Dendrobium huoshanense Improves Lipid Metabolism Disorder by Restoring Gut Flora and Metabolites in Mice Fed a High-Fat Diet

,


Introduction
With changes in lifestyle and aging, lipid metabolism disorders (LMDs) were becoming increasingly prevalent annually.LMD was a major risk factor for metabolic diseases, such as hyperlipidemia, obesity, and atherosclerosis [1] and seriously threatened human health.Terefore, it was crucial to seek efective therapeutic targets to prevent and treat LMD to promote and improve quality of life.
Te gut microbiota was commonly known as an important "metabolic organ," which consisted trillions of microorganisms in the human and animal guts and had a very strong metabolic ability.In recent years, numerous studies had shown that homeostatic gut fora contributed to normal host metabolism.However, disorders in the structure and function of the gut fora, which was induced by HFD [2,3], could promote the onset and progression of LMD [4,5].Studies had shown that the proportion of benefcial gut fora, such as Bifdobacteria and Lactobacillus, was markedly reduced in hyperlipidemic mice fed a HFD, and the loss of these bacteria was strongly correlated with higher lipid levels [6].Bifdobacterium species alleviated LMD and intestinal fora dysbiosis [7].B. pseudolongum had been shown to interact with jackfruit seed-derived resistant starch, signifcantly reducing serum lipid levels and hepatic damage in mice [8].B. pseudolongum treatment signifcantly ameliorated intestinal fora dysbiosis and reduced plasma triacylglycerol (TG) levels, as well as reducing body mass and visceral fat [9].B. animalis subsp.A6 had been shown to inhibit the development of obesity by increasing fatty acid oxidation in adipose tissue [10].In addition, B. pseudolongum, B. animalis, and Lactobacillus salivarius could promote the fecal excretion of bile acids and regulate bile acid-mediated lipid metabolism by producing bile salt hydrolase (BSH), which hydrolyzed conjugated bile acids into free bile acids that were difcult to reabsorb in the intestinal tract [11][12][13].
DH was harvested from the Da-bie Mountains of China, with a high concentration of plants found in Huoshan County, Anhui Province, China.Te plant, known locally as "Mihu," was registered in the 2020 edition of "Pharmacopeia of the People's Republic of China" (the Pharmacopeia) and approved for use as a food material [14].Te main active substances in DH included favonoids, alkaloids, and polysaccharides, which played important therapeutic roles and polysaccharides was an index of the quality control of DH in the Pharmacopeia [14].DH benefted the stomach, promoted fuid production, nourished yin, and promoted clearance [14].Modern pharmacological studies had confrmed that DH had various activities, such as immunoregulation [15], gastric mucosal protection [16], and protection against liver injury [17], which aided in preventing and curing diseases, strengthening the body, and prolonging life.DH polysaccharides had been shown to decrease the levels of total cholesterol (TC), TG, and malondialdehyde and protect against high-cholesterol dietinduced atherosclerosis [18].DH treatment also alleviated ethanol-induced liver injury in HFD mice by restoring perturbed metabolic pathways [19].DH polysaccharides could improve the composition of the gut fora, promoted the proliferation of Lactobacillus and Owenweeksia, and enhanced the function of the intestinal barrier by upregulating the expression of tight junction proteins, mucin-2, beta-defensins, and cytokines [20].After 18 weeks, DH signifcantly lowered serum TG levels and inhibited the progression of atherosclerosis induced by a HFD in LDLR −/− mice [21].In hyperlipidemic rats, the administration of DH also resulted in a signifcant reduction in serum TG levels, as well as TC, and low-density lipoprotein cholesterol (LDL-C) levels and increased high-density lipoprotein cholesterol (HDL-C) levels [22].Although previous studies had shown that the administration of DH had benefcial efects on lipid metabolism and intestinal fora, the mechanism of action of DH in the relationship between lipid metabolism and changes in the intestinal fora remained unclear.
In this study, we evaluated the efects of DH on LMD amelioration in HFD mice.Furthermore, we analyzed whether DH could modulate the structure of the gut fora and its fecal metabolites, as well as their relationship with lipid metabolism to provide theoretical evidence for the development of DH to prevent LMD.

