The effect of curculigo orchioides (Xianmao) on kidney energy metabolism and the related mechanism in rats based on metabolomics

Abstract The Chinese materia medica Xianmao (XM) is widely used in Chinese clinics and the traditional Chinese medicine diets. Although XM is often used to study its kidney‐yang effect, the research on its effect on kidney energy metabolism and its mechanism is still relatively lacking. In this study, rats were given different doses of XM water extract for 4 weeks. Biochemical method was used to detect the content of serum biochemical indexes of liver and kidney function and blood lipid indicators, and HE staining method was used to observe the histopathological of liver and kidney in rats. The kidney Na+‐K+‐ATPase, Ca2+‐Mg2+‐ATPase, SDH (succinate dehydrogenase) enzyme activity, and the content of ATP in rats were measured. Metabolomics technology was used to analyze the potential biomarkers related to the effects of XM on kidney energy metabolism, and then, the metabolic pathways were analyzed. RT‐PCR was used to detect the expression of Ampk, Sirt1, Ppar‐α, and Pgc‐1α mRNA in kidney of rats. The results showed, compared with the blank control group, there was no significant effect on liver and kidney function in XMH, XMM, and XML groups. These significantly increased the kidney Na+‐K+‐ATPase, Ca2+‐Mg2+‐ATPase, SDH enzyme activity, and ATP content in XMH, XMM, and XML groups. Mitochondrial metabolic rate was inhibited in XMH group, but it was significantly increased in XMM and XML groups. The number of mitochondria was increased in XMH, XMM, and XML groups. Overall, these effects may be mediated by TCA cycle metabolism, butanoate metabolism, propanoate metabolism, alanine, aspartate, and glutamate metabolism, retinol metabolism, purine metabolism, pentose phosphate metabolism, aminoacyl‐tRNA biosynthesis, valine, leucine, and isoleucine biosynthesis, and degradation metabolism pathways, as well as by increasing expression of upstream genes Ampk, Sirt1, Ppar‐α, and Pgc‐1α mRNA.


| INTRODUC TI ON
The traditional Chinese medicine is widely used in Chinese clinics, and a variety of molecular mechanisms are associated with functional regulation mediated by Chinese materia medica (Hu et al., 2020;Wang et al., 2019). To balance the yin and yang, the traditional Chinese medicine diets or combining foods with certain Chinese materia medica are purposefully used for the patients . One of the Chinese materia medica, Xianmao (root tubers of Curculigo orchioides, belonging to the family Amaryllidaceae) often utilized with foods to reverse the kidney-yang deficiency symptoms, such as the decline of vital gate fire, frequent urination, cold extremities, sore lower back, waist and knee pain, and soft bones (Chauhan et al., 2010). In addition, XM could active the TRPV1 of rat DRG ganglion cells, which reflected the heat properties of XM .
The physiological function of the body could be affected through the flow of matter and energy. Energy metabolism was considered to be one of the basic forms of the body's material metabolism. The cold and heat properties of the drug were closely related to the body's energy metabolism (Wang et al., 2008). The main active components of XM are curculigo orchioides phenolic glycoside, among which curculigoside and tenoside are higher in content (Yang, 2012), and curculigoside has a certain protective effect on the function of mitochondria . Studies have shown that XM can affect the energy metabolism of normal rats (Fan, 2010). It was found that XM could regulate the metabolism of substances in the body, cyclic nucleotides, and endocrine content (Li et al., 2012), reduce the content of TG and cGMP, and increase the content of cAMP/cGMP, T3, T4, TSH, Ts, Glu, TC, TP, etc. in rat serum (Zhou et al., 2014), to improve the symptoms of kidney-yang deficiency model in rats. In China, although Xiaomao (XM) is often used to study its kidney-yang effect, the research on its effect on kidney energy metabolism and its mechanism is still unknown.
Metabolomics is a technology for analyzing small molecules in organisms based on high-throughput, multivariate data. It can fully reflect the profile and level changes of endogenous metabolites in the body. And it can amplify small differences in upstream gene and protein expression, and further in-depth analysis of the metabolic pathways related to different metabolites can explain the mechanism of action of the research object (Wu et al., 2021). This technology has been widely used in food science , medicine Wang, Chen, et al., 2020;Wang, Gong, et al., 2020;Wang, Jia, et al., 2020) and other fields. Nontargeted metabolomics can comprehensively and unbiasedly reflect the metabolic state of small molecules in organisms, which is conducive to the screening of biomarkers and the construction of dynamic metabolic networks in organisms Wang, Chen, et al., 2020).
In this study, pharmacological experiments combined with nontargeted metabolomics technology were used to explore the effects of XM on kidney energy metabolism and its mechanism. This will improve our understanding of XM and provide a scientific basis for its use.

