Chlorogenic Acid Alleviates Hepatic Ischemia–Reperfusion Injury by Inhibiting Oxidative Stress, Inflammation, and Mitochondria-Mediated Apoptosis In Vivo and In Vitro

CGA improved liver function, oxidative stress, inflammation levels, and liver histopathology injury in rats

To demonstrate whether different concentrations of CGA pretreatment had a protective effect on HIRI and to obtain the optimal CGA concentration, we examined the levels of liver function, oxidative stress markers, inflammation factors, and the degree of liver histopathological damage in rats (Fig. 1). First, we assessed the effect of CGA on liver function by measuring its serum biomarker levels (Fig. 1A). The serum levels of ALT and AST were significantly increased after I/R injury compared with that of the Sham group (p < 0.01). In addition, compared with that of the I/R group, the serum levels of AST were significantly decreased after pretreatment with all doses of CGA; however, only medium and high doses of CGA pretreatment could decrease the ALT levels (p < 0.05). Therefore, CGA pretreatment could reverse liver function injury after HIRI and the most significant decrease was observed in the I/R+CGA-H group (p < 0.05).

Fig. 1figure 1

CGA pretreatment ameliorated liver function, oxidative stress, inflammation levels and the degree of liver histopathological damage in rats.

To examine whether CGA affected oxidative stress in the liver, we measured liver homogenate levels of MDA, SOD and GSH (Fig. 1B). After HIRI, MDA levels increased, and SOD, GSH levels decreased significantly compared with that of the Sham group (p < 0.05). However, after CGA pretreatment, MDA levels decreased and SOD, GSH levels significantly increased compared to that of the I/R group (p < 0.05). This indicates that CGA pretreatment could reduce oxidative stress in the liver and this effect was most significant in the I/R + CGA-H group (p < 0.01).

Next, we investigated the effect of CGA on liver inflammation by measuring the liver homogenate levels of HMGB1, TNF-α, and IL-1β (Fig. 1C). The levels of HMGB1, TNF-α, and IL-1β were significantly increased after HIRI compared to that of the Sham group (p < 0.05). In particular, compared to that of the I/R group, HMGB1 levels were significantly decreased after all doses of CGA pretreatment (p < 0.05); however, only low and high doses of CGA pretreatment resulted in a significant decrease in IL-1β levels (p < 0.01), while only the medium and high doses of CGA pretreatment resulted in a significant decrease in TNF-α levels (p < 0.01). Therefore, CGA pretreatment could significantly decrease aseptic inflammation caused by HIRI and the most significant decrease was observed in the I/R + CGA-H group (p < 0.01).

Later, to investigate the histopathological effect of CGA on the liver, we performed H&E staining on liver tissues (Fig. 1D). The results revealed that hepatocytes had a normal pathological structure in the Sham group (Fig. 1D: a, f); however, those in the I/R group displayed hepatocyte edema, disorganized hepatic cord structure, vacuolation, lamellar necrosis, hemorrhage, and inflammatory cell infiltration (Fig. 1D: e, j). In contrast, hepatocellular lesions were reduced in the CGA groups (Fig. 1D: b–d, g–i). Kruskal–Wallis analyses of the histopathological scores between the different groups revealed that the low and medium CGA doses and I/R groups scored higher than the Sham group and that only the I/R + CGA-H group had a lower score compared to that of the I/R group (p < 0.005; Fig. 1E).

Since the highest CGA dose (100 mg/kg.d) exerted the strongest effect, it was used for subsequent experiments.

CGA Reduced Liver ROS Levels in Rats

To examine the effect of CGA on cellular ROS levels in rat liver tissue, we stained frozen liver sections from each group with a DHE fluorescent probe and DAPI (Fig. 2A, B). Interestingly, compared to that of the Sham group, DHE fluorescence intensity significantly increased in rats subjected to HIRI (p < 0.05) and decreased after CGA pretreatment, compared to that in the I/R group (p < 0.01). The results show that CGA could inhibit ROS production during HIRI in rats, which may in turn reduce oxidative stress-induced liver injury.

Fig. 2figure 2

CGA pretreatment reduced ROS levels in rats.

