Anti-inflammatory activities of Guang-Pheretima extract in lipopolysaccharide-stimulated RAW 264.7 murine macrophages

P. aspergillum was collected from March to May 2014 in Guangzhou, Guangdong Province, China, and identified by Professor Wei Li from the Department of Chinese Medicine Identification in Guangzhou University of Chinese Medicine. P. aspergillum was processed to medicinal grade according to the method of traditional Chinese medicine processing [26], and kept at − 20 °C for later use. A voucher specimen (GPMTCM-2017050301) was deposited in the Guangdong Provincial Museum of Traditional Chinese Medicine. When used, Guang-Pheretima was minced, soaked in 20-times volume of double distilled water (DDW) for 30 min, and then boiled in a reflux condensation system for 1 h. Subsequently, Guang-Pheretima was boiled with fresh DDW again, and the Guang-Pheretima decoction (GPD) was pooled together and filtered through 8 layers of medical gauze. A part of the filtered decoction was lyophilized. The powder was dissolved in Roswell Park Memorial Institution (RPMI) 1640 culture medium to a stock concentration of 1 g/mL, and the stock was filtered through a 0.22 μm filter membrane and stored at − 20 °C. For the remaining filtered decoction, absolute ethanol was added with stirring, up to 80% total volume. The supernatant was then subjected to two rounds of vacuum distillation at 50 °C to remove ethanol. The final protein-free Guang-Pheretima decoction (PF-GPD) was verified by biuret and lyophilized, and the stability and similarity were investigated in our previous study [27]. Although the compositions of PF-GPD are not clearly revealed, we ensured that L-Leucine, L-Lysine and L-valine are existed in PF-GPD by LC/MS/MS, which are 3 key components specified in PHERETIMA in the Pharmacopoeia of People’s Republic of China [21]. The lyophilized PF-GPD powder was dissolved in RPMI 1640 to a stock concentration of 1 g/mL, and the stock was filtered through a 0.22 μm filter membrane and stored at − 20 °C for subsequent experiments.

The RAW 264.7 cell line was purchased from the American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/mL streptomycin, and maintained at 37 °C in an environment containing 5% CO₂. The cells were grown in 25 cm² flasks and passaged when they reached 80% confluence. During passaging, the old medium was removed, and 2 mL fresh medium was added to the flask. The cells were dissociated by pipetting the medium 2–3 times against the flask bottom with a 1000 μL pipette. Thereafter, the cells were sub-cultured in 2 new flasks containing 5 mL culture medium. After passaging, the death rate was below 1% according to Trypan blue test.

Cell viability was measured by a modified method based on previous reports [28, 29]. RAW 264.7 cells were seeded in a 96-well plate at a density of 1 × 10⁴ cells/well, and cultured in RPMI 1640 containing 10% FBS overnight. The cells were treated with differing concentrations of GPD or PF-GPD for 24 h, or pre-treated with GPD or PF-GPD for 2 h followed by 24 h treatment with 1 μg/mL LPS. Thereafter, the medium was aspirated, and 100 μL MTT-PBS solution (500 μg/mL) was added into each well and cultured at 37 °C. Four hours later, the supernatant was aspirated and 150 μL DMSO was added into each well. The plate was placed in a plate-reader for 10 min with low speed oscillation, before the absorbance at 490 nm was read. The cell viability was presented as a ratio. The experiment was performed in triplicate.

The activity of NO synthesis can be deduced from the nitrite content of the culture medium as determined by the Griess reaction [29, 30]. RAW 264.7 cells were seeded in a 24-well plate at a density of 4 × 10⁵ cells/well, and cultured for 24 h. The cells were cultured with RPMI 1640 alone, or treated with 1 μg/mL LPS in the absence or presence of differing concentrations of experimental drugs, and 0.5 μg/mL dexamethasone were performed as positive control. The conditioned medium was collected and centrifuged at 2000×g for 10 min, and the supernatant was stored at − 80 °C. To determine the concentration of nitrite, 50 μL conditioned medium was mixed with 50 μL Griess reagent, and the absorbance was read by a plate reader 15 min later at 540 nm. The nitrite concentration was calculated based on the standard curve of NaNO₂, with fresh RPMI 1640 set as the blank control. The experiment was repeated 4 times.