Materials. DH extract was supplied by Jiuxianzun
Huoshan Dendrobium Co., Ltd., and all ELISA/biochemical kits used in the experiments were purchased from Multisciences Biotech Co., Ltd./Nanjing Jiancheng Bioengineering Institute.

HPLC-MS/MS Analysis of DH.
Te ingredients of the DH extract were analyzed using a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS).Samples for LC-MS/MS detection were prepared as previously described [23].LC-MS/MS analyses were performed as previously reported [24,25].

Establishment of the LMD Model and Treatments.
Tirty-fve male six-week-old mice (weighing 20 ± 2 g) were purchased from Gempharmatech Co., Ltd.(Jiangsu, China).Te mice were housed in the laboratory under the following conditions: temperature: 25 ± 1 °C; relative humidity: 55 ± 10%; and 12 h dark: 12 h light.Te mice were fed a standard diet for one week and had free access to drinking water.One week later, the mice were randomized into fve treatment groups (n = 7) and fed diferent diets.Te control group (CTR) was received normal chow diet (NCD; XT93; 10% calories from fat) with free access to water.Te model group (HFD) was fed a high-fat diet (HFD; XTHF60; 60% calories from fat), with free access to water.Te positive control group (SIM) was fed the HFD plus daily administration (by gavage) of 2.25 mg/kg/day simvastatin, according to previous studies [26,27].Te low-dose DH group was administered HFD plus a daily administration of 300 mg/kg/ day DH (daily LD-DH), and the high-dose DH group was fed the HFD plus daily administration of DH by gavage of 600 mg/kg/day (HD-DH), according to previous studies [28,29].All animals were fed for 14 weeks.Normal chow diet and high-fat diet were purchased from Xietong Pharmaceutical Bio-engineering Co., Ltd.(Jiangsu, China).

Collection of Feces and Tissue Samples.
At the end of the experiment, all mice were starved for 12 h, during which time only water was provided.Fresh fecal samples were obtained from each mouse for gut fora and fecal metabolomic analyses, and all mice were anesthetized and sacrifced.Blood samples were obtained from the orbit, maintained for 30 min at 25 ± 1 °C before centrifugation (6,000g for 10 min) at 4 °C to obtain the blood serum, and stored in a refrigerator at −80 °C.Te livers were removed from each of the sacrifced mice and cleaned with phosphate-bufered saline (PBS).Ten, one part was stored in the refrigerator at −80 °C and the other parts were stored in 4% polyformaldehyde for further analysis.

Biochemical Analysis.
Te serum TC, TG, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were analyzed using commercially available kits.Te frst part of the collected liver tissue sample stored 2 Journal of Food Biochemistry at −80 °C was cleaned with deionized water, and liver tissue (0.5 g) from each mouse was ground with physiological saline using a homogenizer to prepare the liver homogenate, which was then centrifuged (2,000g for 10 min) to obtain the supernatant.Te levels of TC, TG, ALT, and AST in the liver were analyzed using commercially available biochemical kits.All analyses were performed according to the manufacturer's instructions.
2.6.Oil Red and HE Staining of Liver.Next, hepatic histopathologic examinations were conducted by oil red O and HE staining of liver.Oil red O and HE staining of liver was performed as previously described method [30].Image-Pro Plus 6.0 software was used for quantitative analysis.

