GCis formulas were prepared with Renshen (White Ginseng), Fuzi (Hei Shunpian), and Rou Congrong combination (1:1:1). The herbs were acquired from Sinopharm Group Beijing Huamiao Pharmaceutical Co., Ltd. (Beijing, China). The combined extraction liquid was soaked with 10-time distilled water for 30 min, then microboiled for 1 h twice, and mixed with the twice filtrate. Finally, the mixture was concentrated and dried to produce the GCis.

Male C57BL/6J mice (7–8 weeks old, 20–24 g, Experimental Animal Center, Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were housed in cages with a temperature of 22 ± 2°C and humidity of 40 ± 5%, under a 12 h light/dark cycle. Standard mouse chow and water were provided ad libitum. The mice adapted for 7 days and were randomly divided into four groups: Control, Model, GCis-L, and GCis-H (n = 15 per group).

After the 7-day habituation period, except for mice in the control group, the collagenase IV-S (St. Louis, MO, United States) was performed on all animals to produce ICH models. Following intraperitoneal injection of 40 mg/kg pentobarbital, mice were placed in a stereotaxic frame, shaved, and swabbed the incision area with 70% ethanol. A 30-gauge needle was inserted through a burr hole on the skull into the striatum (stereotaxic coordinates: 0.5 mm anterior to the bregma, 2.3 mm lateral to the midline, and 3.7 mm below the skull). ICH was induced by microinfusion pump-mediated injection of 0.03U collagenase type IV-S in 0.5 μL saline at a constant rate of 0.2 μL/min. The needle was slowly removed after an additional 5 min delay to prevent backflow. Body temperature was maintained at 37°C with a rectal probe and a heating blanket until the mice woke up.

Three days before ICH surgery, the mice in treatment groups were intragastrically administered with GCis once daily for 3 days: the GCis-L group received 1.3 g/kg/d, the GCis-H group received 3.9 g/kg/d, and the sham-operated and ICH model groups received distilled water via gavage at the dose of 10 ml/kg. On the day of surgery and after the operation, the mice continue to receive drugs or distilled water for 7 days.

On day 7, the mice were anesthetized intraperitoneally, the viscera (brain, lung, thymus, spleen, bone marrow, and ileum) were rapidly taken out and stored at a −80°C refrigerator, the viscera and body weights were calculated as an index of viscus, and brain edema was measured based on brain weight.

Examinations of neurological deficits of the experimental animals were performed 7 days after surgery. The neurological function was blindly scored on the wire hang test, beam walking test, and elevated body swing test (EBST) as previously described (Wu et al., 2009). The wire hang and beam walking tests were graded from 0 to 5 (0, the worst; 5, the best). Greater scores indicated better neurological functions. EBST was calculated as the percentage of right-biased swings (R) in the total swings recorded (20 times, sum of left- and right-biased swings), R/(L + R).

Twenty-four hours after the last administration, mice were anesthetized and the brain tissue was harvested quickly except for the olfactory bulb, cerebellum, and lower brainstem. Afterward, the brain was dissected into coronal slices (2 mm thick, n = 6). Generally, the slices were digitized and analyzed with ImageJ software, and the hemorrhagic injury volume was presented as a percentage (%) of (total hematoma volume/total brain volume) × 100.

Lung and ileum tissue were harvested and fixed in 4% (v/v) buffered paraformaldehyde, then embedded in paraffin, sectioned at 4 μm thickness, applied to glass slides, and stained with hematoxylin and eosin (H&E) solution. Afterward, the goblet cell was observed by Alcian blue/periodic acid-Schiff (AB/PAS) straining. HE and AB/PAS-stained images were acquired with an upright microscope in ×100 and ×200 magnification (Leica Microsystems, Wetzlar, Germany).

By using the EasyPure^® Genomic DNA Kit (Transgen, Beijing, China), total DNA was isolated from lung tissue. qPCR was conducted on samples to determine the relative amount of bacteria using published bacteria-specific primer sequences (Shim, 2016; Bronchard et al., 2004) for SYBR Green reagent (Bio-rad) on a CFX96Touch RT-PCR system (Bio-rad). The 16Sr RNA/Rpp30 value was calculated by the 2^−dCt method to indicate the bacterial load of lung tissue.

The lactulose/mannitol (L/M) test is widely used to evaluate intestinal permeability in humans and animals, including mice. Animals fasted for 8 h, gavaged with 0.5 ml lactulose and mannitol mixture solution (containing 60 mg/ml fructose and 40 mg/ml mannitol), and housed in metabolic cages individually. Urine samples were collected after 12 h. Lactulose and mannitol concentrations for each urine sample were quantified by UPLC/MS, as previously described (Gervasoni et al., 2018; Xu et al., 2015). Final data were presented as a ratio of lactulose and mannitol.

Intestinal mucus from the duodenum to terminal ileum was collected with a smooth pincette and transferred to an Eppendorf tube, homogenized in 0.5 ml cold PBS, and then centrifuged at 4°C at 3,000 r/min for 10 min. The supernatant was collected, and the content of SIgA was measured according to the manufacturer’s protocol. Then, the protein content of the supernatant was determined by the BCA method. The SIgA level was expressed as nanograms per milligram of total protein.