| Extraction of XM
Xiaomao (XM) was soaked in 10 times water for 60 min, refluxed for 60 min, and then poured out. The residue was extracted by 8 times water reflux for 40 min. The two filtrates were mixed and concentrated into 1 g/ml solution, which was diluted for standby before administration.

| Experimental animals and animal processing
Thirty-two male SD rats, 160 ± 20 g, obtained from Hunan Slack Jingda Experimental Animals Co., Ltd., were allowed to acclimatize for 7 days in the experimental animal science and technology center of Jiangxi University of Chinese Medicine. The temperature was set to 24 ± 2°C. The humidity was set between 55% and 65%, and the lighting condition was set to 12-hr light-dark alternating. Rats In total, 32 rats were randomly divided into the blank control group (Control), high dose of XM group (XMH), medium dose of XM group (XMM), and low dose of XM (XML), with eight rats in each group. The XMH, XMM, and XML groups were administered the crude extracts of 3.15, 1.05, and 0.35 g/kg by gavage for 4 weeks, respectively. The control group was given the same volume of saline. After the experiment, rats were fasting for 12 hr. The feces of the rats were collected before dissection. The kidney tissue was collected and stored in a −80° refrigerator. XM on ALT, AST, UA, BUN, Cr, TG,  TC, TBA,

| Effect of XM on kidney and liver pathological sections
The kidney and liver tissues were dissected, conventionally taken, dehydrated, embedded, prepared, and stained with HE.
Then, they were observed and described under an optical microscope. Different types of lesions in the main description were photographed. 2.7 | Determination of Na + -K + -ATPase, Ca 2+ -Mg 2+ -ATPase, and SDH enzyme activity in rat kidney tissue Physiological saline was added to kidney tissue to make a 10% homogenate, centrifuged for 10 min (4°C, 211 g). The supernatant was obtained to measure the enzyme activity of kidney tissue according to the manufacturer's instructions. Coomassie Brilliant Blue method was used to determine the protein concentration of tissues.

| Determination of ATP content
0.2 g of kidney tissue sample was weighed, and 1 ml of lysate was added. After homogenizing on ice, it was left for about 5 min to fully lysate. Then, the supernatant was centrifuged for 5 min (4°C, 12,000 g), and the supernatant was taken for use. The ATP content of rat kidney was detected according to the manufacturer's instructions.

| Mitochondrial oxygen consumption rate
The kidney mitochondria were extracted by differential centrifugation. The mitochondrial oxygen consumption kit was used to detect the signal value of mitochondria, and the CurveExper 1.4 software was used to draw the mitochondrial oxygen consumption curve of each rat, and the slope of the curve was calculated.

| NAO staining to observe the number of mitochondria
10-mercaptoacridine orange (NAO) is a reagent that can specifically bind to mitochondria. It can be used to detect the specific fluorescent markers of mitochondria and was often used to detect the number of mitochondria (Kan, 2018). After the animal experiment is over, the animal kidney tissue is quickly taken, soaked in glutaraldehyde fixative solution for fixation, and then stored at 4°C for later use. The kidney tissue was embedded in OCT, and frozen section was started after successful embedding. The slices were put in the diluted NAO solution for staining in the dark for 10 min. Finally, the stained slices were mounted with glycerin mounting tablets and observed under a microscope. The fluorescence intensity was calculated with image J software.

| Metabolomics analysis
2.11.1 | Feces and kidney tissue processing 160 mg feces were weighed and put into an EP tube, added 400 μl of double-distilled water, and homogenized thoroughly at low temperature. The samples were centrifuge for 15 min (4°C, 21,130 g), and the supernatant was absorbed. 400 μl methanol was added to the remaining residue and homogenized thoroughly at low temperature. The samples were centrifuge for 15 min (4°C, 21,130 g), and the supernatant was absorbed. 400 μl acetonitrile was added to the remaining residue and homogenized thoroughly at low temperature.
The samples were centrifuged for 15 min (4°C, 21,130 g), and the supernatant was absorbed. The above three supernatants were combined, centrifuged for 15 min (4°C, 24,320 g). The supernatant was taken for test.