Analysis of Intestinal Microfora.
Te fresh fecal samples that had been collected from each mouse before sacrifce were prepared for 16S rRNA sequencing.Briefy, the V3 and V4 hypervariable regions of the 16S rRNA were amplifed (forward primer: ACTCCTACGGGAGGCAGCA; reverse primer: GGACTACHVGGGTWTCTAAT), and amplifcation and quality control of the raw data were performed on the Illumina platform.Sequence denoising or clustering was performed using the DADA2 method, in which data were only deduplicated or clustered based on 100% similarity.Taxonomic composition analysis was performed and visualized using QIIME2.PCoA and NMDS analyses were performed and visualized using the R software.Raw sequencing data were saved in the FASTQ format.
2.8.Metabolic Analysis.Fecal samples, each weighing 100 mg, were placed in 2-mL grinding tubes, each containing 50 mg of zirconia beads.Next, 200 mL of ultrapure water was placed into the grinding tube and homogenized in a grinder (Servicebio KZIII-F) at the maximum speed for 30 s to obtain the fecal homogenate.Ten, 400 µL of methanol : acetonitrile (volumetric ratio of 1 : 1) was added to the homogenized samples.Te solution was vortexed for 30 s, followed by extraction for 30 min in an ultrasonic extractor (KQ-600DE; Kunshan Ultrasonic Instruments Co. Ltd., Kunshan, China) at a low temperature.After the homogenized samples were centrifuged at 13,500g for 20 min, the supernatants were transferred to EP tubes and dried using a nitrogen blower.Finally, the residue was reconstituted with 200 µL of methanol : acetonitrile (1 : 1) and centrifuged at 13,500g for 20.Lastly, a 3-µL aliquot was injected into the UPLC-QTOF/MS (Termo Q Exactive Orbitrap) for analysis [25].
2.9.Statistical Analysis.Diferences between two or more groups were assessed using Student's t-test and one-way analysis of variance (ANOVA), respectively.Data were presented as the mean ± standard deviation (SD).Statistical analyses were performed using SPSS version 22.0.Te fgures were processed using GraphPad Prism software (version 9.0).Except for special instructions, p < 0.05 and p < 0.01 were considered statistically signifcant.
Correlations between the gut fora, fecal metabolites, and lipid metabolic indicators were assessed using Pearson's correlation analysis.

Results
3.1.Qualitative Analysis of DH.Te content of polysaccharides in the DH extract was 426.84 mg/g, which was measured by the phenol-sulfuric acid method, according to "Pharmacopeia of the People's Republic of China" (2020).Te chemical ingredients of the DH extract were analyzed using LC-MS.Hundreds of chemical ingredients were found in the DH extract according to positive and negative ion modes, most of which exhibited sharp symmetrical peaks, indicating good separation.Twenty-one major ingredients were identifed in the DH extract according to the major fragments in the MS spectra, as shown in Table 1.

DH Administration Improved Dyslipidemia in HFD Mice.
Changes in the body weight and serum lipid levels were measured after 14 weeks of feeding (Figure 1).Compared to the CTR group, the body weight and TG and TC levels in the serum of the HFD group were markedly increased, indicating that the hyperlipidemic mouse model was successfully induced by HFD.At the same time, mice treated with diferent doses of DH or SIM exhibited lower body weights and TG and TC levels than HFD mice, indicating that DH efectively improved dyslipidemia in HFD mice.A regulatory efect on liver function was also exhibited in SIM-treated mice.

DH Treatment Reversed Gut Microbial Dysbiosis in HFD
Mice.To study the efects on intestinal fora in HFDinduced hyperlipidemia mice given DH, 16S rRNA sequencing on mouse feces was performed.Tere was no signifcant diference in species richness of gut microbiota among diferent groups of mice (Figure 3    6 Journal of Food Biochemistry hamsteri were markedly higher in the HFD group than in the CTR group.In contrast, compared to the HFD group, B. pseudolongum and B. animalis were markedly higher in the HD-DH group, whereas the abundances of Helicobacter hepaticus and Rothia mucilaginosa were lower (Figures 3(d) and 3(f )).Furthermore, a signifcant diference in the gut microbiota structure in mice was observed by beta diversity analysis based on PCoA and NMDS analysis and intergroup diferences between the HFD and CTR, HFD, and treatment groups (Figures 3(g)-3(j)).Te results indicated that the gut fora structure in the HFD group was signifcantly diferent from that in the CTR group.However, the community structure in the HD-DH group was markedly diferent from that in the HFD group, indicating that DH altered the gut microbiota structure in the HFD group.
In order to explore the metabolic regulation function of gut microbiota, PICRUSt2 software was used to predict the function of gut microbiota (Figure 4).Based on the comparison of the KEGG database, it was found that the main frst-level classifcation of functional pathways including 6 categories, namely, metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases.Among them, there were 28 metabolic pathways in the KEGG secondary-level classifcation of functional pathways, with higher abundance concentrated in metabolism, mainly including amino acid metabolism, lipid metabolism, carbohydrate metabolism, and metabolism of terpenoids and polyketides.Among them, the relative abundance of lipid metabolism in HD-DH was increased than in the HFD group, while amino acid metabolism in HD-DH was decreased than in the HFD group.

