| Yupingfeng San(YPFS)is a commonly used traditional Chinese medicine(TCM)formula in clinical.It is composed of Astragali Radix(AR),Atractylodis Macrocephalae Rhizoma(AMR)and Saposhnikoviae Radix(SR).It was first recorded in Jiu Yuan Fang by Song Zhang during the Song Dynasty.Four dosage forms of YPFS including granules,oral liquid,tea bags and capsules were adopted in Chinese Pharmacopoeia(2020 edition)with a further revised ratio of three herbs(AR,AMR and SR)in 3:1:1.It has been widely used in the treatment of nephrotic syndrome,chronic obstructive pulmonary disease and allergic rhinitis.Modern pharmacological studies have shown that YPFS can regulate the immune system and inhibit the inflammatory response to treat chronic glomerulonephritis,diabetic nephropathy,primary nephrotic syndrome and other kidney injuries,but the therapeutic effect of YPFS on cisplatin induced acute liver and kidney injury is still unclear.Therefore,the purpose of this study is to clarify the chemical basis of YPFS,establish near-infrared fluorescence imaging evaluation method,screen its pharmacodynamic components in the treatment of cisplatin induced acute liver and kidney injury,and study the anti-inflammatory mechanism of the pharmacodynamic monomers obtained by screening,so as to provide scientific basis for further research on the pharmacological effects of YPFS.Objective:In this study,near infrared fluorescence imaging method was established to screen the pharmacodynamic components of YPFS in the treatment of cisplatin induced acute liver and kidney injury and to explore the anti-inflammatory mechanism of the pharmacodynamic components.Methods:1.Characterization of chemical constituents of YPFS.A flavonoid-compound database of YPFS was constructed,and a UHPLC-LTQ-Orbitrap mass spectrometry collection method was established.The MDF method combined with time-segment mode was used to target and filter and identify the flavonoid-containing components in YPFS,and other components were identified by comparison and reference literature.The chemical composition characterization of YPFS was realized.2.Synthesis and imaging of squaraine dyes for monitoring liver and kidney injury.The animal model of cisplatin induced acute kidney injury(AKI)and acute liver injury(ALI)was established.The probes were synthesized by organic synthesis method.The probes were determined by comparing the near-infrared(NIR)imaging effect of liver and kidney.The conventional pharmacological evaluation methods of liver and kidney injury,including biochemical index determination(BUN,CRE,AST and ALT levels),hematoxylin-eosin,were compared with NIR fluorescence imaging methods to establish NIR fluorescence imaging evaluation methods.3.Efficacy of YPFS and its Chinese medicine monomer in treating acute liver and kidney injury induced by cisplatin.The efficacy of YPFS and nineteen TCM monomers in the treatment of cisplatin induced liver and kidney injury was compared by using the established NIR fluorescence imaging method and the conventional pharmacological evaluation method.4.To investigate the anti-inflammatory mechanism of apigenin based on MAPK signaling pathway.Enzyme-linked immunosorbent assay(ELISA)was used to detect the level of TNF-α,IL-6 and IL-βin each group,and the expression of MAPK pathway related proteins was detected by western blot.Results:1.According to literatures,the component database of flavonoids in YPFS was established,and the structural rules of flavonoid aglycone,flavonoid mono-glycosides and flavonoid di-glycosides were summarized,and three MDF screening windows were established.The precursor ion list with time-segment mode was established to get high-selectivity trigger precursor ion list MS~n fragment,and the number of fragments were about2 times higher than that of conventional collection mode.A total of 78 chemical components including flavonoids,chromones,organic acids,coumarins,saponins,lactones and others in YPFS were identified by MDF method and direct matching method.2.Comparing the biochemical indexes and HE staining results of AKI and ALI models with different cisplatin induction time,the cisplatin induction time of 3 days was significantly different from that of normal group.Then,three probes of SQ-676-CD,SQ-686-CD and SQ-847-CD were synthesized to compare their fluorescence imaging effects.The SQ-686-CD has a good kidney and liver penetration depth,so the probe of SQ-686-CD was selected for subsequent fluorescence imaging studies.3.The results showed that YPFS,apigenin(flavonoid group),cimifugin(chromone group),astragaloside I(coumarin and saponin group),isochlorogenic acid B(nucleoside,organic acid and lactone group)were significantly different from the model group in the treatment of cisplatin induced AKI model and ALI model.NIR fluorescence imaging method was used to evaluate the efficacy of apigenin in high,medium and low dose treatment of AKI/ALI model,which was basically consistent with biochemical indexes and HE staining results.The results showed that NIR fluorescence imaging could explain the concentration-dependent therapeutic effect of TCM monomer.4.Compared with the control group,the serum TNF-α,IL-6 and IL-βlevel of mice in the model group was increased(p<0.01),and the serum TNF-α,IL-6 and IL-βlevel of mice after administration of apigenin,and cimifugin was decreased(p<0.05),of which apigenin had the best effect.Compared with control group,the expressions of p-p38,p-JNK and p-ERK proteins in kidney tissue of model group were increased(p<0.01).Compared with model group,the expression of p-p38,p-JNK and p-ERK protein in kidney tissues of apigenin group decreased(p<0.01).Conclusion:The NIR fluorescence imaging method can be used to preliminarily screen the pharmacodynamic components.YPFS and its traditional Chinese medicine monomers have therapeutic effects on cisplatin induced liver and kidney injury,among which apigenin can significantly inhibit inflammation in vivo,and its mechanism may be related to the regulation of MAPK signaling pathway. |
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