https://orcid.org/0000-0001-7256-9675
Abstract
To investigate whether the silent information regulator 1 (SIRT1) was involved in the protective effects of Ganoderma lucidum polysaccharides (GLP) against sepsis-induced cardiac dysfunction.
Lipopolysaccharide (LPS)-induced sepsis model was constructed in C57/BL6J mice. Mice were randomly divided into LPS + GLP + EX-527, LPS + EX-527, LPS + GLP, LPS or control group). The levels of serum inflammatory factor markers were examined by ELISA. H&E staining was performed to assess the inflammation. TUNEL staining and bromodeoxyuridine staining were used to observe cell apoptosis and proliferation, respectively. Expression of apoptosis and proliferation-related proteins was detected by western blot.
GLP treatment could significantly increase the expression of SIRT1, reduce levels of serum inflammatory factors (TNF-α, IL-1α and IL-6) and inflammatory cells in mice heart tissue of sepsis models (all Ps < 0.01). Compared with LPS group, GLP treatment inhibited apoptosis and promoted proliferation of myocardial tissues (all Ps < 0.01). Besides, EX-527 (SIRT1 inhibitor) treatment could partially reverse the protective effects of GLP against sepsis-induced cardiac dysfunction (all Ps < 0.01).
GLP might play a protective role in sepsis-induced cardiac dysfunction through regulating inflammatory response, apoptosis and proliferation via activating SIRT1. Therefore, GLP is expected to be a probable novel strategy for treatment of sepsis.
Introduction
Sepsis is a lethal organ dysfunction that develops into a disordered systemic inflammatory and immune response and is associated with high mortality.[1, 2] Sepsis can trigger damage to various organs, including brain, heart, kidney, lung and liver.[3] Among the complications of sepsis, cardiac dysfunction is the most common and serious and is closely related to the increase of mortality in patients with sepsis.[4] Evidences have showed that patients with sepsis and cardiac dysfunction have a significantly higher mortality rate than patients without cardiac dysfunction.[5, 6] Thus, further clarify the pathogenesis of sepsis-induced cardiac dysfunction and development of novel therapeutic strategies are essential.
As a conservative histone deacetylase, silent information regulator 1 (SIRT1) is closely related to multiple biological processes (such as apoptosis, inflammation, autophagy and senescence).[7–9] Previous studies found that the expression of SIRT1 was reduced in myocardial tissues of sepsis mice, and activating SIRT1 could improve the cardiac dysfunction.[9–11]Ganoderma lucidum, a Chinese medicinal mushroom, has been used in the treatment of various diseases with a history of nearly a thousand years.[12]Ganoderma lucidum polysaccharides (GLP), as the most important biologically active substances of G. lucidum, has anti-inflammation, anti-oxidation, anti-tumour and neuroprotection role in many physiological processes.[13–16] However, the function of GLP on sepsis-induced cardiac dysfunction is unclear. Therefore, this study investigated the role of GLP in sepsis-induced cardiac dysfunction and explored the possible molecular mechanisms for the first time from the perspective of SIRT1. We proposed a novel molecular mechanism of the GLP in protect against sepsis-induced cardiac dysfunction, in which GLP might play a protective role in sepsis-induced cardiac dysfunction via activating SIRT1.
Materials and Methods
Chemicals and reagents
Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). EX-527 was purchased from Selleck Chemicals (Houston, TX, USA). GLP (98% purity) was obtained from Shaanxi Ciyuan Biotech Co., Ltd. (No. CY140220; Xi’an, China). All commercial ELISA test kits were purchased from BioChek (AN Gouda, Holland). SIRT1, caspase-3, caspase-9, Bax, proliferating cell nuclear antigen (PCNA), cyclin D1 and β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
Animals and treatment
C57BL/6 male mice (ageing 6–8 weeks and weighing 20–24 g) were purchased from the Medical Laboratory Animal Center of Guangdong Province (Guangzhou, China). The mice were placed in a pathogen-free environment (22 ± 2°C and 12–12 h/light–dark cycle). This study was carried out according to the Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Guangxi Medical University.
