Six herbs of Danggui-Shaoyao San, Angelica sinensis (Oliv.) Diels (Umbelliferae), Paeonia lactiflora Pall. (Paeoniaceae), Poria cocos (Schw.) Wolf (Polyporaceae), Atractylodes macrocephala Koidz. (Compositae), Ligusticum chuanxiong Hort. (Umbelliferae) and Alisma orientalis (Sam.) Juzep. (Alismataceae) were purchased from the Pharmacy Room of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangzhou, China). LPS (lipopolysaccharide) (Cat No. L2630, sigma, St. Louis, MO, USA), murine IL-4 (Cat No. 96-214-14-20, PeproTech, Suzhou, China), Arg1 (Cat No. 16001-1-AP, Proteintech Group, Wuhan, China), and antibodies against OX42 (Cat No. ab1211), Trem2 (Cat No. ab86491), iNOS (Cat No. ab15323), Arg1 and GAPDH (Cat No. ab8245) were purchased from Abcam (Cambridge, UK).

The extraction of DSS was performed in the Laboratory of Science and Technology Innovation Center, Guangzhou University of Chinese Medicine. Angelica sinensis (Oliv.), Paeonia lactiflora Pall, Poria cocos (Schw.) Wolf, Atractylodes macrocephala Koidz., Ligusticum chuanxiong Hort. and Alisma orientalis (Sam.) Juzep. were mixed with a ratio of 3:16:4:4:8:8. The herbs were soaked in distilled water (1:8, w/v) for 1 h and extracted for 1 h. After the filtrate was collected, distilled water was added (1:6, w/v) for a second extraction. The two filtrates were then mixed and concentrated to 1.28 g/mL DSS extract at 60 ℃.

Analysis of the main active components of the DSS water extract was performed using HPLC-MS/MS in a previous study [26]. It was found that the extract included 78 main constituents. The concentration of albiflorin, paeoniflorin, benzoic acid, gallic acid, ferulic acid, Chuanxiong lactone Ⅰ, Chuanxiong lactone A were 1.76, 12.15, 0.68, 1.17, 0.29, 0.46 and 0.19 mg per gram extractum, and the highest content was paeoniflorin.

A total of 20 male C57BL/6 mice (seven months old) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China) and randomly divided into four groups (five mice/group), (1) normal group (0.9% saline, 10 mL/kg/day, i.g.), (2) high-dose DSS group (6.4 g/kg/day, i.g.), (3) mid-dose DSS group (4.8 g/kg/day, i.g.), (4) low-dose DSS group (3.2 g/kg/day, i.g.). The drug intervention period was continued for seven days. Serum samples were collected from the eyeball blood of mice 1 h after the last treatment administration. The serum samples were heat inactivated at 56 ℃ for 30 min, then divided and stored at –20 ℃ until use. The experiment was carried out in accordance with guidelines established by the National Institutes of Health of the United States for the care and use of laboratory animals and was approved by the Laboratory Animal Ethics Committee of the Guangzhou University of Chinese Medicine (Guangzhou, China).

BV2 microglia cells were obtained from the Science and Technology Innovation Center of Guangzhou University of Chinese Medicine. The cell line has been authenticated based on morphology and functional expression, and mycoplasma testing has been done. Cells were cultured at 37 °C in an atmosphere of 5% CO${}_2$ in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were seeded onto a 96-well plate at a density of 5 $\times$ 10${}^3$ cells per well. For cell subculture, BV2 cells were detached after reaching 90% confluency by using 0.25% Trypsin-EDTA. The BV2 cells underwent various analyses after three generations of passaging.

