Reagents

Glycyrrhizin (purity > 99%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd (China). LPS was purchased from Sigma (St. Louis, MO, USA). ELISA kits for TNF-α, IL-1β, IL-6, IL-8 and IL-12 were purchased from Nanjing Institute of Bioengineering (China). BCA protein quantitative kit and SYBR Green PCR kit were purchased from Smer Fell Science and Technology Co., Ltd. (Shanghai, China). Reverse transcription kit was purchased from Ferments. RIPA, Acrylamide(29: 1), Tris-HCl pH = 8.8 electrophoretic buffer, Tris-HCl pH = 6.8 electrophoretic buffer, SDS, TEMED, PBS phosphate buffer and DEPC were purchased from JRDUN Biotechnology (Shanghai) Co., Ltd. Alexa Fluor 488 Labeled goat anti-rabbit IgG (H + L), DAPI, Goat anti-rabbit-HRP secondary sera, Donkey anti-goat-HRP secondary sera and Goat anti-mouse-HRP secondary sera were purchased from Biyuntian co., Ltd. Occludin(rabbit)and ZO-1 (rabbit) were purchased from Bioss. RMPI-1640 was purchased from Hyclone.

Cell grouping and processing

GREC were acquired from two Boer goats (6 weeks age). Two Boer goats were provided by the Experimental Animal Center of Huazhong Agricultural University (Wuhan, China). Goats were breed in separate cages (24 ± 1 °C, 65% relative humidity) and were free to ingest foods and water. LPS-induced GREC were formulated by adding 5 μm LPS into GREC primary cell culture for 2 h, after inducing, the cells were divided into five groups with the treatment of glycyrrhizin of 0, 60, 90, 120 and 150 μm for 24 h, then cells or the supernatant was collected for further analysis.

Detection of structural integrity of LPS-induced GREC

The fresh LPS-induced GREC with the treatment of glycyrrhizin were washed by PBS for one time, fixed by glutaraldehyde, osmic acid, each fixing followed washing by PBS for three times, dehydrated with different gradients of ethanol 30%, 50%, 70%, 90%, 95% and 100%, each time lasted for 10–15 min, replaced with propylene epoxide for two times, each time last for 10 min, permeated by the ratio of propylene oxide to epon resinand 2:1 and 1:2 for 2 h respectively, whole epon resinand for the night, polymerizates at 37 ℃ for 12 h, 45 ℃ for 24 h and 60 ℃ for 48 h, and finally, the samples were made into cell slice and observed by Transmission Electron Microscope (JEM-2100 F JEOL).

Autophagy examination

First fixed with 2.5% glutaraldehyde for 2 h. Fix with 2% osmium acid for 2 h. Dehydrate with 50%, 70%, 80% and 90% ethanol gradients for 15 min each, and then dehydrate with 100% ethanol 3 times for 20 min each. Replace with acetone 2 times for 15 min each. Impregnate with acetone: embedding agent = 1:2 for 4 h; impregnate with pure embedding agent 2 times for 4 h each. Place the samples in the embedding plate containing pure embedding agent. The plates were polymerized at 65℃ for more than 48 h each. The embedding head was trimmed into a trapezoidal shape and the surface area of the sample was less than 0.2 mm × 0.2 mm. Slicer Sects. 50–70 nm. 3% uranyl acetate-lead citrate double staining. Observe the formation of autophagic structures, mainly including the morphology of autophagosomes and autophagic lysosomes.

Inflammatory mediators assay

The LPS-induced GREC and the treatment of glycyrrhizin mentioned above were centrifuged at 3000 r/min for 20 min and the supernatant was collected. The level of TNF-α, IL-1β, IL-6, IL-8 and IL-12 were measured using ELISA kits according to the manufacture’s instruction (Biolegend, Camino Santa Fe, CA, USA).

The number of Occludin and ZO-1

The LPS-induced GREC (5 × 105 cells/well) were seeded in 24 well plates. Glycyrrhizin of different concentration was subsequently treated in culture medium for 24 h, cells were washed with PBS for three times, each time last for three minutes. The wells were fixed by formaldehyde, permeabilized by Triton X-100, blocked by BSA for 1 h and incubated with primary antibody and secondary antibody, each process with washing with PBS for three times, and finally, the wells were sealed and taken photos by fluorescence microscope.

Western blot analysis

The LPS-induced GREC with the treatment of adding glycyrrhizin were disrupted sufficiently at 4 °C, centrifuged at 12 000 g for 10 min and the supernatant was collected. Total proteins were extracted with the protein extraction reagent and the concentration was measured with the BCA protein reagent. The protein was separated by SDS/PAGE and electrophoretic ally transferred onto a semi-dry rotating membrane. The membrane was blocked in NaCl/Tris containing 5% nonfat dry milk at room temperature for 1 h, and incubated with a primary antibody at 4 ℃ for 12 h and then incubated with a secondary antibody at 37 ℃ for 1 h. Finally, the blots were developed with the ECL Plus Western Blotting Detection System (GE Healthcare, Chalfont St Giles, UK).

Primers design and synthesis

According to the mRNA sequences of NF-κB, TNF-α, IL-1β, IL-6, IL-8 and IL-12 of goats in GenBank, the upstream and downstream primers of mRNA were designed by Primer 5.0 [19, 20]. Primers were synthesized by JRDUN Biotechnology (Shanghai) Co., Ltd., as shown in (Table 1).

Table 1 The primer sequence of targeted genes RNA extraction and reverse transcription

The total RNA was extracted by using Trizol (Invitek, 1596 − 206). The first strand of cDNA was synthesized according to the instructions of Dalian Bao Biological engineering Co., Ltd and was synthesized according to the reverse transcription kit (Fermentas, #K1622) [21]. The reaction system was showed in (Table 2). The reverse transcription was carried out at 37 ℃ for 60 min, 85 ℃ for 5 min and 4 ℃ for 5 min.

Table 2 The system of reverse transcription reaction Real-time PCR

Then the cDNA was subjected to PCR amplification after autoclaving and diluting to 10 μm with distilled water with SYBR Green PCR kits (Thermo, #K0223). The reaction system was shown in (Table 3) and response procedure was that: 95 ℃, 10 min; (95 ℃, 15 s; 60 ℃, 45 s) ×40 cycles; 95 ℃, 15 s; 60 ℃, 1 min; 95 ℃, 15 s; 60 ℃, 15 s.

Table 3 The system of PCR reaction The mRNA expression of NF-κB, TNF-α, IL-1β, IL-6, IL-8 and IL-12

After Real-time PCR amplification, the relative mRNA expression of NF-κB, TNF-α, IL-1β, IL-6, IL-8 and IL-12 were measured with standard mRNA curve.

Statistical analysis

All results were expressed as the means ± SEM with SPSS 16.0. Comparisons between groups were performed with ANOVA followed by Duncan test. Differences were considered to be significant at P < 0.05 or *, extremely significant at P < 0.01 or **.