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UHPLC-MS/MS analysis of cAMP and cGMP in rat plasma as potential biomarkers of Yin-Yang disharmony in traditional Chinese medicine

ABSTRACT: Cyclic 3′,5′-adenosine monophosphate (cAMP) and cyclic 3′,5′-guanosine monophosphate (cGMP) are considered as potential biomarkers for Yin-Yang disharmony in traditional Chinese medicine. However, phosphodiesterase-mediated ex vivo degradation of these molecules in biological samples may result in their underestimation. In the present study, a ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for determination of cAMP and cGMP in rat plasma, with special consideration of their stability ex vivo. Following precipitation of proteins from plasma samples with 0.4 M perchloric acid, the analytes were chromatographed on a Shimadzu Shim-pack-XR-ODS II column with 2.5 mM ammonium acetate and methanol in gradient mode. The MS/MS detection was performed using multiple reaction monitoring in the positive electrospray ionization mode. The lower limit of quantification was 0.27 ng/mL for cAMP and 0.37 ng/mL for cGMP. The method was used to determine the plasma cAMP and cGMP levels in normal and Yin deficiency diabetic rats treated with or without Rehmannia glutinosa. The developed method may be useful for evaluating the regulatory effects of Chinese herbal medicine on the levels of cAMP and cGMP in the body. Graphical abstract Image 1 Highlights • Detailed investigation of ex vivo stability of cAMP and cGMP in rat blood and plasma.• First recommendation of effective strategies to stabilize cAMP and cGMP across pre-analytical procedures.• First application of UHPLC–MS/MS method to the analysis of plasma cAMP and cGMP levels in Yin deficiency diabetic rats.• First demonstration of the feasibility for evaluating the regulation effects of TCM on cAMP and cGMP levels in the body.

SUBMITTER: Wang X

PROVIDER: S-EPMC8424358 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundYang Xin Tang (YXT) is a traditional Chinese herbal preparation which has been reported to improve cognitive function and memory in patients with dementia. As the underlying mechanism of action of YXT has not been elucidated, we examined the effects of YXT and its major herbal components in regulating gene transcription and molecular targets related to Alzheimer's disease (AD).MethodsAqueous and ethanol extracts of YXT and selected herbal components were prepared and validated by standard methods. A series of biochemical and cellular assays were employed to assess the ability of the herbal extracts to inhibit acetylcholinesterase, reduce β-amyloid aggregation, stimulate the differentiation of neural progenitor cells, suppress cyclooxygenase, and protect neurons against β-amyloid or N-methyl-D-aspartate-induced cytotoxicity. The effects of YXT on multiple molecular targets were further corroborated by a panel of nine reporter gene assays.ResultsExtracts of YXT and two of its constituent herbs, Poria cocos and Poria Sclerotium pararadicis, significantly inhibited β-amyloid aggregation and β-amyloid-induced cytotoxicity. A protective effect of the YXT extract was similarly observed against N-methyl-D-aspartate-induced cytotoxicity in primary neurons, and this activity was shared by extracts of Radix Astragali and Rhizoma Chuanxiong. Although the YXT extract was ineffective, extracts of Poria cocos, Poria Sclerotium pararadicis and Radix Polygalae inhibited acetylcholine esterase, with the latter also capable of upregulating choline acetyltransferase. YXT and its components significantly inhibited the activities of the pro-inflammatory cyclooxygenases. Additionally, extracts of YXT and several of its constituent herbs significantly stimulated the phosphorylation of extracellular signal-regulated kinases and cAMP-responsive element binding protein, two molecular targets involved in learning and memory, as well as in the regulation of neurogenesis.ConclusionsSeveral constituents of YXT possess multiple regulatory effects on known therapeutic targets of AD that range from β-amyloid to acetylcholinesterase. The demonstrated neuroprotective and neurogenic actions of YXT lend credence to its use as an alternative medicine for treating AD.

Project description:Ethnopharmacological relevancePsoralea corylifolia Linn. (BGZ) is a commonly used traditional Chinese medicine (TCM) for the treatment of kidney-yang deficiency syndrome (Yangsyn) with good curative effect and security. However, BGZ was also reported to induce liver injury in recent years. According to TCM theory, taking BGZ may induce a series of adverse reactions in patients with kidney-yin deficiency syndrome (Yinsyn), which suggests that BGZ-induced liver damage may be related to its unreasonable clinical use.Aim of the studyLiver injury caused by TCM is a rare but potentially serious adverse drug reaction, and the identification of predisposed individuals for drug-induced liver injury (DILI) remains challenging. The study aimed to investigate the differential responses to BGZ in Yangsyn and Yinsyn rat models and identify the corresponding characteristic biomarkers.Materials and methodsThe corresponding animal models of Yangsyn and Yinsyn were induced by hydrocortisone and thyroxine + reserpine respectively. Body weight, organ index, serum biochemistry, and Hematoxylin and Eosin (HE) staining were used to evaluate the liver toxicity effect of BGZ on rats with Yangsyn and Yinsyn. Transcriptomics and metabonomics were used to screen the representative biomarkers (including metabolites and differentially expressed genes (DEGs)) changed by BGZ in Yangsyn and Yinsyn rats, respectively.ResultsThe level changes of liver organ index, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), suggested that BGZ has liver-protective and liver-damaging effects on Yangsyn and Yinsyn rats, respectively, and the results also were confirmed by the pathological changes of liver tissue. The results showed that 102 DEGs and 27 metabolites were significantly regulated related to BGZ's protective effect on Yangsyn, which is mainly associated with the glycerophospholipid metabolism, arachidonic acid metabolism, pantothenate, and coenzyme A (CoA) biosynthesis pathways. While 28 DEGs and 31 metabolites, related to the pathway of pantothenate and CoA biosynthesis, were significantly regulated for the BGZ-induced liver injury in Yinsyn. Furthermore, 4 DEGs (aldehyde dehydrogenase 1 family member B1 (Aldh1b1), solute carrier family 25 member 25 (Slc25a25), Pim-3 proto-oncogene, serine/threonine kinase (Pim3), out at first homolog (Oaf)) and 4 metabolites (phosphatidate, phosphatidylcholine, N-Acetylleucine, biliverdin) in the Yangsyn group and 1 DEG [galectin 5 (Lgals5)] and 1 metabolite (5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate) in Yinsyn group were significantly correlated to the ALT and AST levels of BGZ treated and untreated groups (receiver operating characteristic (ROC) ≥ 0.9).ConclusionsYinsyn and Yangsyn are the predisposed syndromes for BGZ to exert liver damage and liver protection respectively, which are mainly related to the regulation of amino acid metabolism, lipid metabolism, energy metabolism, and metabolism of cofactors and vitamins. The results further suggest that attention should be paid to the selection of predisposed populations when using drugs related to the regulation of energy metabolism, and the Yinsyn/Yangsyn animal models based on the theory of TCM syndromes may be a feasible method for identifying the susceptible population to receive TCM.

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UHPLC-MS/MS analysis of cAMP and cGMP in rat plasma as potential biomarkers of Yin-Yang disharmony in traditional Chinese medicine

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