Geniposide protected against cerebral ischemic injury through the anti-inflammatory effect via the NF-κB signaling pathway

2.1 Animals

Animals were housed in a humidity-controlled room on a 12 h light/dark cycle at 22°C with free access to food and water. Adult male C57BL/6 mice (8–10 weeks) were used for the experiments. Animals were assigned randomly using a random number table. All assessments were conducted by investigators who were blinded to the experimental group assignment.

A total of 150 adult male C57BL/6 mice (weighing 25–30 g) were used in our current study; 14 (9.3%) mice died before the completion of the experiment and were excluded from the study. Post-mortem examinations did not reveal the occurrence of intracerebral or subarachnoid hemorrhage in any of these animals, and no significant differences were found in the number of deaths in each group.

1. Ethical statement: All experimental procedures were approved by the committee of experimental animals of Hebei Medical University, Shijiazhuang, China (2020-AE006), and conducted in accordance with the National Institutes of Health guidelines.

2.2 Reagents

Geniposide (purity >98% tested by HPLC) was purchased from Chengdu Derick Biotechnology Co. Ltd. (Sichuan, China). Lipopolysaccharide (LPS) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against P-p65 (3033, CST), p65 (6956, CST), A20 (5630, CST), and GAPDH (2118, CST) were purchased from Cell Signaling Technology (Beverly, MA, USA). TRAF6 (38-0900, Invitrogen) was purchased from Thermo Fisher Scientific. Tumor necrosis factor-α (TNF-α; 430907, BioLegend), IL-6 (431307, BioLegend), interleukin-1β (IL-1β; 432601, BioLegend), and iNOS ELISA kits were purchased from BioLegend (CA, USA).

2.3 Geniposide treatment in vivo

Geniposide (Zelang Biotechnology Co, Nanjing, Jiangsu, China) with a purity of more than 98% was dissolved in saline. All the mice were randomly divided into five groups as follows: sham group, middle cerebral artery occlusion (MCAO) group, GD25 (25 mg/kg geniposide) group, GD75 (75 mg/kg geniposide) group, and GD150 (150 mg/kg geniposide) group. Sham and MCAO groups were treated with 0.9% normal saline only through intraperitoneal injection. Geniposide was administered through intraperitoneal injection with doses of 25, 75, or 150 mg/kg twice daily for 3 days before surgery.

2.4 Cerebral ischemia by MCAO

Mice were anesthetized with 1% pentobarbital (100 mg/kg, i.p.). Permanent focal cerebral ischemia was induced by right MCAO using an intraluminal filament occlusion model as previously reported [17]. First, the right carotid artery of mice was exposed to dissect the external carotid artery (ECA) and the internal carotid artery (ICA). ECA was then blocked at the level of MCA branches. Then, a filament (Guangzhou Jialing Biotech Co Ltd, Guangzhou, China) coated with silicone was inserted through the ECA and then advanced into the ICA. Finally, the filament was stopped and ligated, when a slight resistance was felt. Sham-operated mice were subjected to the same way, except for MCAO. During the operation, the body temperature of mice was maintained at 37.5 ± 0.5°C using a heating pad. Regional cerebral blood flow (rCBF) was monitored by a laser-Doppler flowmeter (moor VMS-LDF, Moor Instruments Ltd., UK) before, during, and after MCAO. The middle cerebral artery was occluded with a criterion of <25% of baseline blood flow remaining after MCAO.

2.5 Measurement of neurological deficits

The neurological deficits of mice were conducted before the mice were euthanized at 24 h after MCAO by an examiner blinded to the groups (n = 10 per group). The neurological deficits were scored using a modified scoring system reported previously [18]: 0, no deficits; 1, difficulty in fully extending the contralateral forelimb; 2, unable to extend the contralateral forelimb; 3, mild circling to the contralateral side; 4, severe circling; and 5, falling to the contralateral side.

2.6 Infarct volume

At 24 h after MCAO, mice (n = 5 per group) were anesthetized by pentobarbital, and brains were rapidly removed on the ice. Brains were sliced into five 2-mm-thick coronal sections and then stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich, St. Louis, MO, USA) for 10 min at 37°C. The infarct volume was calculated as follows: ([total infarct volume − {volume of intact ipsilateral hemisphere − volume of intact contralateral hemisphere}]/contralateral hemisphere volume) [19].

