Genipin Attenuates Sepsis-Induced Splenocyte Apoptosis via the Inhibition of Endoplasmic Reticulum Stress

Abstract

Endoplasmic reticulum (ER) dysfunction is characterized by ER stress, which can be triggered by sepsis. Recent studies have reported that lessening ER stress is a promising therapeutic approach to improving the outcome of sepsis. Genipin is derived from gardenia fruit, which is a traditional Chinese medicinal herb for anti-inflammation. Here, mice were treated with genipin (2.5 mg/kg) intravenously to assess its biological effects and underlying mechanism against polymicrobial sepsis. Furthermore, the present study focused on detecting the levels of ER stress-related proteins, including protein kinase R-like ER kinase (PERK), glucose-regulated protein of 78 kDa (GRP78), phosphorylated-eukaryotic initiation factor 2α (p-eIF2α), and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP). The results demonstrated that genipin significantly decreased the serum concentrations of tumor necrosis factor-α and interleukin-6, alleviated histopathological damage to the lungs, livers and spleens, and even improved the survival rates of septic mice. Moreover, sepsis significantly upregulated the protein expression levels of splenic GRP78, PERK, p-eIF2α and CHOP, but their levels were significantly suppressed by genipin. Furthermore, genipin also significantly downregulated cleaved caspase-3 expression levels and reduced sepsis-induced splenocyte apoptosis. In conclusion, genipin potentially improved the survival rate of sepsis and attenuated sepsis-induced organ injury and an excessive inflammatory response in mice. The effects of genipin against sepsis were potentially associated with decreased splenocyte apoptosis via the attenuation of sepsis-induced ER stress to further inhibit ER stress-induced apoptosis.

INTRODUCTION

Sepsis is a potentially lethal illness, caused by a dysregulated host response to infection.¹⁾ Sepsis causes a significant number of deaths annually and represents a long-term public healthcare concern. Moreover, there are no specific treatments that are available to reduce the mortality rate of sepsis beyond certain supportive therapies and routine care.²⁾ Certain sepsis-related pathophysiological events, especially infection, can elicit the accumulation of unfolded or misfolded proteins, which leads to endoplasmic reticulum (ER) dysfunction, also known as ER stress. Once the ER senses the stress, it initiates the unfolded protein response (UPR), which is an intricate defensive response, to rebuild cellular homeostasis.³⁾ Unfortunately, once the regulative capacity of the UPR cannot counteract the stress, apoptosis is initiated and this may lead to sepsis worsening.⁴⁾ Recent research indicates that inhibiting ER stress may act as a potential therapeutic strategy to improve the outcome of sepsis.⁵⁾

The spleen is the largest secondary lymphoid organ, is the main filter for blood-borne pathogens and antigens, and is therefore considered an important organ to resist sepsis.⁶⁾ In clinical practice, splenectomy patients increase the risk to develop sepsis.⁷⁾ The previous work has demonstrated that splenic macrophages fight bacteria by competing for iron,⁸⁾ which enhances the bacterial clearance to limit the further spread of the pathogen. Furthermore, sepsis is characterized by massive apoptosis of lymphocytes in the spleen.⁹⁾ Sepsis-induced lymphocyte apoptosis aggravates the development of immune suppression in septic patients.¹⁰⁾ Immunosuppressive state also leads to an inability of the patient to clear the pathogen. Recent research indicates that genipin inhibits splenic T lymphocyte apoptosis to improve survival,¹¹⁾ but the relationship between ER stress and the effect of genipin on anti-apoptosis remains still unclear.