CGA Reduced the Active Secretion of HMGB1 In Vivo

The expression of IRF-1 protein in the liver tissue of rats in each group was detected by western blotting, and Ac-HMGB1 expression in the liver tissue of rats in each group was detected by IP combined with western blotting. The results showed that IRF-1 and Ac-HMGB1 expression levels in the liver tissue of the I/R+CGA and I/R groups were increased compared with that of the Sham group (p < 0.05). However, the expression levels of IRF-1 and Ac-HMGB1 in the I/R+CGA group were significantly decreased compared with that of the I/R group (p < 0.01, Fig. 3A, B). The results suggest that CGA can effectively inhibit the active secretion of HMGB1 during HIRI in rats.

Fig. 3figure 3

CGA attenuates the expression levels of inflammatory pathway related proteins in rats.

CGA Decreased the Levels of HMGB1/TLR-4/NF-κB Signaling Pathway Components In Vivo

RT-qPCR analysis revealed that the mRNA levels of HMGB1 and TLR-4 in the I/R+CGA group were significantly lower than those in the I/R group (p < 0.01), among which the TLR-4 mRNA levels were not significantly different from that in the Sham group (p > 0.05, Fig. 3C). The results suggest that CGA could inhibit the expression of HMGB1 and its downstream inflammatory pathway receptor, TLR-4, at the transcriptional level during HIRI in rats.

Next, to further determine the mechanism underlying the observed effects of CGA in vivo, we examined the expression of the TLR-4/NF-κB signaling pathway components in the liver tissues of each group (Fig. 3D). Compared with that of the Sham group, the protein expression of TLR-4, MyD88, P65, P-P65, P-IκB-α, IL-1β, and TNF-α was significantly increased after HIRI (p < 0.05), whereas the protein expression of IκB-α decreased. Moreover, the expression of TLR-4, MyD88, P65, P-P65, P-IκB-α, IL-1β, and TNF-α was significantly decreased after CGA pretreatment (p < 0.01) and the expression of IκB-α was significantly increased compared to that of the I/R group (p < 0.01). The results show that CGA could inhibit the NF-κB signaling pathway mediated by TLR-4, and thus reduce the sterile inflammatory response during HIRI in rats.

CGA Attenuated Apoptosis in Rat Liver Tissue

First, we examined the effect of CGA on apoptosis by performing TUNEL staining on liver tissues (Fig. 4A). The results showed that apoptotic cells were significantly increased after I/R, compared to that of the Sham group, but were significantly reduced after CGA pretreatment (p < 0.01). We observed apoptotic cells after HIRI; however, it is unknown whether this is due to a mitochondria-mediated apoptotic pathway? Therefore, we further examined mitochondrial apoptosis pathway-related proteins to prove our hypothesis (Fig. 4B). Compared to that of the Sham group, BCL-2 expression significantly decreased after HIRI (p < 0.01), and its expression significantly increased after CGA pretreatment compared to that of the I/R group (p < 0.05). Conversely, Bax, Cyt-c, cleaved-caspase9, cleaved-caspase3, ENDOG, and AIF expression significantly increased after I/R injury compared to that of the Sham group (p < 0.01); however, their expression significantly decreased after CGA pretreatment compared to that of the I/R group (p < 0.05). The results indicate that CGA significantly attenuated the occurrence of hepatocyte apoptosis during HIRI in rats by inhibiting both the mitochondria-mediated caspase-dependent apoptotic and caspase-independent apoptotic pathways. It remains to be further explored in in vitro experiments whether the underlying cause is related to the reduction of hepatocyte mitochondrial damage by CGA.

Fig. 4figure 4

CGA pretreatment reduced hepatocyte apoptosis and the expression levels of mitochondria-mediated apoptosis pathway related proteins in rats.

CGA Decreased the Levels of HMGB1/TLR-4/NF-κB Signaling Pathway Components In Vitro

L02 cell lines overexpressing stable TLR-4 were screened using lentiviral transfection, and GFP expression was observed under a fluorescence microscope (Fig. 5A). RT-qPCR and western blotting were used to detect TLR-4 expression in the normal L02 cell (NC), empty plasmid transfection (EMT), and TLR-4 overexpression (OE) groups. The results showed that TLR-4 mRNA and protein levels were significantly upregulated in the OE group compared to the NC and EMT groups (p < 0.01), while the results in the EMT and NC groups were not significantly different (P > 0.05, Fig. 5B, C), indicating that the transfection was successful and the empty plasmid had no effect on the results of this experiment.

Fig. 5figure 5

CGA decreased the levels of TLR-4/NF-κB signaling pathway components in vitro.