2.7 Brain edema

Brain edema was evaluated by the standard wet/dry method [20] at 24 h after MCAO (n = 6 per group). The two hemispheres of mice were packaged with tinfoil, respectively, to obtain wet weights on an electronic balance, and dry weights were then obtained after placing them into the oven for 24 h at 100°C. The brain water (BW) content was then calculated as follows:

BW = (wet weight − dry weight)/wet weight × 100%.

2.8 ELISA assay

TNF-α, IL-6, IL-1β, and iNOS levels in the brain homogenate of the ischemic hemisphere were determined using a commercial ELISA kit (NeoBioscience Technology Company, Beijing, China) at 24 h after MCAO. The brain homogenate was then centrifuged at 1,000×g for 15 min at 4°C to collect the supernatant. The procedure was performed following the manufacturer’s instructions. Briefly, 50 mM carbonate coating buffer (pH 9.6) was used to dissolve the antigen (10–20 μg/mL), which was then added into a 96-well enzyme label plate (100 μL/well) for overnight incubation at 4°C. Then, 150 μL of 1% bovine serum albumin was added to each well for 1 h of blocking at 37°C, and 100 μL of serum was added into each well to incubate for 2 h at 37°C. Subsequently, the serum was incubated with 100 μL of diluted horseradish peroxidase (HRP)-labeled secondary antibodies at 37°C for 1 h. Finally, the absorbance value A405 was read on a microplate reader.

2.9 Western blot analysis

Proteins from the mice brains and total proteins from the treated cells were extracted with the radioimmunoprecipitation assay buffer (RIPA) and were determined using the bicinchoninic acid (BCA) protein assay kit, as previously reported [21]. Equal amounts of protein (50 μg) were separated by a 10% SDS/polyacrylamide gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% BSA solution for 1 h and then incubated with primary antibodies (A20, TRAF6, and phosphorylation of NF-κB) at 4°C overnight. On the following day, the membrane was incubated with secondary antibodies. GAPDH was used as an internal control of total proteins. The densitometric values were normalized with respect to GAPDH immunoreactivity to correct for loading and transfer differences among samples. The relative density of blots was determined on an Odyssey infrared scanner (LICOR Bioscience, Lincoln, NE, USA).

2.10 Cell culture and treatments

BV2 cells, a microglial cell line, were cultured in Dulbecco's modified Eagle medium (DMEM) solution (Gibco, USA), supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Solarbio, China), in an incubator under normoxic conditions (37°C, 5% CO₂/95% air). To test the effect of geniposide on cell viability, the cells were divided into a control group, 5 μM group (5 μM geniposide), 10 μM group (10 μM geniposide), 20 μM group (20 μM geniposide), and 100 μM group (100 μM geniposide). To explore the effect of geniposide on inflammation, LPS was administered at a concentration of 200 ng/mL. Cells were classified into different groups: control group, LPS group, and LPS + GD group (100 μM geniposide).

2.11 CCK-8 assay for microglial cell viability

Microglial cells were seeded into 96-well plates with the culture medium at a volume of 100 μL in each well. Cell viability was detected using the CCK-8 assay according to the manufacturer’s instructions. Briefly, 10 μL of the CCK-8 solution was added to each well. The absorbance of each well was read using a microplate reader at a test wavelength of 450 nm, with 620 nm being used as the reference wavelength.

2.12 Prediction of geniposide targets

SwissTargetPrediction (http://www.swisstargetprediction.ch/) is an online analysis software, which can predict the target of small molecules according to the principle of molecular similarity. We utilized this tool to predict the targets of geniposide. A protein–protein interaction (PPI) network was constructed through the STRING (v11.0, https://string-db.org/) database, which is a database of known and predicted PPIs, including direct (physical) and indirect (functional) associations.