Antigen repair was performed with 0.01 M citrate buffer at 90 °C for 30 min. Subsequently, cells were saturated and permeabilized by 0.1% Triton X-100 (Solarbio, Beijing, China) and blocked in goat serum at room temperature. Samples were then incubated overnight at 4 °C with primary antibodies of anti-OX42, anti-iNOS, and anti-Arg1. They were then incubated with goat anti-mouse or goat anti-rabbit IgG at 37 °C for twenty minutes. Finally, the samples were stained with Alexa Fluor 488 anti-rabbit IgG or Alexa Fluor 594 anti-rabbit IgG at room temperature away from light for 1 h. Confocal microscopy (Serial No. 538623, Nikon 80i, Tokyo, Japan) was utilized to capture images. Positive cells were identified from three different perspectives in each sample.

The effect of DSS on the viability of BV2 cells was assessed by cell counting kit-8 (CCK8). The cells were plated into 96-well plates at a density of 2 $\times$ 10${}^5$ cells per well and cultured in medium overnight. Cells were then treated with 10% by volume with different doses of DSS drug-containing serum (0, 3.2, 4.8, and 6.4 g/kg/d) for 12, 24 or 48 h. Subsequently, 20 $\mu$L CCK8 solution ((Lot No. A311-01, Vazyme Biotech Co., Ltd., Nanjing, China) was added into the medium and it was incubated for 45 min. Absorbance was measured at 450 nm using a microplate reader (Serial No. 255927, BioTek, Winooski, VT, USA).

The capacity of BV2 cells to phagocytose A$\beta$ was assessed using flow cytometry. BV2 cells were treated with different concentrations of drug for 24 h. Then, the cells were incubated with 500 nM fluorescently labelled FAM-A$\beta$ for 2 h. After washing three times with cold PBS, samples were assessed by flow cytometry software (FlowJo, v10.6.2, Tree Star, Inc., Ashland, OR, USA).

BV2 cells were plated in six-well plates with fresh medium. Trem2 over-expression lentivirus, Trem2 siRNA, and non-targeting siRNA (control vector) transfection were performed respectively according to the manufacturer’s instructions by Lipofectamine 2000 reagent (Lot No. 2744064, Invitrogen, Carlsbad, CA, USA). BV2 cells were cultured in serum-free medium for 8 h then treated with different concentrations of DSS drug-containing serum (0, 3.2, 4.8, and 6.4 g/Kg/d) for 24 h.

The protein of BV2 cells was obtained by lysis with RIPA buffer. Bicinchoninic acid (BCA) assay was employed to quantify the protein concentration. Samples were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and blocked by 5% BSA for 1.5 h at room temperature. The membranes were then incubated overnight at 4 °C with the Trem2, Arg1, and iNOS antibodies specific for target proteins, and then incubated with the secondary antibodies for 1 h at 37 °C. Detection was performed using the Gel Imaging System (Serial No. 721BR19506, Bio-Rad, Hercules, CA, USA) and quantified by Image Lab 5.2.1 software (Bio-Rad, Hercules, CA, USA).

TRIzol reagent (Lot No. 93289, Sigma, St. Louis, MO, USA) was applied to extract total RNA from BV2 cells with different treatments in each group according to the instruction manual. The ratio of OD260 to OD280 of total RNA was measured by microspectrophotometer and the purity and concentration of RNA were calculated. Samples with a ratio between 1.8–2.0 met experimental requirements. cDNA templates were prepared by performing reverse transcription reactions according to the reverse transcription protocol of PrimeScript™ RT Master Mix (Lot No. RR036B, Takara, Beijing, China). Following $\times$8 dilution, samples were amplified by RT-qPCR (Serial No. 275007042, ABI, CA, USA). Each sample was repeated three times and the average value employed to calculate the relative content of the product. GAPDH was used as the housekeeping gene and the primer list is given in Table 1. The relative mRNA concentrations were measured by E = 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ and the key threshold cycle (CT) value of each reaction was examined.

All data were analyzed by the software package Statistic Package for Social Science (SPSS, v.26.0, Armonk, NY, USA) and data were expressed as mean $\pm$ standard error. Comparison between two groups was analyzed by t-tests, while multiple group comparisons were performed by one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test. A p-value 0.05 was considered to indicate a significant difference.