Mice were randomly divided into four groups (n = 10 per group): (1) control; (2) LPS; (3) LPS + GLP; (4) LPS + EX-527 (SIRT1 inhibitor); (5) LPS + GLP + EX-527 group. Mice were intraperitoneally injected with 15 mg/kg LPS (Sigma-Aldrich, USA) to establish the model of LPS-induced sepsis. The mice in control group were intraperitoneally injected with the same volume of phosphate-buffered saline (PBS). The mice in LPS + GLP group were intraperitoneally injected with GLP (25 mg/kg/day) which was dissolved in dimethyl sulfoxide at 12 h after treatment with LPS. The mice in the LPS + GLP + EX-527 group were intraperitoneally injected with 5 mg/kg EX-527 (Selleck Chemicals, USA) 1 h before treatment with LPS. The dose of GLP (25 mg/kg/day) is referred to previous report.[17] The serum and heart tissue of mice were collected after 3 days of LPS treatment and stored at −20°C for further investigation.
Serum biochemical indicators detection
The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and blood urea nitrogen (BUN) were detected by an automatic biochemical analyser (Beckman Kurt, USA). The antibodies against IL-1α, TNF-α, lactate dehydrogenase (LDH), MIP-1α, IL-6, IL-8, creatine kinase-MB (CK-MB) and MIP-2 in serum were detected by a commercial ELISA test kit (Biochek, Holland). The test procedure was performed according to the manufacturer’s protocol. The absorbance was measured at 450 nm with an ELISA reader spectrophotometer (ELX800, BioTek Instruments, Inc., USA). Concentrations of samples were calculated according to the standard curve and OD value.
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated using TRIzol reagent (Invitrogen, USA). The cDNA of SIRT1 was prepared using the reverse transcription kit (Fermentas, USA). The qRT-PCR analysis was performed using SYBR Green RT-PCR Kit (Thermo, USA). The β-Actin was used as the endogenous control to calculate the relative RNA levels. Data were calculated by the comparative cycle threshold (CT) (2−ΔΔCT) method. The PCR primer sequences were as follows:
SIRT1: sense 5′-ACAGGCTTTAGCGAGTTATT-3′ and antisense 5′-AAGAGGCGAACGAGGG-3′; β-Actin: sense 5′-CATCGTCCACCGCAAATGCTTC-3′ and antisense 5′-AACCGACTGCTGTCACCTTCAC-3′.
Western blot
Expression of target proteins in mice heart tissues was detected using specific primary antibodies to SIRT1, caspase-3, caspase-9, Bax, PCNA, cyclin D1 or β-actin (Cell Signaling Technology) by Western blot. About 25 μg of total proteins were separated on polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore). The membrane was incubated with the primary antibodies at 4°C overnight, followed by corresponding horseradish peroxidase-conjugated-linked secondary IgG antibodies (Santa Cruz, USA; 1 : 10 000 dilutions) for 2 h at room temperature. Super Signal Protein Detection kit (Pierce Biotechnology, USA) was used to detect the signal.
H&E staining
Section of mice heart tissue was dewaxed in xylene and was rehydrated in alcohol solutions. After washing with distilled water, section was stained with Harris haematoxylin (5 min) and counterstained with eosin-phloxine (2 min), respectively.
Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining
After the heart tissues of mice were incubated with protease K (30 min) and washed with PBS, TdT and luciferase-labelled dUTP were added (reacting for 2 h at 37°C). Then, the specific antibody labelled with horseradish peroxidase (HRP) was added (15 min in the dark) for TUNEL staining. Subsequently, tissues were incubated with converter-peroxidase (30 min, 37°C). After mice heart tissues were fixed by using antifade agents (Beyotime Bioengineering Co., China), the apoptosis was observed by an inverted fluorescence microscope (Leica, DM 5000B; LeicaCTR 5000; Germany).
Immunostaining for bromodeoxyuridine (BrdU)
BrdU staining was performed using a BrdU-staining kit (Oncogene Research, Cambridge, MA, USA) in accordance with the manufacturer’s protocol. Cells incorporating BrdU showed dark blue precipitates in their nuclei.
Statistical analysis
Data were analysed by SPSS 22.0 (SPSS Institute, USA) and were expressed as mean ± standard deviation. Multiple groups were compared by one-way analysis of variance, followed by Tukey’s test or least significant difference t-test. P < 0.05 indicated that the difference was statistically significant.
Results
GLP increased the expression of SIRT1 in mice with sepsis
Firstly, C57BL/6 mice were adopted to construct sepsis models. As shown in Figure 1, levels of serum ALT, AST, BUN and inflammatory factor markers (IL-6, IL-1α, IL-8, MIP-1α and MIP-2) in LPS group were significantly higher (all Ps < 0.01) than that in control group. These results indicate the successful construction of sepsis models.
