Yong Tan1, Xinru Liu2, Cheng Lu3, Xiaojuan He1, Jian Li4, Cheng Xiao5, Miao Jiang1, Jing Yang1, Ke Zhou6, Zhongxiao Zhang2, Weidong Zhang7, Aiping Lu8. 1. Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing 100700, China. 2. School of Pharmacy, Second Military Medical University, Shanghai 200433, China. 3. Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing 100700, China; School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Hong Kong. 4. Beijing University of Chinese Medicine, Beijing 100700, China. 5. China-Japan Friendship Hospital, Beijing 100030, China. 6. Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing 100700, China; Hunan University of Chinese Medicine, Changsha 410208, China. 7. School of Pharmacy, Second Military Medical University, Shanghai 200433, China. Electronic address: wdzhangy@hotmail.com. 8. Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing 100700, China; School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Hong Kong. Electronic address: lap64067611@126.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Kidney-yang deficiency syndrome (KYDS) is a diagnostic pattern in traditional Chinese medicine (TCM) and clinical data showed that the unbalance in adrenal cortical hormone is the key issue in KYDS patients. The processed Ranunculaceae aconitum carmichaeli debx (bai-fu-pian in Chinese, BFP) is one of the most commonly used Chinese herbs for treating KYDS. The present study was conducted to explore the therapeutic biomarkers of the BFP in treating hydrocortisone administration induced KYDS rats. MATERIALS AND METHODS: Thirty male Sprague-Dawley rats were randomly divided into five groups with six in each group. KYDS in rats was induced by i.p. injection of hydrocortisone at the dose of 10mg/kg per day for 15 days as described previously. The rats with KYDS were administered orally, starting from the day of hydrocortisone administration stopped, with BFP extract at the dose of 0.32g/kg, 0.64g/kg and 1.28g/kg per day respectively for 15 days. The blood samples were collected for the liquid chromatography quadruple time-of-flight mass spectrometry (LC-Q-TOF-MS) test, as well as radioimmunoassay to determine the concentrations of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and adrenocorticotrophic hormone (ACTH). The metabolic responses to BFP administration were investigated by using the principal components analysis (PCA) and orthogonal partial least squares analysis (OPLS). Bioinformatics analyses were performed by using the Ingenuity Pathway Analysis (IPA). Variance analysis and linear regression analysis were used in this study. RESULTS: The signs and concentrations of cAMP, cGMP and ACTH in the model rats were similar to those previously described about KYDS rats and BFP treatment can reverse the changes. Seventeen significantly changed metabolites among different groups were identified. Thirteen metabolites were identified in the KYDS rats comparing to healthy rats with nine up-regulated and four down-regulated. After BFP treatment at three dosages, five up-regulated metabolites including phosphate, betaine, (4-hydroxyphenyl) acetaldehyde, 5-hydroxyindol-3-acetic acid and 5'-phosphoribosyl-N-formylglycinamide were dose-dependently reversed. The network analysis with IPA showed that four canonical pathways including superpathway of methionine degradation, purine nucleotides de novo biosynthesis II, tyrosine synthesis and serotonin receptor signaling involved the therapeutic mechanism of BFP in treating the KYDS rats. CONCLUSIONS: Five therapeutic biomarkers (phosphate, betaine, (4-hydroxyphenyl) acetaldehyde, 5-hydroxyindol-3-acetic acid and 5'-phosphoribosyl-N-formylglycinamide) and two corresponding canonical pathways (amino acid metabolism and purine nucleotide metabolism) were identified to be involved in the therapeutic mechanism of BFP treating the KYDS.
ETHNOPHARMACOLOGICAL RELEVANCE: Kidney-yang deficiency syndrome (KYDS) is a diagnostic pattern in traditional Chinese medicine (TCM) and clinical data showed that the unbalance in adrenal cortical hormone is the key issue in KYDS patients. The processed Ranunculaceae aconitum carmichaeli debx (bai-fu-pian in Chinese, BFP) is one of the most commonly used Chinese herbs for treating KYDS. The present study was conducted to explore the therapeutic biomarkers of the BFP in treating hydrocortisone administration induced KYDS rats. MATERIALS AND METHODS: Thirty male Sprague-Dawley rats were randomly divided into five groups with six in each group. KYDS in rats was induced by i.p. injection of hydrocortisone at the dose of 10mg/kg per day for 15 days as described previously. The rats with KYDS were administered orally, starting from the day of hydrocortisone administration stopped, with BFP extract at the dose of 0.32g/kg, 0.64g/kg and 1.28g/kg per day respectively for 15 days. The blood samples were collected for the liquid chromatography quadruple time-of-flight mass spectrometry (LC-Q-TOF-MS) test, as well as radioimmunoassay to determine the concentrations of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and adrenocorticotrophic hormone (ACTH). The metabolic responses to BFP administration were investigated by using the principal components analysis (PCA) and orthogonal partial least squares analysis (OPLS). Bioinformatics analyses were performed by using the Ingenuity Pathway Analysis (IPA). Variance analysis and linear regression analysis were used in this study. RESULTS: The signs and concentrations of cAMP, cGMP and ACTH in the model rats were similar to those previously described about KYDS rats and BFP treatment can reverse the changes. Seventeen significantly changed metabolites among different groups were identified. Thirteen metabolites were identified in the KYDS rats comparing to healthy rats with nine up-regulated and four down-regulated. After BFP treatment at three dosages, five up-regulated metabolites including phosphate, betaine, (4-hydroxyphenyl) acetaldehyde, 5-hydroxyindol-3-acetic acid and 5'-phosphoribosyl-N-formylglycinamide were dose-dependently reversed. The network analysis with IPA showed that four canonical pathways including superpathway of methionine degradation, purine nucleotides de novo biosynthesis II, tyrosine synthesis and serotonin receptor signaling involved the therapeutic mechanism of BFP in treating the KYDS rats. CONCLUSIONS: Five therapeutic biomarkers (phosphate, betaine, (4-hydroxyphenyl) acetaldehyde, 5-hydroxyindol-3-acetic acid and 5'-phosphoribosyl-N-formylglycinamide) and two corresponding canonical pathways (amino acid metabolism and purine nucleotide metabolism) were identified to be involved in the therapeutic mechanism of BFP treating the KYDS.