peak 20)were possible pharmacological substances for the prevention and treatment of DR by Bushen Huoxue prescription.Based on the multi-dimensional spectrum-effect relationship,this paper explores the pharmacodynamic basis of Bushen Huoxue prescription for prevention and treatment of DR,and provides a basis for further research and clinical application of Bushen Huoxue prescription,and lays a foundation for the development of new Chinese medicine for DR treatment. |
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