2.13 Gene ontology (GO) analysis

GO analysis consists of three parts: molecular function, biological process, and cellular component. Enrichment analysis was performed via the David 6.8 (https://david.ncifcrf.gov/) database, which provides a comprehensive set of functional annotation tools to elaborate the biological function of the long list of genes. Briefly, to start DAVID, first click on “Functional Annotation” under “Shortcut to David tools” at the left of the home page. Then, we upload the file containing the targets of geniposide, which were predicted through SwissTargetPrediction. After the file was uploaded, we select “Functional Annotation Clustering” in the center of the page. The GO terms obtained in the drawing are arranged in descending order according to the −log10 (P value) of enrichment, and we used the first 30 results.

2.14 Statistics

All data were expressed as mean ± SEM. For statistics normally distributed, comparisons were assessed by one-way ANOVA followed by Student–Newman–Keuls tests and least significant difference (LSD) tests for multi-group comparisons and two-tailed Student’s t-test for two-group comparisons. For neurological deficits, Mann–Whitney U test was used for comparisons between the two groups. Fisher’s exact test was applied for the GO analyses. P < 0.05 was considered statistically significant.

3.1 Geniposide protected against cerebral ischemic injury in mice

The flow chart of our experiment is shown in Figure 1a. At 24 h after MCAO, mice displayed characteristics of circling to the left or even not walking spontaneously. The higher the neurological defect scores, the more serious the cerebral ischemic injury. As shown in Figure 1b, no visible neurological defect was observed in mice of the Sham group, while severe neurological defect was observed in mice of the MCAO group. The neurological defect scores in mice of the 150 mg/kg GD group were markedly reduced, compared with the MCAO group. However, there was no significant difference among the groups of MCAO, 25 mg/kg GD group, and 75 mg/kg GD group.

Figure 1

Geniposide protected against cerebral ischemia injury at 24 h after MCAO in mice. (a) Schematic diagram of the experimental design. (b) Effect of geniposide on the neurological deficit scores at 24 h after MCAO (n = 10 per group) (*P < 0.05, nonparametric Mann–Whitney U test). (c) Effect of geniposide on the brain edema at 24 h after MCAO (n = 6 per group) (*P < 0.05, one-way ANOVA). (d) Effect of geniposide on the infarct volume at 24 h after MCAO (n = 5 per group) (*P < 0.05, one-way ANOVA). Data are expressed as mean ± SEM.

Brain edema was measured at 24 h after MCAO using wet/dry methods, as shown in Figure 1c. In the ischemic hemisphere, 75 and 150 mg/kg GD groups displayed ameliorated BW content (79.67 ± 0.46% vs 82.28 ± 0.53% and 79.00 ± 0.57% vs 82.28 ± 0.53%, P < 0.05, respectively) when compared with the MCAO group. However, no obvious difference was observed among the groups of MCAO and 25 mg/kg GD group.

Infarct volume was evaluated at 24 h after MCAO by staining, through which viable tissue is stained red and ischemic tissue is stained pale, as shown in Figure 1d. Reduced infarct volume was observed in mice of 75 and 150 mg/kg GD groups (46.28 ± 3.04% vs 54.73 ± 2.87% and 45.10 ± 0.24% vs 54.73 ± 2.87%, P < 0.05, respectively) when compared with the MCAO group. No obvious difference in the cerebral infarction volume was observed in the MCAO and 25 mg/kg GD group.

These results indicated that geniposide exerted a neuroprotective effect after cerebral ischemic injury. Based on the above-mentioned results, we selected 150 mg/kg GD for the following study.

3.2 Target prediction and construction of the PPI network of geniposide

To predict the potential targets of geniposide, we queried geniposide targets using the SwissTargetPrediction tool. One hundred geniposide targets were predicted with SwissTargetPrediction (Supplemental materials). To construct an interaction network, we studied the PPI relationship via the STRING database. This network consisted of the above 100 terms, including 100 nodes and 400 edges (Figure 2). As shown in Figure 2, the interactions mainly exist in inflammatory-related proteins, such as MAPK, MMP9, Akt1, ESR1, HSP90, and so on, which suggested that the neuroprotective effect of geniposide may be mainly attributed to its anti-inflammatory effect.

Figure 2

The PPI network of geniposide targets predicted by SwissTargetPrediction.

