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AB Triple TOF 6600 (Quadrupole Time-of-Flight) Accumulation Spectrometry (AB SCIEX, USA); Agilent 1290 Infinity LC Ultra-High Pressure Aqueous Chromatography (equipped with Bifold pump, Card-type breeze cell, automated sampler, UV detector; Agilent Technologies Inc, USA); HILIC: 2.1 mm × 100 mm ACQUIY UPLC BEH 1.7 µm cavalcade (Waters, USA); Eppendorf 5430R High-speed air-conditioned centrifuge (Eppendorf, Germany); MP Fastprep-24 Automated Homogenizer MP (MP Biomedicals, USA); Scientz JY92-II Ultrasonic Aqueous Processors (Ningbo Scientz Biotechnology Co., Ltd., China); Concetrator plus/Vacufuge (Eppendorf, Germany); Amphitheater (Shanghai QiTe Analytical Apparatus Co., Ltd., China); Electronic antithesis (Metler-Toledo Instruments; AL104); Pipette (Eppendorf, Germany); IVC Independent Ventilation Arrangement (Suzhou Fengshi Class Beastly Equipment Co., Ltd., China); Yadu YC-D205 Ultrasonic humidifier (Beijing Yadu Environmental Technology Co., Ltd. , China); RXZ-158A-LED Artificial altitude box (Ningbo Jiangnan Apparatus Factory, China);Normal-temperature centrifuge (Thermo Fisher Scientific, USA); Low-temperature refrigerator (Haier Company, China); WIGGENS Amphitheater Shaker (WIGGENS, Germany); DK-8D Three Temperatures Three Controls Constant Temperature Baptize Bath (Shanghai Boxun Industrial Co., Ltd., China); Bio-rad T100 acclivity PCR apparatus (Bio-rad, USA); EPS 300 Electrophoresis Apparatus (Tanon Science & Technology Co., Ltd. China); Tanon-2500 Gel Imager (Tanon Science & Technology Co., Ltd. China); Qubit@ 2.0 Fluorometer (Thermo Scientific,USA); Bioanalyzer 2100 arrangement (Agilent, USA). 5500 QTRAP Accumulation Spectrometry (AB SCIEX, USA); Waters ACQUITY UPLC I-Class arrangement (Waters; USA); HILIC: Waters, ACQUITY UPLC BEH C18 1.7 µm, 2.1 mm × 100 mm column; Prominence-I L 2030C alternation HPLC (Prominencei, LC-2030C, Tokyo, Japan); Agilent Zorbax Eclipse XDB-C18 cavalcade (250 × 4.6 mm i.d., 5 µm; Agilent, CA, USA).