DH Signifcantly Altered the Profle of Fecal Metabolites from Intestinal
Microbiota in HFD Mice.Gut metabolites afected by the intestinal microbiota may alter host metabolism.Diferential fecal metabolites in mice were evaluated using nontargeted metabolic profle analysis (Figure 5).Score plots of PLS (Figures 5(a showed that the CTR, HFD, and HD-DH groups were signifcantly diferent from each other, suggesting that the levels of gut metabolites in the CTR group were changed markedly by a HFD, and that this elevation in metabolites could be reversed by DH.A total of 60 critical metabolites from feces showed signifcant changes among the three groups based on the threshold of VIP (>1.5) and p (<0.05) between the HFD and HD-DH groups (Figure 6(a)).Among these, 17 metabolites were signifcantly increased and two metabolites were markedly decreased in the HFD group than the results from the CTR group, which were all reversed by DH (Figure 6(b)), which is closely related to lipid metabolism.Tese metabolites were found to be mainly associated with bile secretion (deoxycholic acid and acetylcholine), steroid hormone biosynthesis (cholesterol sulfate), fatty acid biosynthesis (stearic acid), urine and nucleotide metabolism ((S)-ureidoglycine, guanosine, and xanthosine), arginine and proline metabolism (agmatine, D-ornithine, citrulline, guanidoacetic acid, and cis-4-hydroxy-D-proline), lysine degradation (5-aminopentanoic acid and glutaric acid), aminoacyl-tRNA biosynthesis (L-methionine and L-valine), glutathione metabolism (gamma-glutamylalanine), and arachidonic acid metabolism (20-HETE) (Figure 7).In conclusion, bile acid, lipid, and amino acid metabolism in the HFD group were altered by DH.

Mechanism of DH against Dyslipidemia Revealed by
Pearson's Correlation Analysis.Pearson's rank correlation analysis was used to analyze the correlations among the 22 altered fecal metabolites, biochemical parameters, and gut microbiota in the HFD and HD-DH groups.Te altered gut microbiota and biochemical parameters of species, such as B. pseudolongum and B. animalis, showed signifcant negative correlation with the TG, TC, ALT, and AST levels in the serum and liver.Desulfovibrio_C21_c20 and B. bifdum were signifcantly negatively correlated with the TC levels (Figure 8(a)).In addition, Rothia mucilaginosa, Helicobacter hepaticus, and Clostridium celatum were signifcantly positively correlated with the TG, TC, ALT, and AST levels in the serum or liver.Tese results suggest that the aforementioned intestinal bacteria, such as B. pseudolongum and B. animalis, may be critical for the benefcial efects of DH.Next, the correlations between the 22 altered metabolites and biochemical parameters were evaluated (Figure 8(b)).Te results showed that the metabolites stearic acid, xanthosine, deoxycholic acid, 20-HETE, and cholesterol sulfate were negatively correlated with these biochemical parameters.Te fecal metabolites except for the abovementioned 5 metabolites showed signifcantly positive correlation with these biochemical parameters.Correlations between gut fora species and fecal metabolites were also observed (Figure 8(c)).Te results showed that xanthosine was markedly positively correlated with B. pseudolongum, whereas stearic acid, deoxycholic acid, and cholesterol sulfate were signifcantly positively correlated with B. animalis.B. pseudolongum and B. animalis were markedly negatively correlated with cis-4hydroxy-D-proline, gamma-glutamylalanine, N-acetyl-Lglutamine, L-methionine, guanidoacetic acid, (S)-ureidoglycine, acetylcholine, 2-methylserine, agmatine, 5-hydroxypentanoic acid, L-valine, L-prolinamide, or citrilline.However, Rothia mucilaginosa or Clostridium celatum showed signifcant positive correlation with metabolites, such as L-methionine, guanidoacetic acid, D-ornithine, (S)-ureidoglycine, glycylleucine,