| Mass spectrometry conditions
The mass spectrometer was operated in positive and negative ion mode with Dual electrospray ion source. The positive ion capillary voltage was 4,000 V, and the negative ion capillary voltage was 3,500 V. Atomizer pressure was 30 psig. Drying airflow was 10 L/ min. Drying gas temperature was 300°C. Fragmentor voltage was 175 V. Cone voltage was 65 V.

| RT-PCR analysis of Ampk, Sirt1, Pgc-1α, and Pparα in rat kidney tissues
TRIzol reagent was added to kidney tissue to extract total RNA. GADPH was used as an internal reference for fluorescence quantitative PCR amplification to detect the expression of Ampk, Sirt1, Pgc-1α, and Pparα related genes in rat kidney tissues of each group.
The amplification reaction conditions: 95°C for 10 min, 95°C for 15 s, 60°C for 60 s, 40 cycles in total. The primer sequence was showed in Table 1.

| Data analysis and processing
PLS-DA was used to generate molecular formulas of potential biomarkers. Compounds satisfying p < .05, FC > 2 and VIP > 1.0 were selected as biomarkers for preliminary screening. The M/Z value and retention time of the compound obtained from the analysis were combined with METLIN (http://www.metlin.scipps.edu) and HMDB (www.hmdb.ca) databases to identify the structure of the compound.
The identified compound name was input into MetaboAnalyst analysis platform for enrichment and topological analysis, thereby the related metabolic pathways were related screened for potential biomarkers.
All data are expressed as mean ± SD. Statistical significance of results was performed with one-way analysis of variance (ANOVA), using the statistical software SPASS17.0. p < .05 was considered statistically significant.

Gene name
Primer sequence

Fragment length (bp)
Annealing temperature (°C) TA B L E 1 Primer sequence

| Effects of XM on ALT, AST, UA, BUN, Cr, TG, TC, TBA, HDL-C, and LDL-C
Compared with the blank control group, ALT, AST, BUN, Cr, TG, TC, TBA, HDL-C, and LDL-C were not significantly affected by the treatment of XM (p > .05), and UA was significantly decreased (p < .01) ( Figure 1).

| Histopathological examination
Compared with the blank control group, the liver and kidney histopathology of the rats in the XMH, XMM, and XML groups were not abnormal ( Figure 2, Figure 3).

and SDH enzyme activity in rat kidney tissue
Compared with the control group, three different doses of XM all significantly increase the Na + -K + -ATPase, Ca 2+ -Mg 2+ -ATPase, and SDH activity in the kidney (p < .05, p < .01). All three doses of XM can significantly increase the ATP content of kidney tissue (p < .01) ( Figure 4).

| Mitochondrial oxygen consumption rate
Compared with the blank control group, the slope of the curve in the XMH group decreased significantly (p < .05), indicating that the XMH group may inhibit the metabolism of mitochondria. Compared with the blank control group, the slope of the curve of the XMM group and the XML group increased significantly (p < .05, p < .01), indicating that the mitochondrial metabolism rate increased at these concentration ( Figure 5).

| NAO fluorescent stain
Compared with the blank control group, the mitochondrial fluorescence intensity was significantly increased in the XMH, XMM, and XML groups (p < .01). It suggested that the number of kidney mitochondria increased after the intervention of XM at different concentrations ( Figure 6, Figure 7).

| Metabolomics analysis
To obtain as much compound information as possible, positive ion and negative ion modes were used for data collection and representative differential metabolites were obtained.