Genipin is one of the major bioactive molecules of the gardenia fruit, which is a traditional Chinese medicinal herb and is used as an anti-inflammation herb for hundreds of years.¹²⁾ Genipin has previously been demonstrated to have several pharmacological actions, including anti-cytotoxic effects, anti-oxidative damage and anti-fibrinolytic activity.^13–16) Genipin also suppresses tumor necrosis factor (TNF)-α production to ameliorate acute liver dysfunction.¹⁷⁾ Moreover, it has recently been reported that genipin improves the survival of septic mice and attenuates lung and liver injury.¹⁸⁾ However, the underlying mechanisms of how genipin protects against sepsis remain unclear. Therefore, a cecal ligation and puncture (CLP) model was first constructed to induce polymicrobial sepsis in our study. Subsequently, the protective mechanisms of genipin on sepsis were investigated by analyzing the survival rate, organ injury, inflammatory response, splenic ER stress and splenocyte apoptosis.

MATERIALS AND METHODS

Animals

Male C57BL/6J mice (weight, 25–30 g; age, 8–12 weeks; Beijing Vital River Laboratories, China) were housed in cages on a 12/12-h light/dark cycle. Standard chow diet and purified water were freely available. Mice were acclimatized for 3 d before subsequent experimentation. Mice were randomly assigned to a specific treatment group, which was described in the following experimental design. These protocols adhered to the Guide for the Care and Use of Laboratory Animals (8th edition, 2011) and obtained the permission of the Scientific Investigation Board of Tianjin Medical University (No. IRB2019-D05, 2019).

Experimental Design

Following CLP or sham surgery, mice were assigned to the following three groups: i) Saline-treated sham mice (sham group); ii) Saline-treated CLP mice (CLP group); and iii) Genipin-treated CLP mice (genipin group). In these experiments, genipin (2.5 mg/kg; FUJIFILM, Japan) or 0.9% saline (vehicle) was administrated to mice intravenously at 0 and 20 h following the end of the operation. In the survival experiment, mortality was recorded up until 120 h following CLP. Next, in the biochemical studies, under deep anesthesia with 3.5% isoflurane, all mice were sacrificed via cervical dislocation following sample collection at a predetermined time.

Cecal Ligation and Puncture

The CLP model was produced to induce polymicrobial sepsis as previously described.¹⁹⁾ Briefly, under anesthesia with 2.5% isoflurane, the cecum was exposed using a 1-cm-long abdominal incision. The cecum was ligated and was subsequently punctured with a 20 G needle. A small quantity of bowel content was extruded through the punctured holes and subsequently the incision was sutured after the cecum was put back into the abdominal cavity. In the sham-operated animals, the cecum was just exposed but there was no ligation and puncture. Immediately following surgery, the mice were subcutaneously administered with 0.9% saline (1 mL) and warmed on a thermal blanket until they recovered.

Assessment of Cytokine Levels

At 24 and 36 h following CLP, whole blood samples were collected and centrifuged to isolate serum. The serum concentrations of interleukin (IL)-6 and TNF-α were quantified using mouse enzyme-linked immunosorbent assay (ELISA) kits according to the kit’s protocol (Dakewe, China).

Histopathologic Evaluation

Liver, spleen and lung samples were harvested at 36 h following CLP and fixed with formalin, embedded in paraffin, sliced into 8 µm thick and stained with hematoxylin–eosin (H&E). As previously described,¹⁸⁾ the histological characteristics of the liver and lung were evaluated using a scoring method with an optical microscope (Leica Microsystems GmbH, Germany). The histological characteristics of the spleen were analyzed by reference to the previous scoring method.²⁰⁾

Western Blotting

The spleen was isolated and homogenized in protein extraction solution to collect the total protein. The concentration of total protein was determined using the bicinchoninic acid protein assay kit (Beyotime, China). Total proteins were transferred to polyvinylidene fluoride membranes (Merck KGaA, Germany) following separation. The membranes were blocked with 8% skimmed milk powder in Tris-buffered saline with 0.1% Tween-20 (TBS/T) and were then incubated with primary antibodies (1 : 1000 dilution). The next morning, the blots were incubated for 1 h with the appropriate secondary antibodies (1 : 2000 dilution). Subsequently, the membranes were visualized using an enhanced chemiluminescence detection system (Applygen Technologies, Inc., China). The intensity of the protein bands was analyzed using ImageJ software. The primary antibodies were as follows: glucose-regulated protein of 78 kDa (GRP78), protein kinase R-like ER kinase (PERK), phosphorylated-eukaryotic initiation factor 2α (p-eIF2α), CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), and cleaved (c)-caspase-3 (Cell Signaling Technology, Inc., U.S.A.). The intensity values were standardized to β-actin (Applygen Technologies, Inc., China).

Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate (dUTP) Nick End-Labeling (TUNEL) Staining

TUNEL staining was performed to evaluate the severity of splenocyte apoptosis as previously described.^21,22) Spleen samples were harvested at 36 h following CLP, fixed with 4% paraformaldehyde for 24 h, dehydrated with 30% sucrose solution for 24 h, embedded in optimal cutting temperature compound (−80 °C) and then cut using a cryostat. Sections (5-µm thick) were permeabilized with 1% Triton X-100 for 20 min. The slides were again washed and incubated with TUNEL reaction mix (Beyotime, China) for 1 h at 37 °C. Finally, the slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Sections were imaged using a confocal microscope (Zeiss LSM 800; Zeiss GmbH, Germany). As previously described,²³⁾ The number of TUNEL-positive splenocytes was quantified at ×400 magnification.

Statistical Analysis

The survival curve was calculated using the Kaplan–Meier estimate and the log-rank test. Statistical analysis was performed using a one-way ANOVA (three groups) or the unpaired t-test (two groups). For all analyses, GraphPad Prism 5 software was used and p < 0.05 was considered to indicate a statistically significant difference.

RESULTS

Genipin Improves Survival Rates of Septic Mice

To investigate the therapeutic efficacy of genipin on improving the sepsis survival rate, the present study used a CLP model to induce sepsis in mice. The results demonstrated that survival rates were 100% in the sham group. Moreover, 2.5 mg/kg genipin significantly improved the survival rates of septic mice compared with the saline-treated CLP mice (50 vs. 20%; Fig. 1). In addition, Fig. 1 showed the peak time of death was at 36 h after CLP. These data are consistent with that presented in a previous study.¹⁸⁾ This suggested that genipin may improve the sepsis survival rate.

Fig. 1. Genipin Improves the Survival Rate of Septic Mice

Mice were assigned to the following three groups (n = 20 mice/group): i) Saline-treated sham group (sham + saline); ii) saline-treated CLP group (CLP + saline); and iii) genipin-treated CLP group (CLP + genipin). Mice were intravenously administered with genipin (2.5 mg/kg) or saline (0.9%) at 0 and 20 h following the sham or CLP operation. The survival rate was monitored once every 12 h, up to 120 h, following the completion of surgery. * p < 0.05 vs. CLP + saline group (log-rank test).

Genipin Mitigates Inflammatory Response and Alleviates Organ Damages in Septic Mice

In the saline-treated CLP group, the serum level of IL-6 and TNF-α showed a continuous increase at 24 and 36 h after CLP. Compared with the saline-treated CLP mice, the serum concentrations of IL-6 and TNF-α were significantly inhibited by genipin at 36 h after CLP (Fig. 2A). Based on the survival and cytokine test, 36 h was selected as the optimal time point for further tests. In the sham mice, H&E staining sections of the liver, spleen and lung disclosed an approximately normal histological structure. In the saline-treated CLP group, sections exhibited evidence of organ injury, including inflammatory cell infiltration, vascular congestion and hepatocellular necrosis. Cellular necrosis is particularly noticeable in the white pulp of the spleen, while the red pulp was congested. However, compared with the CLP group, genipin treatment resulted in a distinct reduction in organ injury (Figs. 2B, C).