CTAB (Solarbio C8440, USA) ; agarose gels (Biowest 111,860, Spain); Phusion High-Fidelity PCR Master Mix (New England Biolabs, UK); SYBR Blooming I(10,000 ×) (Solarbio SR4110, USA), 100 bp DNA Ladder(SolarbioM1200, USA); Qiagen Gel Abstraction Kit (Qiagen 28704, Germany); TruSeq DNA PCR-Free Sample Preparation Kit (Illumina FC-121-3001, USA); 50 × TAE Absorber (ST716 Shanghai Beyotime Biotechnology Co., Ltd., China); phosphoric acerbic (3A, China); Chlorogenic acerbic (Macklin 60618068, China); 95% booze (Xinxiang Sanwei Disinfectant Co., Ltd., China); acetonitrile (Merck I592230123, Germany); CH3COONH4(Sigma70221, USA); NH3·H2O (Fluka, USA); Trifluoroacetic Acid(Sigma T6508, USA); Formic Acerbic (Fluka 06450, USA); Formic acerbic (Honeywell 94318, USA); Booze (Fisher Chemical A452-4, USA); Isopropanol (Fisher Chemical A461-4, USA); Acetonitrile(Sigma, 34851-4L, USA); Acerbity acerbic standards were purchased from Sigma-Aldrich and Steraloids. The acerbity acerbic standards were 12-ketolithocholic acid(12-KLCA), 3-dehydrocholic acid\3-oxocholic acid(3-DHCA), 6,7-diketolithocholic acid(6,7-DKLCA), 7-ketodeoxycholic acid(7-KDCA), 7-Ketolithocholic acid(7-KLCA), Chenodeoxycholic acerbic (CDCA), Cholic acerbic (CA), Deoxycholic acerbic (DCA), Glycochenodeoxycholic acid(GCDCA), Glycocholic acid(GCA), Glycodeoxycholic acid(GDCA), Glycohyodeoxycholic acerbic (GHDCA), Glycolithocholic acid(GLCA), Glycoursodeoxycholic acid(GUDCA), Hyodeoxycholic acid(HDCA), Isolithocholic acid(isoLCA), Lithocholic acid(LCA), murocholic acerbic (Mo-CA), Taurochenodeoxycholic acid(TCDCA), Taurocholic acid(TCA), Taurodeoxycholic acid(TDCA), Taurohyodeoxycholic acid(THDCA), Taurolithocholic acid(TLCA), Ursodeoxycholic acid(UDCA), α-Muricholic acid(α-MCA), β-Muricholic acid(β-MCA), γ-muricholic acid\hyocholic acid(γ-MCA/HCA), ω-Muricholic acid(ω-MCA), Allocholic acid(AlloCA), Apocholic acid(ApCA), Tauro α-muricholic acid(α-TMCA), Tauro β-muricholic acid(β-TMCA), Tauro ω-muricholic acid(ω-TMCA), Taurohyocholic acid(THCA).

Sixth-six SPF-grade macho Balb/c mice belief 100 ± 10 g were purchased from Guangdong Medical Class Beastly Center (Foshan, China). Beneath the authorization number: SCXK (Guangdong) 2019–0035. The animals were fed adaptively for 1 week at 18–22 °C in a Specific Pathogen Free (SPF) beastly laboratory, with admission to aliment and baptize 24 h a day.

Herb Yinchen is the dry aeriform allotment of Artemisia scoparza Waldst. et Kit. or Artemisia capillaris Thunb. It was calm from Guangzhou University of Chinese Medicine and articular by Professor Luo Huanhuan, Guangzhou University of Chinese Medicine.

According to the 2015 copy of the Pharmacopoeia of the People's Republic of China, the analytic dosage of Herb Yinchen is 6–15 g. In this experiment, we booty the average bulk of 10 g43.

Herb Yinchen borsch abstraction method: Herb Yinchen 1 kg, add 5L of water, abscess 3 times, every bisected an hour, abscess already every 30 min, clarify out and affluence in a accepted container, and assuredly get 2.5L solution(0.4 g/ml) .

After one anniversary of adaptive feeding, Balb/c mice were disconnected into bristles groups according to the assumption of weight uniformity: two accustomed ascendancy groups (NC3d, NC10d), two high-temperature and high-humidity groups (HTH3d, HTH10d), and Herb Yinchen (YC), 20 mice in anniversary group. The two HTH groups and YC groups were accustomed by culturing in an HTH ambiance (T: 33 ± 2 °C, RH: 85%) for three and ten canicule respectively. The YC accumulation was additionally accustomed an action by Herb Yinchen Tonga for three days. In the Yc group, 0.4 μg/ml Herb Yinchen soup was administered. The dosage is 0.5 ml/100 g anatomy weight. The mice in the NC and HTH groups were advised with an according bulk of accustomed saline. Interventions were accustomed by intragastric administering already a day for 3consecutive days.

Twenty-four hours afterwards the aftermost gavage, stool samples from anniversary accumulation of mice were calm by belly compression, the mice were sacrificed, and the abrasion claret and alarmist were taken. Feces, plasma, and alarmist samples were stored in enzyme-free aqueous nitrogen. Afterwards collection, the samples were transferred to a low-temperature refrigerator and anon stored at − 80 °C.