Successful construction of sepsis models.The levels of serum ALT (A), AST (B), BUN (C), IL-1α (D), IL-6 (E), IL-8 (F), MIP-1α (G) and MIP-2 (H) were measured by an automatic biochemical analyser or ELISA. Data were presented as mean ± standard deviation (n = 10 per group). **P < 0.001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen.
After sepsis models were treated with GLP, the expression of SIRT1 in mice heart tissue was detected by RT-PCR and western blot. As shown in Figure 2, the expressions of SIRT1 in LPS group were significantly lower (all Ps < 0.01) than that in control group. GLP treatment significantly increased (all Ps < 0.01) the expression of SIRT1 as compared with the LPS group. In addition, treatment with EX-527 (SIRT1 inhibitor) significantly decreased (all Ps < 0.01) the expression of SIRT1 as compared with the LPS + GLP group. Taken together, the results indicated that GLP increased expression of SIRT1 in mice heart tissue of sepsis models.
GLP increased the expression of SIRT1 in mice with sepsis.(A) RT-PCR analysis of the expressions of SIRT1 at mRNA levels in each group. (B) Western blot analysis the expressions of SIRT1 at protein levels in each group. Data were presented as mean ± standard deviation (n = 10 per group). **P < 0.001. GLP, Ganoderma lucidum polysaccharides; LPS, lipopolysaccharide.
GLP protected against sepsis-induced myocardial inflammation
To further verify the role of GLP in sepsis, we next examined levels of serum inflammatory factor markers, CK-MB and LDH by ELISA. Results showed that GLP treatment could significantly reduce levels of serum IL-1α, IL-6, TNF-α, CK-MB and LDH, whereas EX-527 partially reversed the role of GLP (all p < 0.01, Figure 3A). HE staining of mice heart tissue showed that an obvious decrease in inflammatory cells was observed in LPS + GLP group compared to LPS group. However, treatment with EX-527 could increase (all p < 0.01) inflammatory cells of mice heart tissue as compared with the LPS + GLP group (Figure 3B). These results suggested that GLP may protect against sepsis-induced myocardial inflammation by activating SIRT1.
GLP protected against sepsis-induced myocardial inflammation.(A) ELISA analysis the level of serum IL-6, IL-1α, TNF-α, CK-MB and LDH in each group. (B) H&E staining (×200) of mice heart tissue in each group. Data were presented as mean ± standard deviation (n = 10 per group). **P < 0.001. CK-MB, creatine kinase-MB; ELISA, enzyme-linked immunosorbent assay; GLP, Ganoderma lucidum polysaccharides; LPS, lipopolysaccharide; LDH, lactate dehydrogenase.
GLP attenuated apoptosis in mice with sepsis
We next examined the effects of GLP on cardiomyocyte apoptosis by TUNEL staining and western blot. Results showed that the number of apoptotic myocardial cells and the expression of apoptotic-related proteins (caspase-3, caspase-9 and Bax) in the LPS + GLP group were evidently decreased as compared with the LPS group (Figure 4). Besides, the effect of GLP against apoptosis was abolished by EX-527 treatment. These results indicated that GLP may attenuate apoptosis in mice with sepsis by activating SIRT1.
GLP attenuated apoptosis in mice with sepsis.(A) TUNEL staining results of myocardial tissues of mice in each group. (B) Western blot analysis of caspase-3, caspase-9 and Bax expression in each group. Data were presented as mean ± standard deviation (n = 10 per group). **P < 0.001. GLP, Ganoderma lucidum polysaccharides; LPS, lipopolysaccharide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling.
GLP promoted proliferation of myocardial tissues in mice with sepsis
The role of GLP on cell proliferation was investigated by BrdU staining. The BrdU-positive cells in LPS + GLP group (Figure 5A) were higher than that in LPS group. In addition, the effect of GLP on cell proliferation was partially reversed by treatment with EX-527. Consistently, the expression of cyclin D1 and PCNA (specific marker of cell proliferation) in LPS + GLP group were significantly increased (all Ps < 0.01, Figure 5B) as compared with LPS group. EX-527 treatment significantly reduced the expression of cyclin D1 and PCNA (all Ps < 0.01) as compared with the LPS + GLP group. The above results indicated that GLP may promote the proliferation of myocardial tissues in mice with sepsis by activating SIRT1.