Fig. 2. Effect of Genipin on Serum Cytokine Levels and Organ Injury in Septic Mice

(A) Serum IL-6 and TNF-α concentrations were quantified using ELISA kits (n = 4–6 mice/group). The concentrations in the sham samples were lower than the lower limit of the kit detection range, so data were not shown. (B) Lung, liver and spleen samples were obtained 36 h following CLP and were stained using H&E. Scale bars, 100 µm. (C) Summary of lung, liver and spleen histological scores (n = 4–5 mice/group). Data are presented as the mean ± standard deviation (S.D.) with significance: * p < 0.05, ** p < 0.01. ns, not significant.

Genipin Attenuates Splenic ER Stress in Sepsis

A variety of harmful stimuli occurring in sepsis, such as infection, trauma, ischemia, or hypoxia, can lead to ER stress and trigger UPR to counteract this stress.⁴⁾ Data from our previous study demonstrated that GRP78 and CHOP, the specific ER stress marker, were markedly upregulated in the spleen following CLP.²⁴⁾ Recently, Tanaka et al. also reported that genipin suppressed the upregulation of GRP78 and CHOP in ER stress-induced Neuro2a cells.²⁵⁾ To further confirm whether genipin inhibited the ER stress response in sepsis in the present study, alterations in the classical PERK signaling pathway of ER stress in the spleen following CLP were investigated. At 36 h after CLP, spleens were collected for Western blotting analysis. GRP78 and PERK are usually activated simultaneously in ER stress and subsequently PERK phosphorylates eIF2a, which leads to the shutdown of cellular protein synthesis, especially CHOP.^26,27) Compared with the sham mice, the protein expression levels of GRP78, PERK, p-eIF2α and CHOP were significantly upregulated in the CLP mice (Fig. 3). Notably, compared with the saline-treated CLP group, genipin treatment attenuated the levels of the aforementioned proteins. These findings suggested that CLP-induced sepsis was sufficient to induce ER stress in the spleen, which could be attenuated using genipin therapy.

Fig. 3. Genipin Attenuates ER Stress-Related Protein Expression Levels in the Spleen

(A) Representative bands of GRP78, PERK, p-eIF2α, and CHOP. (B) Histograms represent the semi-quantification of these protein expression levels (n = 4–7 mice/group). Data are presented as the mean ± S.D. with significance: * p < 0.05 and ** p < 0.01.

Genipin Diminishes Splenocyte Apoptosis in Septic Mice

Lymphocyte apoptosis serves an important role in immunosuppression in sepsis.²⁸⁾ Immunosuppression impairs the clearance of invading microorganisms, which leads to sepsis progression.^4,29) Apoptosis can also be induced by ER stress-related molecules, especially CHOP, which is a bridge between ER stress and apoptosis.³⁰⁾ As previously stated, genipin treatment downregulated CHOP protein expression levels in the spleen. Therefore, it was hypothesized that genipin treatment may contribute to the reduction of splenocyte apoptosis in sepsis. To assess the number of apoptotic splenocytes, TUNEL staining was used. The results demonstrated that the number of TUNEL-positive splenocytes was significantly increased in the saline-treated CLP mice compared to the saline-treated sham mice. As expected, genipin significantly reduced the number of TUNEL-positive splenocytes compared to the saline-treated CLP mice (Fig. 4A). Furthermore, the protein expression levels of c-caspase-3, a proapoptotic protein, also increased in the spleen of CLP mice, whereas genipin significantly reduced the levels of the c-caspase-3 compared with the saline-treated CLP mice (Fig. 4B). Taken together, these findings suggested that genipin diminished splenocyte apoptosis in septic mice.

Fig. 4. Genipin Attenuates Sepsis-Induced Splenocyte Apoptosis

(A) Representative images of TUNEL splenocyte staining. Scale bar, 20 µm. The histogram represents the quantification of TUNEL staining positive splenocytes. (B) Representative bands and relative quantitative analysis of the c-caspase 3 expression levels, three mice per group. The results are presented as mean ± S.D. with significance: * p < 0.05, ** p < 0.01.