Discussion
In this study, not only were the levels of TC, TG, AST, and ALT reduced in the serum and liver, but the hepatic steatosis and lipid accumulation were increased in the liver of the HFD group, which was reversed by DH.Tese fndings suggested that DH could inhibit lipid disorders and restore liver function in the HFD group.
To elucidate the modulatory mechanism of DH in LMD, the structure of the mouse gut microbiota was explored using high-throughput 16S rRNA gene sequencing.Interestingly, diferent doses of DH signifcantly altered the structure of the intestinal fora and inhibited the structural dysbiosis of the gut fora in the HFD group.Furthermore, HD-DH upregulated the relative abundance of Allobaculum and Bifdobacterium at the genus level and B. pseudolongum and B. animalis at the species level.
B. animalis subsp.lactis BB-12 inhibited obesity by regulating intestinal fora in two phases in human foraassociated rats [31].In overweight and obese children, the body mass index and serum levels of TC and LDL-C were reported to decrease signifcantly after 12 weeks of treatment with supplementary probiotics, including B. animalis CP-9 in a double-blind, randomized, placebo-controlled trial [32].B. animalis attenuated the infammatory response and reduced lipid accumulation in mice with nonalcoholic fatty liver disease (NAFLD) To further explore the mechanism of DH in LMD, the metabolite profles of the intestinal microbiota were analyzed using fecal metabolomics.Te results showed that DH could alter the fecal metabolite profles of HFD mice.
Especially, DH signifcantly promoted excretion of fecal deoxycholic acid, stearic acid, and cholesterol sulfate.Te excretion levels of fecal metabolites, such as deoxycholic acid, xanthosine, and cholesterol sulfate promoted by DH, were negatively correlated with biochemical parameters.Desoxycholic acid, a secondary bile acid, was converted from primary intestinal bile acids by gut bacteria, such as Bifdobacterium spp.Bifdobacterium could lead to higher levels of deoxycholic acid [35].Evidence indicated that secondary bile acids, such as deoxycholic acid and lithocholic acid, were toxic bile acids associated with LMD and gastrointestinal diseases [36].Increasement of fecal excretion of deoxycholic acid, stearic acid, and cholesterol sulfate, could reduce hepatic cholesterol and decrease lipogenesis.B. animalis subsp.lactis F1-7 reduced the TG, TC, LDL-C, and HDL-C levels in the serum, and the TC and TG levels in the liver of HFD mice, while total bile acids were increased in feces, which improved hyperlipidemia in HFD mice by downregulating FXR [37].Tose results were consistent with our research that DH promoted proliferation of B. animalis and the excretion of fecal bile acid and improved lipid metabolism.
In addition, most amino acid metabolic pathways were enriched in HFD mice and DH supplementation suppressed the metabolic levels of these amino acids in the gut microbiota, which was consistent with the previous research [38].Te metabolite levels of amino acid metabolism (such as L-valine, cis-4-hydroxy-D-proline, and gamma-glutamylalanine) inhibited by DH positively correlated these biochemical parameters.Evidence suggested branchedchain amino acids including L-valine could cause insulin resistance and glucose metabolism disorder [39].HFD could induce disorders of lipid metabolism and amino acid metabolism.Sporisorium reilianum polysaccharide  signifcantly alleviated obesity via gut microbiota-related lipid metabolism and amino acid metabolism [40].Te active peptide of Walker ameliorated hyperlipidemia by altering amino acid metabolism [41].In addition, B. pseudolongum prevented liver injury in mice by regulating the gut microbiota composition and liver metabolitesrelated phenylalanine metabolism, and alanine, citrate cycle, and so on [42].Tese results were similar to our study.
In addition, B. pseudolongum and B. animalis had a positive correlation with deoxycholic acid, xanthosine, and cholesterol sulfate and had a negative correlation with these metabolites from amino acid metabolism, while Helicobacter hepaticus, Rothia mucilaginosa, and Clostridium celatum were in reverse.Helicobacter hepaticus, Rothia mucilaginosa, and Clostridium celatum might promote amino acid metabolism.Tamarind xyloglucan oligosaccharides  In this study, Pearson's correlation analysis showed that DH might directly enhance the excretion of deoxycholic acid, stearic acid, and cholesterol sulfate, as well as alter amino acid metabolism and other pathway metabolism, to lower the levels TG and TC and regulate lipid metabolism via the metabolic ability of the gut fora.