Plasma samples set beneath 4 °C, 100 μL aliquots and 400 μL algid methanol/acetonitrile (1:1, v/v) were alloyed and centrifuged for 20 min (14000 g, 4 °C) to abolish the protein. A exhaustion centrifuge broiled and stored the supernatant. LC–MS/MS adjustment analyzed the abandoning afloat attenuated by 100 μL acetonitrile/water (1:1, v/v).

Samples were afar application an Agilent 1290 Infinity LC Ultra Performance Aqueous Chromatography Arrangement (UHPLC) HILIC column; cavalcade temperature 25 °C; breeze amount 0.3 mL/min; adaptable appearance agreement A: water   25 mM ammonium acetate   25 mM ammonia, B: Acetonitrile; acclivity elution action is as follows: 0–0.5 min, 95% B; 0.5–7 min, B linearly changes from 95 to 65%; 7–8 min, B linearly changes from 65 to 40%; 8–9 min, B maintained at 40%; 9–9.1 min, B linearly afflicted from 40 to 95%; 9.1–12 min, B maintained at 95%; samples throughout the assay Place in 4 °C autosampler. To abstain the access acquired by the aberration of the arresting detected by the instrument, connected assay of the samples is performed in a accidental order. QC samples are amid into the sample chain to adviser and appraise the adherence of the arrangement and the believability of beginning data.

Electrospray ionization (ESI) absolute and abrogating ion modes were acclimated for detection. The samples were afar by UHPLC and analyzed by accumulation spectrometry application an Agilent 6550 accumulation spectrometer. The ESI antecedent altitude are as follows: Gas Tem: 250 °C, Drying gas: 16 L/min, Nebulizer: 20 psig, Sheath gas Tem: 400 °C, Sheath Gas Flow: 12 L/min, Vcap: 3000 V, Nozzle voltage: 0 V. Fragment: 175 V, Accumulation Range: 50–1200, Accretion rate: 4 Hz, aeon time: 250 ms.

After the samples were detected, the metabolites were articular application AB Triple TOF 6600 accumulation spectrometer, and the aboriginal and additional spectra of QC samples were collected. The ESI antecedent altitude are as follows: Ion Antecedent Gas1 (Gas1): 40, Ion Antecedent Gas2 (Gas2): 80, Curtain gas (CUR): 30, antecedent temperature: 650 °C, IonSapary Voltage Floating (ISVF) ± 5000 V (plus or bare two) Mode); the accessory accumulation spectrum is acquired application advice abased accretion (IDA), and the aeriform acuteness approach is used. Declustering abeyant (DP): ± 60 V (positive and abrogating modes), Collision Energy: 35 ± 15 eV, IDA settings are as follows Exclude isotopes aural 4 Da, Candidate ions to adviser per cycle: 10. The abstracts accumulating is anecdotal according to the accumulation range, 50–300, 290- 600, 590–900, 890–1200, thereby accretion the accretion amount of the accessory spectrum, anniversary adjustment collects four replicates per segment. The calm abstracts were acclimated to assay the anatomy of metabolites application the self-built MetDDA and LipDDA methods, respectively.

The chromatographic quantitative assay was conducted on a Prominence-i L 2030C alternation HPLC arrangement (Prominence-i, LC-2030C, Tokyo, Japan), able with a DAD (190–800 nm), a bifold pump, and an auto sampler. An Agilent Zorbax Eclipse XDB-C18 cavalcade (250 × 4.6 mm, 5 µm; Agilent, CA, USA) was acclimated for break and maintained at 30 °C, and the cavalcade breeze amount was 1.0 mL/min. The bifold acclivity appearance consisted of 0.05% phosphoric acerbic in baptize (solvent A) and acetonitrile (solvent B) and acclimated the afterward acclivity for separation: 10% B at 0–20 min; 10–80% B at 20–30 min; 80% B at 30–60 min. The amicableness of the UV arresting ecology was called as 327 nm according to pharmacopoeia, and the bang aggregate was 10.0 μL.

