Elisas

We used commercially available enzyme-linked immunosorbent assay kits (ELISAs from Cloud-Clone and Cusabio, Wuhan, China) to quantify the amount of GFAP, MBP, Iba1, Col I, Col IV, CS, LAM, FN, HA, Col VI, and LUM (see Table 3) in samples of protein extracts from human brain tissue (see Figure 3B). We isolated protein out of the brain samples using 300 μl Triton buffer containing 0.2% protease and 0.2% phosphatase inhibitors. The brain solutions were incubated on ice for 30 min. After centrifuging at 13,000 rpm and 4°C for 5 min, we diluted the solutions to 1 ml with Triton buffer to ensure that we could perform all ELISAs. Subsequently, we decanted the supernatant and measured the protein concentration with a Bradford assay. The analysis was performed using a microplate spectrophotometer (ELISA-reader) at a wavelength of 450 and 405 nm for measuring the absorbance. The received optical density results for the standard dilutions were then utilized to create standard curves using the software MARS Data Analysis from BMG Labtech and the 4- or 5-parameter best fit. By comparing with the standard series and the determined values for antigen concentration (protein concentration), we calculated the content of the protein in ng/total protein in mg in each sample.

Blood samples were centrifuged at 3500 rpm and 4 °C for 15 min, and the serum was stored at − 80 °C until further analyses. Tissue pre-treatment methods including ileum and liver were the same as above. Expressions for fibroblast growth factor 15 (Fgf15), LPS, claudin-1 and ZO-1 were measured by enzyme-linked immunosorbent assays (ELISAs) according to the manufacturer’s instructions (Cloud-Clone Corp.). The levels of inflammatory factors in serum were measured using a V-PLEX Pro inflammatory panel following the manufacturer’s instructions.

The amounts of RNases in serum were determined using commercially available ELISAs (all Cloud-Clone Corp., Houston, TX, USA) according to the manufacturer’s instructions. Briefly, a total of 100 µL of standards or samples were added to the wells followed by the addition of 100 µL detection reagent A. Serum was diluted for the measurements of RNase levels (RNase 1 [1:1000]; RNase 3 [1:200]; RNase 7 [1:2]). The plate was incubated for 1 h at 37 °C. After three wash steps with the supplied wash solution, 100 µL detection reagent B was added to each well. The plate was incubated for 30 min at 37 °C. 90 µL substrate solution was added to each well before the reaction was halted with stop solution after 10 min. The absorbance was measured at 450 nm on a microplate reader (Sunrise Tecan, Crailsheim, Germany). The lower detection limit of the immunoassay for RNase 1, 3 and 7 was 30 pg/mL, 16 pg/mL and 0.58 ng/mL, respectively.