Conclusion
DH treatment was found to reduce lipid levels and improve liver function in HFD mice.DH most likely exerts an antihyperlipidemic efect by shaping the intestinal fora and its metabolites, which are closely related to lipid metabolism (Figure 9).Tus, this study provides evidence that DH, a traditional medicinal and edible herb, may be an important candidate for preventing and improving lipid metabolism by regulating the gut fora.In the future, the clinical efcacy of DH in improving lipid metabolism disorders will need to be evaluated for the development and utilization of DH.
Damage in HFD Mice.Liver function disorders were analyzed in the fve groups of mice.Increased hepatic steatosis and lipid deposition in the liver were observed in the HFD group than the CTR group (Figures 2(a)-2(d)).In contrast, DH treatment signifcantly decreased hepatic steatosis and lipid deposition (Figures2(a)-2(d)).In addition, the liver index of the HFD mice was signifcantly increased compared to the CTR group, while the liver index of mice treated with DH was signifcantly decreased (Figure2(e)).Compared to the CTR group, the HFD group had higher levels of serum AST and ALT, hepatic TC, TG, AST, and ALT, which were reversed by DH (Figures2(f )-2(k)).Tese results suggested that DH improved liver function damage induced by HFD.
(a)).At the phylum level, the relative abundances of Actinobacteria, Proteobacteria, TM7, Tenericutes, and Fusobacteria of HFD mice were decreased with DH treatment (Figure3(b)).In addition, the relative abundances of bacterial species at the genus and species levels in diferent treatment groups were Journal of Food Biochemistry compared.Te abundances of Allobaculum and Rikenella in the HFD group were markedly lower than the corresponding values in the CTR group.In contrast, Clostridiaceae clostridium, Helicobacter, and Odoribacter were more abundant in the HFD group.In the HD-DH treatment group, the abundances of Allobaculum and Bifdobacterium were markedly higher than those in the HFD group, whereas the abundances of Clostridiaceae clostridium and Streptococcus were lower (Figures3(c) and 3(e)).At the species level, the abundances of Clostridium celatum and Lactobacillus

Figure 1 :Figure 2 :
Figure 1: Administration of DH improved dyslipidemia in mice fed HFD.(a) Schematic diagram of DH treatment.(b) Body weight of mice at the end of experiment, and the levels of TG and TC in serum were tested using commercial biochemical kits.## p < 0.01and * * p < 0.01 vs. HFD or CTR groups.

Figure 3 :
Figure 3: Efects of DH on intestinal fora in HFD mice.(a) Te chao1 index for alpha diversity analysis.(b) Relative abundance of diferential specifc taxa at the phylum level.(c) Intestinal fora composition profle at the genus level.(d) Intestinal fora composition profle at the species level.(e) Relative abundance of specifc taxa at the genus level.(f ) Relative abundance of specifc taxa at the species level.(g) PCoA and (i) NMDS analysis for beta diversity analysis.(h) Adonis analysis for intergroup diferences.(j) Anosim analysis for intergroup diferences.

Figure 5 :
Figure 5: Te profle of fecal metabolites from gut microbiota in HFD mice was markedly changed by DH.(a) Positive PLS-DA score plot.(b) Positive OPLS-DA plot.(c) Negative PLS-DA score plot.(d) Negative OPLS-DA plot.(e) Te permutation test following PLS-DA model in positive mode.(f ) Te permutation test following OPLS-DA model in positive mode.(g) Te permutation test following PLS-DA model in negative mode.(h) Te permutation test following OPLS-DA model in negative mode.(i) Positive PCA score plot.(j) Negative PCA score plot.

Figure 7 :
Figure 7: Statistics on the number of diferentially expressed molecules in the KEGG pathway.

Figure 9 :
Figure 9: Efects of DH in the mice with lipid metabolism disorders.Te pink and green arrows correspond to the HFD and HD-DH groups, respectively.Te upward arrows indicate upregulation efects and the downward arrows represent downregulation efects.

Table 1 :
Ingredients identifed in the DH extract.