| Principal component analysis (PCA)
Metabolic profiles of feces and kidney tissue samples were obtained for each group. In the PCA score plot for feces, principal components showed a R 2 X = 0.526, Q 2 = 0.0996. As shown in Figure 8, there was clear separation among rats in the XMH, XMM, and XML. In the PCA score plot for kidney tissues, principal components showed a R 2 X = 0.59, Q 2 = 0.228. As shown in Figure 9, there was clear separation among rats in the XMH, XMM, and XML, but there were greater variation and more discrete aggregation, suggesting that the effect of XM on individual rats was highly variable. PCA could be used for a preliminary assessment of the metabolite profiles of rats in different F I G U R E 1 Effects of XM on serum liver, kidney function, and blood lipid in rats groups; however, owing to the variation within groups (and inability to highlight the differences between groups), further PLS-DA was needed.
3.6.2 | Partial least squares discrimination analysis (PLS-DA) of feces in different groups As shown in Figure 10a, an PLS-DA mode for feces in the XMH and Control was established in the positive mode. The samples of feces in the XMH and Control groups were completely spatially separated, with R 2 X = 0.513, R 2 Y = 0.997, Q 2 = 0.904, indicating that the mode had good predictive ability. As shown in Figure 11a, an PLS-DA mode for feces in the XMH and Control was established in the negative mode. The samples of feces in the XMH and Control were completely cating that the mode had good predictive ability.
As shown in Figure 10c, an PLS-DA mode for feces in the XMM and Control was established in the positive mode. Samples of feces in the XMM and Control groups were completely spatially separated, with R 2 X = 0.495, R 2 Y = 0.998, Q 2 = 0.921, indicating that the mode had good predictive ability. As shown in Figure 11c, an F I G U R E 2 Effects of XM on kidney histopathology in rats

PLS-DA mode for feces in the XMM and Control was established
in the negative mode. Samples of feces in the XMM and Control groups were completely spatially separated, with R 2 X = 0.494, As shown in Figure 10e, an PLS-DA mode for feces in the XML and Control was established in the positive mode. Samples of feces in the XML and Control groups were completely spatially separated, with R 2 X = 0.506, R 2 Y = 0.994, Q 2 = 0.883, indicating that the mode had good predictive ability. As shown in Figure 11e, an PLS-DA mode for feces in the XML and Control was established in the negative mode. Samples of feces in the XML and Control groups were completely spatially separated, with R 2 X = 0.531, R 2 Y = 0.994, and Q 2 gradually declined, indicated that the mode was highly robust and there was no overfitting.

| PLS-DA of kidney tissues in different groups
As shown in Figure 12a, an PLS-DA mode for kidney tissues in the XMH and Control was established in the positive mode. The samples of kidney tissues in the XMH and Control groups were completely indicating that the mode had good predictive ability. As shown in and Q 2 gradually declined, indicated that the mode was highly robust and there was no overfitting.

| RT-PCR
The above results showed that high, medium, and low doses of XM could promote kidney energy metabolism in rats, so we chose one of the dose groups (chose medium dose of XM) to carry out the experimental verification of its upstream mRNA related to energy metabolism. RT-PCR results showed that the relative expression levels of Ampk, Sirt1, Pgc-1α, and Pparα in XM group were significantly higher than those in the blank control group (p < .05, p < .01) ( Figure 15).

| Effects of different doses of XM on liver and kidney function in rats
When the high dose of XM extract is 15 g/kg and the low dose is 5 g/ kg, continuous administration for 60 days would cause certain adverse reactions to the physiological and biochemical functions of the liver (Chen, 2011). It was found that when the dosage was 100 times of the clinical dosage, the long-term administration for 3 months showed no obvious toxicity to the liver and kidney function (Xiang et al., 2006;Zhang et al., 2005). When the alcohol extract of XM was administered continuously for 30 days at a dose of 120 g/kg, there