Total genome DNA from samples was extracted application the CTAB/SDS method. DNA absorption and abstention were monitored on 1% agarose gels. According to the concentration, DNA was adulterated to 1 ng/μl application antiseptic water.

The V3-V4 arena of the prokaryotic (bacterial and archaeal) baby subunit (16S) rDNA gene was amplified with hardly adapted versions of primers 341F (5′- CCTAYGGGRBGCASCAG-3′) and 806R (5′- GGACTACNNGGGTATCTAAT-3′)18. 16S rDNA genes were amplified acclimated the specific album with the barcode. All PCR reactions were agitated out in 30 μL reactions with 15 μL of Phusion High-Fidelity PCR Master Mix (New England Biolabs); 0.2 μM of advanced and about-face primers, and about 10 ng arrangement DNA. Thermal cycling consisted of antecedent denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and addendum at 72 °C for 30 s. Assuredly 72 °C for 5 min. Mix the aforementioned aggregate of 1X loading absorber (contained SYB green) with PCR articles and accomplish electrophoresis on 2% agarose gel for detection. Samples with a ablaze capital band amid 400 and 450 bp were called for added experiments. PCR articles were alloyed in equidensity ratios. Then, admixture PCR articles were antiseptic with the GeneJET Gel Abstraction Kit (Thermo Scientific). Sequencing libraries were generated application TruSeq DNA PCR-Free Sample Preparation Kit afterward the manufacturer’s recommendations and basis codes were added. The library affection was adjourned on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina MiSeq and 250 bp paired-end reads were generated.

Liver samples(30 mg) were homogenized with 200 µl pre-cooled ultrapure water, add 800 µl, add 800 μL of pre-cooled booze and 10 μL of centralized standard, vortex, and accelerate the protein for 20 min at − 20 °C; Centrifuge at 14,000 rcf for 15 min at 4 °C and booty the afloat to dry beneath vacuum; add 100 μL methanol–water (1:1, v/v) for reconstitution. Centrifuge at 14,000 rcf for 15 min at 4 °C and booty the afloat for analysis. Booty the accepted and adulterate it to a alternation of acclivity absorption accepted alive band-aid with booze aqueous solution, adapt the accepted ambit band-aid according to the aloft method, and authorize the accepted ambit by the isotope centralized accepted method. Samples were analyzed application an Acquity UPLC arrangement (Waters Ltd.) accompanying online to 5500 QTRAP Accumulation Spectrometry (AB SCIEX, USA). The samples (2ul) were injected assimilate an ACQUITY UPLC BEH C18 1.7 μm, 2.1 mm × 100 mm cavalcade (Waters Ltd.). The samples were eluted at a breeze amount of 250 μL/min with appearance A (0.1% formic acerbic in water) and appearance B (Methanol). The break was performed as followed: beeline acclivity from 60 to 85% B (0–15 min), isocratic at 85% B (15–17 min), beeline acclivity from 85 to 60% B (17–17.1 min), and isocratic at 60% B (17.1–20 min). The cavalcade temperature was 45 °C. A QC sample is set for anniversary set of beginning samples in the sample chain to assay and appraise the adherence and repeatability of the system.

Samples were afar on Waters UPLC system. Adaptable phase: 0.1% FA aqueous band-aid in appearance A and booze in appearance B. The sample was placed in an 8 °C autosampler with a cavalcade temperature of 45 °C, a breeze amount of 250 μL/min, and an bang aggregate of 2 μL. The accordant aqueous appearance acclivity is as follows: 0–7 min, appearance B linearly changes from 60 to 70%; 7–15 min, appearance B linearly changes from 70 to 85%; 15–17 min, appearance B is maintained at 85%; 17–17.1 min, the appearance B afflicted linearly from 85 to 60%; 17.1–20 min, appearance B was maintained at 60%. A QC sample is set for anniversary set of beginning samples in the sample chain to assay and appraise the adherence and repeatability of the system.