3.3 GO analyses of geniposide

To investigate the biological function and potential mechanism of geniposide, GO analyses were performed via the DAVID database. Biological process analysis was mainly enriched in terms of inflammatory response and negative regulation of apoptotic progress. Molecular function analysis was primarily enriched in terms of protein binding (Figure 3). These findings suggested that geniposide participated in biological functions that were closely related to the inflammatory response.

Figure 3

GO pathway enrichment of geniposide targets predicted by SwissTargetPrediction. GO analysis included three parts: the biological process (a), cellular component (b), and molecular function (c), which, respectively, describe the biological processes involved, the cellular environments in which they are located, and the molecular functions of potential gene products. Enrichment analysis was performed via the DAVID 6.8 database. The enriched terms of GO analysis are arranged in descending order according to −log10 (P value). BP: biological process; MF: molecular function; CC: cellular component.

3.4 Geniposide downregulated the expression of inflammatory cytokines

To confirm the accuracy of target prediction and GO analysis, we thus investigated the expression of pro-inflammatory cytokines in the brain at 24 h after cerebral ischemia. The results showed that the expression of TNF-α, IL-6, IL-1β, and iNOS was upregulated after MCAO (Figure 4). Importantly, after geniposide treatment, the expression of IL-6 and iNOS was significantly reduced, which suggested that geniposide exhibited an anti-inflammatory effect after cerebral ischemia.

Figure 4

Geniposide suppressed the overproduction of pro-inflammatory cytokines at 24 h after MCAO in mice. The expression of TNF-α (a), IL-6 (b), IL-1β (c), and iNOS (d) in the penumbra of mice brains was detected by ELISA (n = 4–5 per group) (*P < 0.05, one-way ANOVA).

3.5 Geniposide upregulated the expression of A20 and inhibited the activation of NF-κB

We further explored the expression of A20, TRAF6, and the phosphorylation of NF-κB, which was the upstream signal of IL-6 and iNOS, in the penumbra of the brain tissue. As shown in Figure 5, the expression of A20, TRAF6, and the phosphorylation of NF-κB was increased after MCAO. Importantly, geniposide ameliorated the ischemia-induced upregulation of TRAF-6 and p-NF-κB significantly. Besides, geniposide significantly increased the expression of A20 when compared with the MCAO group.

Figure 5

Geniposide inhibited inflammation by upregulating A20 expression and downregulating TRAF6 expression and NF-κB phosphorylation. The expression of A20, TRAF6, p-NF-κB 65, and p-NF-κB in the penumbra of mice brains was detected by western blot (a). Quantification of the expression of A20 (b), TRAF6 (c), and phosphorylation of NF-κB (d) by Image-pro-plus (n = 4 per group) (*P < 0.05, one-way ANOVA).

As IL-6 and iNOS were mainly produced by activated microglia, we used microglia cell lines, BV2 cells, to examine the inhibitory effect of geniposide on microglia activation and its underlying mechanisms.

To evaluate whether geniposide affected the cell viability of BV2 cells, the cell viability was assessed by CCK8 assay. As shown in Figure 6a, geniposide exerted no significant toxic effects on BV2 at four concentrations (5, 10, 20, and 100 μM) after 24 h treatment. We then selected 100 μM in subsequent studies.

Figure 6

Geniposide inhibited LPS-induced inflammation by upregulating A20 expression and downregulating TRAF6 expression and NF-κB phosphorylation in BV2 cells. The cell viability was examined by CCK8 assay (a). The expression of A20, TRAF6, p-NF-κB 65, and p-NF-κB in BV2 cells was detected by western blot (b). Quantification of the expression of A20 (c), TRAF6 (d), and phosphorylation of NF-κB (e) by Image-pro-plus. The results shown are representative of three independent experiments (*P < 0.05, one-way ANOVA). Data are expressed as mean ± SEM; n = 4–6 per group.

LPS (200 ng/mL) was then used to activate microglia. As shown in Figure 6b–e, the expression of A20, TRAF6, and the phosphorylation of NF-κβ was increased after LPS treatment. Importantly, geniposide ameliorated the LPS-induced upregulation of TRAF-6 and p-NF-κβ significantly. Moreover, geniposide significantly increased the expression of A20 when compared with the LPS group.