| Effects of different doses of XM on the activities of ATPase, SDH enzyme, and ATP content in rats
ATPase exists on the cell membrane of tissue cells and organelles. It is a kind of protease on biological membrane, which plays an important role in material transmission, energy conversion, information transmission, etc. (Sudar et al., 2008). Na + -K + -ATPase is a complex membrane protein, which can use the energy produced by ATP hydrolysis to transport three Na + ions out of the cell and transfer two K + ions into the cell at the same time. This enzyme produces a lift on the cell membrane to maintain the resting potential of the cell (Ma et al., 2019). The Na + ion gradient drives many transport processes through cotransportation (such as glucose cotransporter), exchangers (Na + /Ca 2+ exchanger), and drives amino acids and vitamins into cells (Lingrel, 2010). It was found that when the activity of Na + -K + -ATPase decreased, the amount of Na + ions transported out of the cell decreased, which would lead to intracellular Na + overload, and then activate the Na + -Ca 2+ exchange protein on the membrane, At the same time, it would also lead to the release of calcium ions in mitochondria, which would lead to intracellular Ca 2+ overload, and the decrease in Mg 2+ activity will not only reduce the content of Mg 2+ in cells, but also reduce the activities of other enzymes related to energy metabolism and inhibit energy generation (Bao et al., 2004). Therefore, when the activities of Na + -K + -ATPase and Ca 2+ -Mg 2+ -ATPase are inhibited, it not only affects the hydrolysis of ATP, but also leads to the obstacle of intracellular ion transport, aggravating the obstacle of energy supply and application (Zhai et al., 2016).
ATP is the energy material directly used by the body, and its content  (Acevedo et al., 2013), converting succinic acid to fumarate, dehydrogenating FDAH, and then oxidizing FDA transmitter to generate energy (Wang, Jia, et al., 2020). The decrease in SDH activity will inhibit TCA cycle and oxidative phosphorylation, lead to mitochondrial dysfunction, and ultimately affect the efficiency of energy synthesis (Ekekcioglu et al., 1999). This study results showed that the activities of Na + -K + -ATPase, Ca 2+ -Mg 2+ -ATPase, and SDH were increased in high, medium, and low doses of XM groups, which indicated that XM could promote the activity of ATPase, increase energy consumption, and increase energy production by strengthening TCA cycle, thus promoting energy metabolism. The content of ATP increased in high, F I G U R E 1 3 PLS-DA analysis and permutation test of kidney tissues in the negative mode. (a) PLS-DA score plot of XMH and Control groups, (b) permutation test of XMH and Control groups, (c) PLS-DA score plot of XMM and Control groups, (d) permutation test of XMM and Control groups, (e) PLS-DA score plot of XML and Control groups, (f) permutation test of XML and Control groups medium, and low dose groups, which indicated that in the experimental conditions, the energy production of rats may be greater than energy consumption.

| Effects of different doses of XM on mitochondrial metabolic rate and mitochondrial number in rats
Mitochondria are the main sites for ATP synthesis, and more than 80% of the energy required for life activities comes from mitochondria (Liu et al., 1988). It is found that low dose aconite can promote the metabolism of mitochondria, and when the dose reaches a certain concentration, the metabolism of mitochondria will be inhibited (Zheng, 2015), indicating that the drug dose may affect the meta-

| Analysis of metabolic pathway
4.4.1 | TCA cycle, butanoate metabolism, propanoate metabolism, alanine, aspartate, and glutamate metabolism In this experimental condition, the high, medium, and low doses of XM groups were enriched the TCA cycle, butanoate, propanoate metabolism, alanine, aspartate, and glutamate metabolism, and the involved differential metabolism was succinic acid in which the content was increased. TCA cycle is an important pathway of aerobic Combined with the biochemical results of succinate dehydrogenase activity, it is speculated that XM may promote kidney energy synthesis by increasing succinate in TCA cycle pathway.