DISCUSSION

Despite new progress in antimicrobial therapy, resuscitative strategies and ventilator management, sepsis remains to be a critical illness.³¹⁾ Therefore, it is urgent to explore effective therapeutic approaches. Genipin is one of the bioactive ingredients of the gardenia fruit, which is a well-known antipyretic-detoxification Chinese medicinal herb. Genipin has been shown to exhibit anti-inflammatory, antioxidant and bacteriostatic activity.^12,32,33) For instance, recent study has reported that genipin inhibits carrageenan-induced rat paw edema and attenuates the concentration of nitric oxide in exudate.¹²⁾ Genipin also ameliorates lipopolysaccharide (LPS)-induced acute lung injury via activation of phosphoinositide 3-kinase (PI3K)/AKT signaling and significantly suppresses proinflammatory cytokines via interference with toll-like receptor signaling in RAW264.7 cells.^18,33) These researches imply that genipin may improve the outcome of LPS or CLP-induced sepsis. Therefore, the CLP model was constructed in mice and the mice subsequently received intravenous administration of genipin. The protective effect of genipin on sepsis was assessed by analyzing the survival rate, organ injury, inflammatory response, splenic ER stress and splenocyte apoptosis. The results demonstrated that genipin significantly improved the 120-h survival rate of CLP mice. Second, genipin attenuated pathological damage to the lungs and livers, such as the infiltration of inflammatory cells and necrosis. Meanwhile, genipin lessened the apoptosis of lymphocytes in spleen tissue, particularly the white pulp. Moreover, genipin also decreased the serum levels of IL-6 and TNF-α. But interestingly, TNF-α only was significantly inhibited by genipin at 36 h, not at 24 h. The reason for this observation might be that genipin limited the differentiation of monocytes into macrophages,³⁴⁾ which were the major source of TNF-α. A previous study has identified that the peak time of recruited monocytes was at 24 h after CLP, and monocytes were essential for defense against sepsis.³⁵⁾ Thus, monocytes might be recruited with the same number in two groups at 24 h after CLP, so that genipin failed to attenuate TNF-α production at this point. Reciprocally, monocytes had differentiated into macrophages at 36 h in the CLP group, but the differentiation was limited by genipin and led to attenuating TNF-α production at 36 h after CLP in the genipin group. This inference specifically deserves further investigation. Taken together, these results demonstrated that genipin potentially has the ability to protect against sepsis.

The ER is the main subcellular compartment, which is mainly responsible for lipid and protein synthesis. A variety of harmful stimuli that occur in sepsis, such as bacterial infection, trauma and hypoxia, may upset ER homeostasis and trigger ER stress.^24,36) Numerous studies have reported that ER stress serves an important role in the pathogenesis of sepsis, particularly in the inflammatory immune response.^11,24,37) For example, ER stress impairs the activity of lymphocytes and enhances immunosuppression during sepsis.^11,24) Our previous study demonstrated that GRP78 and CHOP proteins were markedly upregulated in the spleen following CLP.²⁴⁾ In our study, the upregulation of GRP78 and CHOP protein expression levels was again confirmed in the spleen after CLP. Moreover, the results demonstrated that PERK and p-eIF2α protein expression levels were also significantly upregulated.

GRP78 is an important chaperone protein in the ER and functions to ensure correct protein folding.³⁸⁾ The upregulation of GRP78 expression implies that the cell is experiencing ER stress. PERK is an ER-located transmembrane stress sensor and is usually bound to GRP78 in its inactive form.³⁸⁾ When ER stress occurs, PERK is activated via its separation from GRP78.³⁹⁾ Subsequently, activated PERK phosphorylates eIF2α to inhibit protein synthesis.²⁶⁾ These changes trigger UPR to resist stress and restore ER homeostasis. Therefore, the upregulated PERK and p-eIF2α levels in the present study may potentially indicate that the PERK signaling pathway has been activated. In sharp contrast, genipin inhibited the protein expression levels of splenic GRP78, PERK and p-eIF2α in this study. This finding is consistent with the chemical chaperone which attenuates ER stress activity to ameliorate CLP-induced sepsis.^40–42) For instance, the chemical chaperone Tauroursodeoxycholic acid significantly attenuates ER stress and lymphocyte apoptosis during polymicrobial sepsis.⁴²⁾ The 4-phenylbutyric, an inhibitor of ER stress, improves organ function and survival in CLP rats.⁴⁰⁾ Taken together, these results suggest that attenuating ER stress with genipin could be a potential drug for treating sepsis.