Ileum tissue samples were weighed to 30 mg, and 200 μL water MP homogenate was added, followed by vortexing for 60 s. Then, an 800 μL methanol acetonitrile solution (1:1, V/V) was added, followed by vortexing for 60 s , at low-temperature ultrasonic for 30 min, twice. Precipitated protein was placed at −20°C for 1 h, 14,000 rpm. Finally, after centrifuging at 4°C for 20 min, the supernatant was freeze-dried, and samples were stored at −80°C. While plasma samples were slowly dissolved at 4°C, 100 μL of each sample was taken, and 400 μL of pre-cooled methanol acetonitrile solution (1:1, V/V) was added to the plasma samples, followed by vortexing for 60 s, precipitation of protein, drying as above, and preservation of samples at −80°C.

Samples were analyzed by UPLC-Q-TOF/MS (Agilent 1290 Infinity LC/AB SCIEX). The peak area and retention time were extracted by Multiquant software, the retention time was corrected with neurotransmitter standard, and metabolites were identified. Information on all ion pairs of neurotransmitters is shown in Table 1.

Total RNA of ileum and bone marrow (n = 5) were extracted using TRIzol^® reagent (Invitrogen, Thermo Fisher Scientific Inc., USA). The integrity of the 28 and 18 s ribosomal RNA was determined on a 1% agarose gel to assess the degradation degree of RNA. While the concentration and purity of the isolated RNA were estimated by NanoDrop spectrophotometric measurements (ND-2000, NanoDrop, Thermo Fisher Scientific Inc.), the RNA integrity (RIN) was measured in the Agilent 2100 bioanalyzer (Agilent Technologies Inc., Germany). The RNA-Seq was performed at Applied Protein Technology (Shanghai, China). The directional cDNA libraries were prepared, the constructed library was qualified and quantified using the Qubit 2.0 fluorometer and the Agilent 2100 bioanalyzer, and the Illumina HiSeq platform was used for sequencing. The raw reads obtained from HiSeq sequencing are processed to obtain high-quality sequences (Clean Reads) by removing low-quality sequences and removing adaptor contamination. All subsequent analyses are based on Clean Reads. The gene expression level was measured in units of fragments per kilobase of transcript sequence per millions of base pairs sequenced (FPKM). p-value <0.05 and |log₂ foldchange|>1 were used for selecting differentially expressed genes (DEGs). The volcano plot and cluster heatmap were performed on the normalized gene expression data to visualize the resulting expression intensity values of the ileum transcripts (Xu et al., 2019).

In our study, the up-DEGs and down-DEGs in Mod vs. Sham and down- or upregulation in GCis vs. Mod were filtered out as reverse-regulated DEGs. The STRING tool (STRING; https://string-db.org/) was used to generate PPIs among the reverse-regulated DEGs. Then, Cytoscape was used to visualize the network. To explore gene function and regulatory networks of reverse-regulated DEGs, the enrichment analysis of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways mapping were conducted.

Brain, lung, and ileum tissues were homogenized in RNA lysis buffer. Total RNA was extracted using the Eastep total RNA extraction kit (Promega, Beijing). Equal amounts of RNA were reversely transcribed into cDNA with FastQuant RT kit (with gDNase) (Tiangen, Beijing) in accordance with the manufacturer’s instructions. Relative gene expression was determined using specific quantitative primers (see Table 1). Polymerase chain reactions were performed on a CFX96 touch system (Bio-Rad, United States) with SuperReal PreMix Plus (SYBR Green) kit (Tiangen, Beijing). The mRNA expression levels were normalized to GAPDH using the ^ΔΔCt relative to control groups.

For comparison of multiple groups, one-way analysis of variance (ANOVA) followed by the least-significant difference method was applied (GraphPad Prism 7 Software, Inc., La Jolla, CA, United States). p-value < 0.05 was considered statistically significant. Data were analyzed by SPSS 22.0 software (International Business Machines Corp, Armonk, NY, United States, RRID: SCR_019096). The achieved data were expressed as the mean ± SEM.

On day 7, the spleen, thymus index, white blood cell (WBC), and lymphocyte (LY%) were significantly reduced within the groups of the GCis formula in comparison with the control group (p < 0.01) (Figures 3A–D). After 1 week of treatments, the spleen, thymus index, and LY% of the GCis formula group (p < 0.01) were significantly increased than the model group, suggesting that the GCis formula can improve the peripheral immunity in the lung infection mice induced by ICH.

DEGs were screened out by p-value ≤ 0.05 and |log₂ foldchange|≥1. The volcano plot showed the relative changes in gene levels (Figures 3E,F). In the model vs. sham group, 855 DEGs were identified (552 upregulation and 303 downregulation). In the GCis formula vs. model group, 420 DEGs were identified (61 upregulation and 359 downregulation). A hierarchical cluster was used to obtain the sample clustering results (Figures 3G,H). There were significant differences in gene expression between the model and sham groups, GCis and model group, and high similarity in gene expression between the same group.

Based on 61 GCis formula DEGs, the GO and KEGG pathway enrichment was analyzed (Figures 3I,J). The enriched pathways were mainly related to immune response, including synthesis and release of immune cells, B-cell-mediated immunity, lymphocyte-mediated immunity, adaptive immune response based on somatic recombination of immune, and Toll-like receptor signaling pathway.