CCK8 results showed no toxic effect of 12.5–200 µM CGA on L02 cells, while 12.5–25 µM CGA promoted their proliferation (Fig. 5D). To determine the optimal CGA concentration, cells were divided into NC, EMT, OE, and OE + CGA (12.5, 50, or 200 µM) groups according to the CCK-8 assay results. The OE + CGA groups were administered 12.5, 50, and 200 µM CGA and cultured with TLR-4 overexpressing cells for 48 h. The protein expression of TLR-4 was detected by western blotting, revealing that the TLR-4 expression was significantly lower in the 50 and 200 µM CGA groups than in the OE group (p < 0.01). TLR-4 expression was particularly significant in the CGA 200 μM group (p < 0.01, Fig. 5E); therefore, CGA 200 µM was selected for subsequent experiments.

Next, we measured the protein expression of the TLR-4/NF-κB signaling pathway components in L02 cells (Fig. 5F). Compared to the protein levels in the NC group, TLR-4, MyD88, P65, P-P65 and P-IκB-α protein levels were significantly increased in the OE and H/R groups (p < 0.01), whereas the IκB-α expression was decreased (p < 0.05). Interestingly, these expression patterns were significantly reversed in the OE + CGA and H/R + CGA groups compared to that of the OE and H/R groups (p < 0.05). These results further indicate that CGA could reduce HIRI-induced sterile inflammation by inhibiting the TLR-4/NF-κB signaling pathway.

CGA Reduced ROS Expression, Mitochondrial Damage, and Apoptosis In Vitro

To further prove our hypothesis, we established a hepatocyte hypoxia model for 6 h and reoxygenation model for 12 h to simulate HIRI, and examined ROS levels, mitochondrial damage, and apoptosis related indicators. First, L02 cells were divided into NC, H/R, and H/R+CGA low, medium, and high dose groups, the latter were pretreated with 12.5, 50 and 200 µM CGA for 48 h. The H/R and H/R+CGA groups were established as H/R models, and cleaved-caspase3 protein was detected in each group by western blotting. The results showed that the levels of cleaved-caspase3 protein in the H/R and H/R+CGA groups were significantly higher than those in the NC group (p < 0.05), and the levels of cleaved-caspase3 protein in the H/R+CGA 50 and 200 µM groups were significantly lower than those in the H/R group (p < 0.05), most significantly in the 200 µM group (p < 0.01, Fig. 6A). The results suggest that both medium and high doses of CGA may have protective effects on H/R-induced apoptosis, with the best effect observed at a CGA concentration of 200 µM. Therefore, 200 µM CGA was chosen for subsequent experiments to explore the specific underlying mechanism.

Fig. 6figure 6

CGA reduced ROS expression, mitochondrial damage, and apoptosis in vitro.

Next, L02 cells were stained with a DCFH-DA fluorescent probe and detected by flow cytometry (Fig. 6B). The ROS fluorescence intensity was higher in the H/R + CGA and H/R groups than in the NC group (p < 0.01). Furthermore, the ROS fluorescence intensity was decreased in the H/R + CGA group compared to that of the H/R group (p < 0.01). The results suggest that CGA inhibits the production of ROS in hepatocytes during H/R.

Next, MitoTracker Red CMXRos and Annexin V-FITC fluorescent probes were used to detect the mitochondrial damage and apoptosis of L02 cells (Fig. 6C). Compared to that of the NC group, MitoTracker fluorescence intensity in the H/R + CGA and H/R groups was significantly lower, whereas the apoptotic cells were significantly higher (p < 0.01). Conversely, MitoTracker fluorescence intensity was increased, and apoptosis was lower in the H/R + CGA group than in the H/R group (p < 0.01). We also detected the expression of mitochondrial apoptosis pathway-related proteins (Fig. 6D), and found that BCL-2 expression was significantly lower in the H/R group than in the NC group (p < 0.05). However, BCL-2 was significantly upregulated in the H/R + CGA group (p < 0.05) compared to that of the H/R group. Compared with that of the NC group, Bax, Cyt-c, cleaved-caspase9 and cleaved- caspase3, ENDOG, and AIF expression was significantly increased in the H/R group (p < 0.05); however, their expression was significantly decreased in the H/R + CGA group compared to that in the H/R group (p < 0.05). The results show that CGA could alleviate hepatocyte mitochondrial damage by reducing ROS production in hepatocytes during HIRI, thereby inhibiting the mitochondria-mediated apoptotic pathway and reducing hepatocyte injury.

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