The productions of PGE₂, TNF-α, IL-1β and IL-6 in RAW 264.7 cells under different conditions were measured by enzyme-linked immunosorbent assay (ELISA) [31]. RAW 264.7 cells were seeded in a 96-well plate at a density of 1 × 10⁴ cells/well, and cultured overnight in RPMI 1640 containing 10% FBS. The cells were pre-treated with GPD or PF-GPD for 2 h, or pre-treated with indomethacin (2.5 μL/mL) or dexamethasone (0.5 μL/mL) as a positive control for 2 h, and then treated with 1 μg/mL LPS for 24 h. The medium was then centrifuged at 2000×g at 4 °C, and the supernatant was used to determine the levels of the above factors in accordance with the instructions of the ELISA kits.

In the presence of the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) and oxygen, iNOS can synthesize NO using the nitrogen atom from the L-arginine end [32], and COX-2 is a key enzyme during the synthesis of PGE₂ [33]. Hence, we studied the relationship between the anti-inflammatory activity of Guang-Pheretima with iNOS and COX-2. From the results of the previous studies, we found that GPD at non-cytotoxic concentrations was not able to suppress the release of inflammatory factors in LPS-induced inflammatory model in RAW 264.7 cells. Thus, we continued the subsequent experiments only with gradient concentrations of PF-GPD.

RAW 264.7 cells were cultured in 6-well plates (1 × 10⁶ cells/well) overnight. The cells were pre-treated with differing concentrations of PF-GPD for 6 h (RPMI 1640 as control), and treated with 1 μg/mL LPS for 24 h except for the control group. Total proteins were extracted on ice with RIPA lysis buffer (Pierce, Rockford, IL, USA) with 10 min incubation, and the lysates were centrifuged at 12000×g for 10 min at 4 °C. Supernatants were collected and assayed for protein concentration with BCA assay kit based on a bovine serum albumin (BSA) standard curve. The samples were adjusted to the same protein concentration, and 30 μg protein from each sample was subjected to sodium dodecyl sulfonate- polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane at 120 mA. The membrane was blocked at room temperature for 1 h with 5% skim milk solution containing 0.05% Tween-20. Subsequently, the membrane was washed three times with 0.05% Tween-20, and incubated with a primary antibody overnight at 4 °C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. After washing three times, the membrane was incubated with enhanced chemiluminescence (ECL) reagent for 2 min and exposed to a film. The relative levels of the target proteins were determined by a gel imaging system.

In order to further investigate the effects of Guang-Pheretima on the LPS-induced RAW 264.7 cell inflammation model, the relative mRNA levels of iNOS and COX-2 were determined by Real-Time PCR. RAW 264.7 cells were cultured in 6-well plates (1 × 10⁶ cells/well) overnight, pre-treated with different concentrations of PF-GPD or dexamethasone (0.5 μg/mL) or indomethacin (2.5 μg/mL) for 6 h (RPMI 1640 as control), and treated with 1 μg/mL LPS for 18 h (except for the control group). Total RNA was extracted with Trizol (Invitrogen, USA) and then reverse-transcribed to complementary deoxyribonucleic acid (cDNA) according to the manufacturer’s instructions (Reverse Transcriptase M-MLV, TAKARA, Japan). Samples were then amplified using qPCR (ABI 7500, Applied Biosystems, Foster City, CA, USA) according to the instructions of the fluorescence quantification kit (SYBR® Premix Ex Taq™, TAKARA, Japan). The PCR conditions were as follows: 95 °C for 30 s, 35 cycles of 95 °C for 3 s and 60 °C for 34 s. The PCR primers for iNOS and COX-2 are listed in Table 1. All data was collected and calculated as 2 ^-△△Ct.

In order to observe translocation of NF-κB and degradation of IκB-α, RAW 264.7 cells were cultured in 6-well plates (1 × 10⁶ cells/well) overnight, pre-treated with different concentrations of PF-GPD for 6 h (RPMI 1640 as control), and treated with 1 μg/mL LPS for 1 h (except for the control group). Cytosolic proteins were extracted with NE-PER lysis buffer according to the manufacturer’s instructions, followed by Western Blot analysis using the method described in section 2.6. All antibodies were purchased from Cell Signaling Technology.

After analyzing the indicator constituents in the PF-GPD with LC-MS/MS (Fig. 1), the cytotoxicity in the different concentrations of GPD and PF-GPD against RAW 264.7 cells in the presence or absence of 1 μg/mL LPS was assessed with MTT assay (Fig. 2a and b) (P < 0.01); PF-GPD was not cytotoxic at any of the tested concentrations (Fig. 2c and d) (P > 0.05). Based on the above results, 5–20 μg/mL GPD and 40–320 μg/mL PF-GPD were used for the subsequent experiments.