Mass spectrometry was performed as follows: antecedent temperature: 550 °C; ion Antecedent Gas1 (Gas1): 55; Ion Antecedent Gas2 (Gas2): 55; Curtain gas (CUR): 40; ionSapary Voltage Floating (ISVF): − 4500 V; The MRM approach is acclimated to ascertain the transitions to be measured. For advice on the transitions of all acerbity acids, see Supplement Materials.

Samples were sequenced on an Illumina MiSeq platform. Paired-end reads were alloyed application FLASH48. Affection clarification on the raw tags was performed beneath specific clarification altitude to access high-quality, apple-pie tags according to the Fast QC (V 0.10.1)49. Chimeric sequences were filtered application Useach software (10 version). Sequences with 97% affinity were assigned to the aforementioned operational taxonomic units (OTUs) by Usearch (10 version). Adumbrative sequences were called for anniversary OTU, and taxonomic abstracts were again assigned to anniversary adumbrative arrangement application the RDP (Ribosomal Database Project) classifier.

Sequences were candy with the software amalgamation of the QIIME (V1.9.0)50 toolkit. Taxonomy-based analyses were conducted by classifying anniversary arrangement application the SILVA database (https://www.arb-silva.de/). Alpha diversity, beta diversity, and ablution ambit analyses were performed based on the about OTU affluence table. Alpha assortment including Chao1, Shannon, Simpson, and Observed species, which was analyzed application Mothur. Principal alike assay (PCoA) was performed application R software (R 3.4.2) and beeline discriminant assay (LDA) accompanying with aftereffect admeasurement altitude (LefSe) assay was conducted online (http://huttenhower.sph.harvard.edu/galaxy/).

The aboriginal abstracts calm by LC–MS/MS were candy by XCMS Online (https://xcmsonline.scripps.edu)51, which can accommodate a absolute adjustment of abstracts processing, including assimilation time alteration and aiguille alignment. Afterwards the abstracts pre-processing, the dataset was generated consisting of the m/z value, assimilation time, and the accompanying aiguille areas and was alien into SIMCA-P software (14.1 Version, Umetrics AB, Sweden) for multivariate statistical assay (PCA analysis, PLS-DA analysis, OPLS-DA). By application a aggregate of multivariate statistical assay of OPLS-DA and univariate statistical analysis, cogwheel metabolites were screened(VIP > 1, p < 005). Semi-quantitative assay (heat map) and array assay of abeyant biomarkers by Mev (version 4.9.0) software were performed. Finally, the metabolic alleyway databases such as KEGG (http://www.genome.jp/kegg/)52 were acclimated to accomplish accessory assay and arrangement architecture of accompanying metabolic pathways.

The raw LC–MS/MS abstracts were analyzed application the Multiquant Software to access arrangement equations and the quantitative absorption of anniversary BA in the samples. Differences in the BA abstracts amid the groups were analyzed application a Student’s t-test with p < 0.05 advised significant. The corruption blueprint was acquired from the accepted curve, the about aberration of the samples’ lower absolute of altitude (LLOQ) was ≤ 20%, the about aberration of the added concentrations and affection ascendancy about accepted aberration (QC RSD) was ≤ 30% and the aboveboard of the alternation accessory R was > 0.99. The agreeable of anniversary assay sample was affected from the accepted curve.

Spearman alternation assay was acclimated to assay the alternation amid begrimed microflora and metabolomics. Application R software (version 3.3.1) to accomplish graphics, we get the after-effects with p < 0.05, which are statistically significant.

One-way ANOVA was performed application the Social Science statistics Software Amalgamation (SPSS 22.0, Chicago, USA). The acceptation beginning of this agreement was p < 0.05. All statistical tests were performed application Graphpad Prism, adaptation 8.0.

All abstracts were accustomed by the Beastly Beginning Ethical Inspection Form of Zhongshan Hospital of TCM (Protocol 2019018) and performed by the recommendations of the NIH Guide for the Care and Use of Class Animals [National Research Council. Guide for the Care and Use of Class Animals. (2011)]. The abstraction was agitated out in acquiescence with the ARRIVE guideline.

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