GLP promoted proliferation of myocardial tissues in mice with sepsis.(A) BrdU staining results of myocardial tissues of mice in each group. (B) Western blot analysis of PCNA and cyclin D1 expression in each group. Data were presented as mean ± standard deviation (n = 10 per group). **P < 0.001. BrdU, bromodeoxyuridine; GLP, Ganoderma lucidum polysaccharides; LPS, lipopolysaccharide.
Discussion
Although significant progress has been made in the treatment strategy of sepsis recently, the incidence and mortality rate of sepsis are still rising.[18] GLP, a polysaccharide extract from G. lucidum, has been verified to have anti-tumour and immuno-modulating activities.[19] However, the role of GLP on sepsis still remains unknown. Therefore, we investigated the role of GLP in sepsis-induced cardiac dysfunction and explored the possible molecular mechanisms in this study.
GLP has been reported to possess various pharmacological properties. Chen et al. found that GLP had hepatoprotective and anti-inflammatory effects on mice with liver injury through inhibiting the activation of NLRP3.[20] Fan et al. demonstrated that GLP could protect against doxorubicin-induced cardiotoxicity through inhibiting doxorubicin-induced pro-inflammatory cytokines production, apoptosis and oxidative stress.[12] A study performed in IEC-6 cells (one non-transformed small-intestinal epithelial cell) showed that GLP treatment accelerated the wound repair by stimulating the cell migration, differentiation and proliferation.[21] A previous study also showed that GLP could promote the neural progenitor proliferation and cognitive function in the mice model of Alzheimer’s disease.[22] Consistent with the previous studies, we found that GLP treatment could significantly reduce the levels of serum inflammatory factors, inhibited apoptosis and promoted the proliferation of myocardial tissues compared with the LPS group. The above results indicated that GLP may play a protective role in sepsis-induced cardiac dysfunction by regulating cell proliferation, apoptosis and inflammatory response.
We next investigated the possible molecular mechanisms from the perspective of SIRT1 by using a SIRT1 inhibitor (EX-527). The function of SIRT1 in cardiac dysfunction of sepsis had been confirmed. Evidence showed that reduced SIRT1 signalling could exacerbate the sepsis-induced myocardial injury and attenuate the protective role of liver X receptor agonist.[10] We found that the expressions of SIRT1 in the LPS-induced sepsis model mice were significantly lower than that in normal mice. Besides, GLP treatment significantly increased the expression of SIRT1 compared with LPS group. These results indicated that SIRT1 might be involved in the protective role of GLP on sepsis-induced cardiac dysfunction. Several studies had showed that SIRT1 play a crucial role in regulating cell apoptosis and inflammatory response.[8, 11, 23, 24] In this study, we observed that treatment with SIRT1 inhibitor (EX-527) could increase inflammatory cells, promoted apoptosis and inhibited proliferation of mice heart tissue compared with the LPS + GLP group. The above results indicated that GLP might play a protective role in sepsis-induced cardiac dysfunction by regulating inflammatory response, apoptosis and proliferation via activating SIRT1. However, the protective mechanism of GLP in sepsis-induced cardiac dysfunction needs to be determined in further investigations.
Conclusions
We investigated the protective effects of GLP in sepsis-induced cardiac dysfunction and explored possible molecular mechanisms from the perspective of SIRT1 for the first time. Results showed that GLP treatment significantly reduced the levels of serum inflammatory factors and increased the expression of SIRT1. In addition, GLP could protect against sepsis-induced cardiac dysfunction via regulating inflammatory response, apoptosis and proliferation. Moreover, treatment with EX-527 (SIRT1 inhibitor) could partially reverse the protective effects of GLP against sepsis-induced cardiac dysfunction. Combining the above results, GLP may play a protective role in sepsis-induced cardiac dysfunction via activating SIRT1. Therefore, GLP is expected to be a probable novel strategy for treatment of sepsis.
Acknowledgements
None.
Author Contributions
Conception and design: X.S. and Z.X.; Administrative support: X.S.; Data collection and collation: Z.X., G.Y. and T.Q.; Data analysis and interpretation: Z.X. and Y.L.; Drafting article: Z.X. All the authors have read and approved the final manuscript.
Funding
None.
Conflict of Interest
The authors declare that they have no conflict of interest.
Ethics Approval
This study was carried out according to the Guide for the Care and Use of Laboratory Animals and was approved by the Animal Care and Use Committee of Guangxi Medical University (date, 3 September 2019; number, GXM19163).
Data Availability
The data underlying this article are available in the article.
References