BV2 cells were treated either with LPS at a concentration of 100 ng/mL or IL-4 at a concentration of 20 ng/mL in DMEM for 24 h to induce either the M1 or M2 phenotype. As shown in Fig. 1A, the results of immunofluorescence double-labeling revealed that activated BV2 microglia cells with large and round cellular bodies, disappeared cellular projections and amoeboid shape were observed after either LPS or IL-4 treatment. LPS up-regulated the M1 microglia biomarker iNOS, which indicated an alteration of microglia polarization toward M1 status. While IL-4 up-regulated the M2 microglia polarization marker Arg1, which promoted microglia polarization toward a M2 phenotype (Fig. 1B). Moreover, LPS enhanced the protein expression of iNOS and decreased the expression of Trem2 in the M1 polarization state. In contrast, IL-4 remarkably increased the protein expression of Arg1 and Trem2 in the M2 polarization state (Fig. 1C,D). Taken together, Trem2 was highly expressed in the M2 polarization phenotype and poorly expressed in the M1 polarization phenotype.

Fig. 1.

Trem2 expression in different polarization states of BV2 cells. (A,B) Cell morphology changes and immunofluorescence of iNOS (red)/Arg1 (red) and double immunofluorescence labeling with OX-42 (green). (C,D) Western blot analysis of iNOS, Arg1, and Trem2 protein expression in control, LPS (100 ng/mL), and the IL-4 (20 ng/mL) group. GAPDH served as the loading control. Scale bar: 10 $\mu$m, **p 0.01 compared with the control group.

To further confirm the effect of Trem2 on BV2 microglia activation, the Trem2 over-expression lentiviral vector and Trem2 siRNA were used to transfect BV2 cells. The expression of microglia activation marker proteins was then evaluated by Western blot. Results showed that Trem2 over-expression significantly reduced the protein expression level of iNOS, whereas increased expression of Arg1 in BV2 cells (Fig. 2A,B). In contrast, Trem2 silencing obviously enhanced iNOS expression and inhibited Arg1 production. Results further revealed that Trem2 induced a shift of M1 microglia towards the M2 phenotype (Fig. 2C,D). The effect of Trem2 on the A$\beta$ scavenging capacity of BV2 cells was then assessed by flow cytometry. This showed that Trem2 silencing remarkably suppressed A$\beta$ clearance in BV2 cells, which further confirmed that Trem2 enhanced the A$\beta$-scavenging capacity of BV2 cells (Fig. 2E,F).

Fig. 2.

Effect of Trem2 on microglia polarization and A$\beta$-scavenging capacity of BV2 cells. (A,B) Western blot analysis of Trem2, Arg1, and iNOS in Trem2 over-expression. GAPDH served as loading control. (C,D) Western blot analysis of Trem2, Arg1, and iNOS in Trem2-siRNA. (E) Flow cytometry was used to determine the phagocytosis capacity of A$\beta$ by BV2 microglia. The M1 range representative FITC positive event. (F) Comparison of mean FITC fluorescence intensity among the different groups. **p 0.01, compared with the control-vector group.

To investigate the effect of DSS on BV2 cell viability, BV2 cells were cultured with different concentrations of DSS-containing serum (3.2 g/kg/d, 4.8 g/kg/d, and 6.4 g/kg/d) for different durations (12 h, 24 h and 48 h). As shown in Fig. 3A, compared with the normal group, cell viability significantly increased in the low, medium, and high dose DSS-containing serum groups. These findings showed that DSS-containing serum increased BV2 cell viability in a dose- and time-dependent manner.

Fig. 3.

DSS enhanced cell viability in a dose-dependent manner and increased Trem2 expression, which promoted the conversion of microglial cells to the M2 state. (A) Cell viability was detected by CCK8 Assay. (B,C) Western blot analysis of Trem2 in control and DSS treated groups. GAPDH served as control. (D,E) mRNA level of the M2 biomarker Arg1 and IL-10 treated in four different groups for 48 h using RT-qPCR technology. *p 0.05, **p 0.01, compared with the normal group.