In normal physiological conditions, CHOP activity remains at a very low level in the cytoplasm. However, CHOP is markedly elevated by activating the PERK signaling pathway during ER stress.^36,43) As a transcription factor, CHOP translocates into the cell nucleus to induce an apoptotic response via downregulating the transcription of the Bcl-2 antiapoptotic gene and upregulating the transcription of the Bad/Bak proapoptotic genes.^43,44) Tribbles pseudokinase 3 (TRB3), a proapoptotic protein, is also upregulated by CHOP.⁴⁵⁾ TRB3 inhibits protein kinase B (PKB) activity to modulate caspase-3 activation indirectly.⁴⁶⁾ C-caspase-3 is the active form of caspase-3 and triggers a downstream reaction that leads to apoptosis.⁴⁷⁾ Therefore, CHOP is an important transcription factor that bridges ER stress and apoptotic processes. In the present study, CHOP and c-caspase-3 were significantly activated in the spleen at 36 h following CLP. Furthermore, via detection of TUNEL-positive splenocytes, the results demonstrated that splenocyte apoptosis significantly increased in CLP mice. Combined with the results assessing the PERK signaling pathway, these findings indicated that splenocyte homeostasis was possibly impaired by ER stress, which may further trigger ER stress-induced apoptosis in sepsis.

Apoptosis is a distinctive and important mode of programmed cell death that is tightly regulated. Abnormal apoptosis usually leads to the progression of serious illnesses, such as sepsis, ischemia and cancer. In 1999, Hotchkiss et al. reported extensive lymphocyte apoptosis occurred in patients with sepsis.²⁸⁾ Moreover, another study then demonstrated that the extensive depletion of immune cells was caused by abnormal apoptosis.¹¹⁾ These results demonstrated that abnormal apoptosis may contribute to the impairment of the immune response in sepsis. Therefore, medical interventions that inhibit the mechanism of apoptosis have become an important focus in the investigation of sepsis therapeutics. A previous study reported that genipin significantly inhibited LPS-induced hepatocyte apoptosis to alleviate liver dysfunction.²³⁾ Furthermore, Tanaka et al. have reported that genipin has a protective effect on tunicamycin-induced ER stress in Neuro2a cells.²⁵⁾ Consequently, this study further analyzed the effect of genipin on ER stress-induced cell apoptosis in the spleen following CLP. Genipin significantly downregulated the protein expression levels of splenic CHOP and c-caspase-3 in CLP mice. The number of TUNEL-positive splenocytes was also significantly decreased by genipin. These data indicated that genipin may attenuate splenocyte apoptosis via the downregulation of the CHOP-mediated apoptotic signaling pathway during ER stress-induced apoptosis.

In summary, the results of the present study demonstrated that genipin significantly alleviated organ damage, attenuated the inflammatory response and improved the survival rates in septic mice. These results have indicated that genipin potentially has a protective effect against sepsis. Furthermore, it was indicated that the potential protection mechanism of genipin against sepsis occurs via the attenuation of ER stress, the downregulation of CHOP protein expression and the reduction of splenocyte apoptosis. Therefore, genipin may be a novel drug that can be used to treat sepsis. However, basic and clinical research still needs to be performed in the future.

Acknowledgments

This research was supported by the National Natural Science Foundation of China (Nos. 81871546, 82172122, and 81471841) and the Tianjin Key Medical Discipline (Specialty) Construction Project (No. TJSYXZDXK010).

Conflict of Interest

The authors declare no conflict of interest.

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