To evaluate the effect of GPD and PF-GPD on the synthesis of the inflammatory mediator NO, we performed a Griess test to measure the concentration of NaNO₂ in the conditioned medium of LPS-stimulated RAW 264.7 cells. The results indicated that the concentration of NaNO₂ increased in LPS-stimulated RAW 264.7 cells (P < 0.01), suggesting that LPS induced the production of NO in the cells. At all tested concentrations GPD did not affect LPS-induced NO production (P > 0.05), whereas 40–320 μg/mL PF-GPD significantly attenuated LPS-induced elevation of NaNO₂ in a dose-dependent manner (P < 0.01) (Fig. 3).

PGE₂ and TNF-α are important mediators of inflammation, while IL-1β and IL-6 are pro-inflammatory cytokines that are secreted in the early stages of inflammation. Thus, we examined the levels of PGE₂, TNF-α, IL-1β and IL-6 in the conditioned medium of LPS-stimulated RAW 264.7 cells by ELISA. The ELISA results showed that GPD at the tested concentrations was not able to reduce PGE₂, TNF-α, IL-1β nor IL-6 levels in the conditioned medium, whereas PF-GPD significantly inhibited LPS-stimulated secretion of PGE₂, TNF-α, IL-1β and IL-6 compared to LPS-stimulated cells without treatment (P < 0.01) (Fig. 4a-d). In addition, we found that high concentrations of GPD (20 μg/mL) did not decrease LPS-induced secretion of TNF-α in RAW 264.7 cells, instead, it significantly elevated TNF-α secretion (P < 0.05) (Fig. 4b). The possible reason for this result is that the active component in GPD that could inhibit TNF-α secretion was too dilute at the above concentrations to have an effect, and the diluted active component was not able to suppress TNF-α secretion that was induced by other toxic substance(s) in GPD.

iNOS and COX-2 are key enzymes in the synthesis of NO and PGE₂, respectively. In order to explore the mechanism behind the inhibitory effect of PF-GPD on the LPS-induced secretion of NO and PGE₂ in RAW 264.7 cells, we analyzed the expression of iNOS and COX-2 proteins in the inflammation model by Western Blot. The results showed that at 80–320 μg/mL of PF-GPD could markedly decrease the relative expression level of iNOS (P < 0.01), while PF-GPD at a high concentration (320 μg/mL) could recover the expression of iNOS to a level comparable to that of normal control (NC) cells (Fig. 5a). On the other hand, PF-GPD significantly reduced the expression of COX-2 in the cells, as compared to the LPS treatment group (P < 0.01) (Fig. 5b). Taken together, PF-GPD inhibited LPS-induced expression of iNOS and COX-2 in RAW 264.7 cells, and the inhibitory effect was dose dependent.

Based on the Western Blot results, in order to test the hypothesis that PF-GPD downregulates the expression of iNOS and COX-2 proteins by inhibiting iNOS and COX-2 transcription, we assessed the effect of PF-GPD on the synthesis of iNOS and COX-2 mRNA in LPS-stimulated RAW 264.7 cells by real-time PCR. The results indicated that PF-GPD at all tested concentrations (40–320 μg/mL) markedly reduced the relative level of iNOS mRNA (P < 0.01) (Fig. 6a) and COX-2 mRNA (P < 0.01) (Fig. 6b).

It has been extensively reported that the nuclear translocation of NF-κB is a common hallmark of inflammation and cancer [17]. The engagement of NF-κB to the nucleus activates transcription, and it may lead to overexpression of iNOS and other inflammation-related factors. To investigate the effect of PF-GPD on the nuclear translocation of NF-κB and the underlying molecular mechanism, we performed Western blotting to examine the relative levels of nuclear and cytosolic NF-κB p65 as well as the degradation of IκB-α in LPS-stimulated RAW 264.7 cells with or without PF-GPD treatment. The results showed that LPS stimulation caused degradation of IκB-α compared with negative control cells (P < 0.05) (Fig. 7a), whereas PF-GPD blocked LPS-induced degradation of IκB-α (P < 0.01), and inhibited NF-κB p65 translocation from the cytoplasm (Fig. 7b) to the nucleus (Fig. 7c), thus interrupting the transcription and translation of inflammatory genes.