To verify the effect of DSS on Trem2 expression in BV2 cells, Trem2 protein expression levels were examined in BV2 cells treated with different concentrations of DSS-containing serum for 12 h, 24 h, and 48 h. As shown in Fig. 3B,C, the results of Western blot showed that DSS enhanced Trem2 expression in BV2 cells and these effects were dose and time dependent. Next, at the 48 h time point, the effect of DSS on the M2-associated mRNA expression levels of Arg1 and IL-10 were determined and it was found that DSS-containing serum dramatically increased the expression of Arg1 and IL-10 mRNA (Fig. 3D,E). Alternatively, it was observed that DSS-containing serum significantly reduced the M1-associated mRNA levels of iNOS and TNF-$\alpha$ (Fig. 4D,E). These findings showed that DSS inhibited the alteration of microglia polarization toward the M1 phenotype and induced microglia into the M2 anti-inflammatory phenotype via increasing the expression of Trem2.

Fig. 4.

DSS enhances the phagocytosis capacity of microglial cells in a dose-dependent manner. (A) Fluorescence intensity of cells was measured by flow cytometry to determine the phagocytosis capacity of A$\beta$ by BV2 microglia treated in normal group and low/middle/high DSS concentration groups. The M1 range representative FITC positive event. (B) Comparison of mean FITC fluorescence intensity among the above four groups. (C–F) mRNA level of iNOS and pro-inflammatory cytokines IL-1$\beta$, TNF-$\alpha$, and IL-6 using RT-qPCR technology in different groups. *p 0.05, **p 0.01, compared with the normal group.

The effect of DSS on the A$\beta$-scavenging capacity of BV2 cells treated with a corresponding concentration of DSS-containing serum for 48 h was analyzed by flow cytometry. Results Showed that DSS increased the A$\beta$-scavenging capacity of BV2 cells in a dose-dependent manner (Fig. 4A,B). Furthermore, it was found that DSS remarkably decreased mRNA expression of IL-1$\beta$ and IL-6 (Fig. 4E,F). This indicated that DSS administration significantly decreased the amount of A$\beta$ deposits and mRNA expression of A$\beta$-mediated inflammatory factors in BV2 cells.

Different concentrations of DSS-containing serum were used to treat BV2 cells transfected with siRNA-Trem2 and it was found that DSS increases the A$\beta$-scavenging capacity when the BV2 cells were transfected with siRNA-Trem2 (Fig. 5A,B). Importantly, it was found that DSS up-regulated the expression levels of Trem2 (Fig. 6A,B). Moreover, it was observed that high doses of DSS-containing serum significantly increases Arg1 and IL-10 levels in BV2 cells transfected with siRNA-Trem2 (Fig. 6C,D). These results strongly suggested that Trem2 silencing inhibited DSS-mediated microglia polarization toward a M2 phenotype and A$\beta$-scavenging capacity of BV2 cells.

Fig. 5.

DSS did not recover A$\beta$-scavenging capacity in BV2 cells after silencing gene Trem2. (A) Different concentrations of DSS serum were used in normal BV2 cells and BV2 cells to silence Trem2 by siRNA and flow cytometry was used to detect the phagocytic function on A$\beta$. The M1 range representative of a FITC positive event. (B) Comparison of mean FITC fluorescence intensity among the above six groups. ##p 0.01, compared with control-vector group, *p 0.05, **p 0.01, compared with the Trem2-siRNA group.

Fig. 6.

Trem2 silencing inhibited DSS-mediated microglia polarization toward M2 phenotype and A$\beta$-scavenging capacity of BV2 cells. (A,B) Western blot analysis of Trem2 in BV2 cells treated with different concentrations of DSS-containing serum after transfection with siRNA-Trem2. (C,D) mRNA level of the M2 phenotype biomarker Arg1 and IL-10 of the different groups using RT-qPCR. ##p 0.01, compared with control-vector group, **p 0.01, compared with the Trem2-siRNA group.