(W0930-04-24) Kidney Metabolomics Demonstrated the Energy Supply Upregulation by Radix Aconiti Lateralis Preparations in Rats

Purpose: Radix Aconiti Lateralis (Fuzi) is an extensively used Traditional Chinese Medicine (TCM) to treat cardiovascular diseases, rheumatism arthritis, bronchitis, pains, diarrhea, and hypothyroidism etc^1,2. Although it is believed that Fuzi goes into the heart and kidney vessels, reinforcing fire and Yang, dispelling wind and damp, and restoring from collapse according to the TCM theory³, the pharmacological mechanisms of such clinical indications remain largely unknown. In this study, we aimed to systematically elucidate the heart and kidney metabolic function of Fuzi preparations using metabolomics analyses.

Methods: To prepare the Fuzi preparations, 5g concentrated granules of Heishunpian or Paofupian (Purapharm^© with batch numbers A190097510 for Heishunpian and A1701434 & A190056510 for Paofupian) were dissolved in 10 mL boiling water followed by sonication for 30 min. To prepare the corresponding alkaloid mixtures for the above-mentioned Fuzi preparations, each of the six toxic alkaloids (namely aconitine (AC), hypaconitine (HA), mesaconitine (MA), benzoylaconine (BAC), benzoylhypaconine (BHA), benzoylmesaconine (BMA)) was dissolved in 0.1 M acetate buffer (pH=5.0)⁴ followed by mixing them at the ratio based on their contents in the corresponding Fuzi preparations. An equivalent dose of 30 g/kg Heishunpian consisted of AC at 1.44 μg/kg, HA at 260.8 μg/kg, MA at 5.34 μg/kg, BAC at 0.87 mg/kg, BHA at 1.17 mg/kg, and BMA at 2.62 mg/kg, while 30 g/kg of Paofupian consisted of 0.23 μg/kg HA, 0.09 μg/kg MA, 0.31 mg/kg BAC, 0.41 mg/kg BHA, and 1.54 mg/kg BMA. As shown in Figure 1 forty Sprague Dawley male rats were randomly assigned into four treatment groups (G1~G4) and one control group (G5) with eight per group to receive either Heishunpian/Paofupian or their corresponding alkaloid mixtures once daily for 15 days. After sacrificing the rats on Day 16, the heart and kidney were collected and weighed followed by being stored under -80℃ for further analyses. The organ index was calculated based on the body weight and organ weight of each rat. Cardiac/renal serum biomarkers such as Creatinine (CREA), Blood Urea Nitrogen (BUN) and Total Protein (TP) in rat serum were also monitored. Our previously validated UPLC-Q-Exactive Orbitrap MS-based metabolomics analysis platform⁵ was used to quantify the endogenous metabolites in the collected heart and kidney samples. Endogenous metabolites with Variable Importance of Projection (VIP) >1 were selected for further analyses of the cardiac/renal metabolism pathways affected by Fuzi preparations or their toxic alkaloids via Principal Component Analysis (PCA) or Partial Least Squares Discriminant Analysis (PLS-DA) using MetaboAnalyst 4.0.

Results: There were 564 endogenous metabolites identified from the collected heart and kidney samples. As shown Figure 2, highly accurate PCA and PLS-DA analyses (R²＞0.96, Q²＞0.56) indicated that the distance between each Fuzi treatment group and control group in kidney was greater than that in the heart, suggesting that the Fuzi treatment might significantly altered more endogenous substances in kidney than that in heart. Our pathway enrichment analysis further verified that the altered endogenous substances were mainly related to fatty acid-related metabolic pathways in rat kidneys. Further insight into mRNA expression level of effectors on fatty acid-related metabolic pathways (Figure 3a&3b) revealed that cluster of differentiation 36 (Cd36), fatty acyl-CoA synthetase (Acs), fatty acid synthase (Fas) were significantly changed in rat kidneys but not in rat hearts, which confirmed kidney as the main organ reacting to Fuzi treatment. As shown in Figure 3b, Fuzi treatment could block the intake of extracellular fatty acid via downregulatingCd36 (3.5~7.4% of control). Such inhibition could possibly promote the intracellular fatty acid synthesis⁶ as evidenced by the sharp elevated expression levels of Fas (3~5 times of control) and Acs (4 times of control), key enzymes in fatty acid synthesis and activation, after Fuzi treatment. In the meantime, the unaltered mRNA expression on fatty acid oxidation pathway indicated a relatively steady level of fatty acid consumption in healthy rats. The activated synthesis of fatty acid resulted in an elevated kidney index (from 0.76±0.05 to 0.82±0.03, p＜0.01) only after the intake of Paofupian granule and detectable decrease of CREA after treatment of both Heishunpian granule (from 12.13±2.36 IU/L to 10.13±3.72 IU/L, p＜0.05) and Paofupian granule (from 12.13±2.36 IU/L to 9.88±2.75 IU/L, p＜0.05), could possibly be due to an increase of fat to muscle ratio⁷.

Conclusion: We for the first time utilized metabolomic analyses to demonstrate that both Fuzi preparations and their major toxic alkaloids could significantly elevate the synthesis and accumulation of fatty acids in rat kidney. Since the synthesis of fatty acid may serve as a source of energy, the upregulation of fatty acid synthesis by Fuzi preparation can be the underline mechanism for its clinical indication of reinforcing fire and Yang and restoring from collapse.

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Acknowledgements:

Funding: This research was funded by the Health and Medical Research Fund (15161541) by the Food and Health Bureau, Hong Kong SAR, P. R. China, and by National Natural Science Foundation of China (81903909), P. R. China.
Conflicts of Interest: The authors declare no conflict of interest.


Figure 1. Animal grouping and sampling schedule for treatment of Radix Aconiti Lateralis and their corresponding toxic alkaloid mixtures in rats.


Figure 2. Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) revealed metabolic profiling differences between each treatment group in (a) heart and (b) kidney after oral administrations of Heishunpian, Paofupian and their corresponding alkaloid mixtures in rats.


Figure 3. mRNA expression levels of genes related to fatty acid synthesis/metabolism and glucose metabolism in heart (a) and kidney (b), fatty acid-related metabolic pathways in heart (c) and kidney (d) of rats received 15-day oral administrations of 30 g/kg Heishunpian/Paofupian granule or its equivalent alkaloid mixtures (n=3, *p < 0.05, **p < 0.01, ***p < 0.001).