| Retinol metabolism
Retinol is an important cofactor in activating mitochondrial protein kinase C (PKC) (Kim & Hammerling, 2020), which activates PKC through redox. After the activation of PKC, it can increase the production of acetyl CoA, then stimulates PDH (pyruvate dehydrogenase) complex (Patel & Korotchinka, 2006;Patel & Roche, 1990), and then increase the utilization rate of pyruvate (Acin-Perez et al., 2010), promoting oxidative phosphorylation in mitochondria, resulting in increased oxygen consumption and ATP synthesis in mitochondria. In addition, retinol can be used as the carrier of mitochondrial electron, which can accelerate the electron transfer after binding with PKC, and then promote the process of oxidative phosphorylation (Hammerling, 2016). In the conditions of this experiment, the high, medium, and low doses of XM groups were enriched in the retinol metabolic pathway, and the related differential metabolite was retinaldehyde in which the content was increased.
Retinol can be converted to retinaldehyde after oxidation, which indicates that retinol may be oxidized to retinaldehyde in the process of activating mitochondrial protein kinase or acting as mitochondrial electron transport carrier.  (Higashi et al., 2015;Wang et al., 2011;Xu et al., 2015). In healthy rats, absorption and oxidation of branched chain amino acids occur in many different tissues, Once it enters cells, BCAA can be stored in amino acid pools, integrated into proteins, or sent to mitochondria for oxidation, and can also be used for the synthesis of ketones and glucose (Neinast et al., 2019 and NH 3 under the action of adenylate deaminase (Liu & Mao, 1999).
On the one hand, IMP can synthesize AMP again after obtaining amino group, which is also a part of purine nucleotide cycle. On the other hand, IMP can decompose into xanthine under the action of hypoxanthine nucleotide dehydrogenase and finally produce uric acid to be excreted out of the body . In this experimental condition, both medium dose and low dose of XM could affect purine metabolism pathway, and their differential metabolites were xanthosine and AMP, respectively. Xanthosine is derived from the oxidative decomposition of xanthine nucleotides. AMP is not only a synthesis product of purine nucleotides, but also a product of its oxidative decomposition. In addition, AMP can obtain highenergy phosphoric acid groups through oxidative phosphorylation to generate ATP. In this experiment, the content of xanthosine nucleoside decreased, and the content of AMP increased. It is speculated that after XM intervention, it can promote the circulation of purine nucleotides, reduce the oxidation decomposition of IMP into xanthine, and increase IMP to resynthesize AMP.

| Pentose phosphate metabolism
It is reported (Yao et al., 2020) (Ha et al., 2016;Herzig & Shaw, 2018;Lee & Kim, 2018;Shaw et al., 2004). PGC-1α can not only increase energy consumption, but also increase the biogenesis and respiration rate of mitochondria, absorb, and use substrates to produce energy (Lehman et al., 2000;Stpierre et al., 2003;Wu et al., 1999). SIRT1 is a nicotinamide adenine dinucleotide (NAD + )-dependent deacetylase, which can sense the energy metabolism state of living cells in tissues and affect the energy metabolism of tissues (Zhang et al., 2009). SIRT1, AMPK, and PGC-1α can interact to form a network that can sense energy changes (Wen et al., 2016). AMPK can increase the activity of SIRT1 by increasing the level of NAD + , activate the deacetylation of SIRT1 downstream proteins (such as PGC-1α), and increase the activity of PGC-1α transcription, thereby regulating energy metabolism and mitochondrial synthesis (Chau et al., 2010;Scarpulla, 2011;Tang, 2016). AMPK can regulate the expression of downstream target molecule PPARα, forming AMPK/PPARα signaling pathway, which plays a very important role in maintaining energy metabolism (Wang et al., 2019). The results of this experiment showed that XM can increase the expression of SIRT1, AMPK, PGC-1α, and PPARα mRNA, indicating that XM may affect the body's energy metabolism in the following three ways: (a) XM may increase NAD + level by activating AMPK and then activate SIRT1. SIRT1 can catalyze the acetylation of PGC-1α, promote the biosynthesis of mitochondria, thereby affecting energy metabolism; (b) XM may directly activate PGC-1α through AMPK, promote the synthesis of mitochondria, and increase the body's energy metabolism; (c) XM may promote energy metabolism through AMPK/PPARα signaling and then affect glycolipid metabolism.

| CON CLUS ION
In conclusion, XM can enhance the kidney tissue energy metabolism. It may increase energy metabolism by changing the rate of mitochondrial metabolism and the number of mitochondria. And these effects may be mediated by TCA cycle metabolism, butanoate, propanoate metabolism, alanine, aspartate, and glutamate metabolism, retinol metabolism, purine metabolism, pentose phosphate metabolism, aminoacyl-tRNA biosynthesis, valine, leucine, and isoleucine metabolism pathways, as well as by increasing expression of upstream genes Ampk, Sirt1, Pparα, and Pgc-1α mRNA.

This study was supported by Jiangxi University of Traditional
Chinese Medicine 1050 Youth Talent Project (5142001007)

CO N FLI C T S O F I NTE R E S T
The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article.

E TH I C A L A PPROVA L
The study was approved by the Experimental Animal Ethics Sub-

Committee of the Academic Committee of Jiangxi University of
Traditional Chinese Medicine and complies with the animal research guidelines of the China Ethics Committee (JZLLSC2019-0082).

DATA AVA I L A B I L I T Y S TAT E M E N T
All datasets presented in this study are included in the article.