Liensinine alleviates septic heart injury by targeting inflammation, oxidative stress, apoptosis, and autophagy

Sepsis is one of the most commonly discussed worldwide health issues, with high rates of morbidity and mortality [1]. Severe sepsis can trigger a systemic inflammatory response, which can result in organ failure and death in a short time. Of all organs, the heart is one of the most vulnerable target organs during sepsis [2]. Previous research has revealed that sepsis-induced heart damage is often accompanied by uncontrolled inflammation, oxidant/antioxidant imbalance, excessive apoptosis, and autophagy. There are currently no licensed medications to treat septicemic cardiac insufficiency; as a result, finding a medication that may specifically restrict these pathological alterations for the prevention of cardiac dysfunction induced by sepsis is critical. In recent years, the use of natural products has become increasingly important to various diseases. Liensinine is an isoquinoline alkaloid isolated from Nelumbo nucifera Gaertner′s seed embryo. It has been shown to have anti-inflammatory, antioxidant, and apoptotic properties [3]. Therefore, we conducted follow-up experiments on these aspects to verify whether liensinine could protect against LPS-induced septic heart injury in mice.

CK (creatine kinase) and LDH (lactate dehydrogenase) are often regarded as criteria for heart damage. As shown in Supplementary Figure S1, serum CK and LDH were utilized to assess the severity of cardiac damage. In comparison to the control group, LPS significantly increased CK and LDH in serum. After treatment with liensinine, both CK and LDH were decreased compared to the LPS group. H&E staining was also utilized to evaluate pathological alterations in the hearts of mice. The results showed that after intraperitoneal injection of LPS, the myocardial cell space in mice increased, the vasculature of the myocardial interstitium was dilated, the structure was significantly disturbed, and the number of inflammatory cells infiltrating the organ was increased ( Figure 1A). These pathological alterations, on the other hand, were alleviated after liensinine treatment. These findings indicate that a sepsis model has been successfully constructed and that liensinine protects mice against LPS-induced septic cardiac damage.

Figure 1 .

Liensinine mitigates cardiac damage and inflammation in septic mice and alleviates oxidative damage by altering the Keap 1/Nrf2 signaling pathway

(A) H&E was used to stain the heart tissue. Scale bar: 200 μm. The red arrow indicates increased myocardial cell space, and the black arrow indicates inflammatory cell infiltration. The mRNA extracted from heart tissues was reverse transcribed into cDNA, and the mRNA expression levels of IL-1β (B), iNOS (C), TNF-α (D) and IL-10 (E) in the heart were measured by qRT-PCR. (F) Proteins were extracted from heart tissues to detect the expression levels of inflammation-related proteins: iNOS (G), COX-2 (H), and p-NF-κB p65 (I). The activities of MDA (J), SOD (K), CAT (L) and GSH-Px (M) were measured using the supernatant of heart tissue homogenates. (N) Proteins from heart tissues were extracted, and the expression levels of key proteins of the Keap 1 (O)/Nrf2 (P)/HO-1 (Q) axis were determined. * P<0.05, ** P<0.01, *** P<0.001 vs control group; # P<0.05, ## P<0.01, ### P<0.001 vs LPS group.

In a sepsis model, LPS induces systemic inflammation and causes cardiomyocytes to release large amounts of pro-inflammatory cytokines involved in the initiation and control of the inflammatory response [4]. Then, the impact of liensinine on the expression of inflammatory cytokines caused by septic cardiac damage was investigated by qRT-PCR. As shown in Figure 1B–E, LPS stimulated the expressions of interleukin-1 beta ( IL-1β), inducible nitric oxide synthase ( iNOS), and tumor necrosis factor-alpha ( TNF-α) and inhibited the expression of interleukin-10 ( IL-10). The release of these cytokines was dramatically influenced by liensinine therapy versus the LPS group. Compared with the LPS group, a decrease in the mRNA expression of IL-1β, iNOS, and TNF-α and an increase in IL-10 level occurred in the three groups after treatment with low, medium, and high doses of liensinine. These findings imply that liensinine treatment can attenuate the inflammatory cytokine response elicited by LPS during sepsis-associated cardiac damage.

iNOS and cyclooxygenase-2 (COX-2) are important enzymes in the progression of inflammation and oxidative stress. NF-κB is a key transcription factor that controls the expression of inflammatory mediators, including iNOS and COX-2 [5]. Therefore, we assume that LPS may stimulate NF-κB, which consequently elevates the production of iNOS and COX-2 in sepsis-induced cardiac damage. To this end, we detected the protein production of iNOS and COX-2, as well as NF-κB activation, to test whether these signals are the targets of liensinine inhibition in LPS-induced cardiac inflammatory response. We discovered that the LPS group had higher levels of iNOS, COX-2, and p-NF-κB p65 than the control group. This effect was reversed after liensinine treatment, and the expression levels of iNOS, COX-2, and p-NF-κB p65 were decreased. As a result, liensinine reduced the expressions of the inflammatory proteins iNOS and COX-2, accompanied by the activation of the NF-κB pathway ( Figure 1F–I).

When the body is activated by external stimuli, it produces oxidative stress to protect itself, but too much oxidative stress leads to the onset and progression of human diseases. As a result, oxidative stress inhibition has substantial scientific implications for heart damage in sepsis [6]. Malondialdehyde (MDA) is activated during lipid peroxidation, leading to tissue damage. Figure 1J shows that compared to the control group, LPS dramatically elevated MDA level in mice. Additionally, compared to the LPS group, the MDA activity was decreased after liensinine treatment. Changes in the antioxidant system in cardiac tissue were also examined to further establish the impact of liensinine on LPS-induced cardiac oxidative stress. Figure 1K–M illustrates that compared to those in the control group, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were all dropped considerably following LPS injection. The activities of SOD, CAT, and GSH-Px were increased after liensinine treatment compared to the LPS group. These findings demonstrate that liensinine protects mice against oxidative stress caused by LPS by boosting SOD, CAT, and GSH-Px activity in the heart.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial transcription factor that controls the expressions of several antioxidant genes and helps reduce oxidative stress [7]. As a result, we wanted to determine whether liensinine treatment targets Nrf2 to balance oxidative stress in the hearts of mice. In the heart tissue of LPS-treated mice, high level of kelch-like ECH-associated protein 1 (Keap 1), low level of Nrf2, and high amount of the downstream oxidative stress factor heme oxygenase 1 (HO-1) were identified, as illustrated in Figure 1N–Q. These findings imply that liensinine protects the heart from LPS-induced oxidative damage caused by sepsis by activating the Keap 1/Nrf2 signaling pathway.

Apoptosis is widely considered to be the decisive process of myocardial inhibition induced by sepsis [8]. Significant apoptosis occurred in the LPS group compared to the control group, as indicated in Figure 2A, but this was reversed in the liensinine group. In LPS-treated cardiac tissue, the number of apoptotic cells was considerably higher than that in the control group; however, the number of apoptotic cells was reduced following liensinine (40 mg/kg, BW) administration compared with that in the LPS group ( Figure 2B,I). At the same time, we quantified key proteins involved in cardiac cell apoptosis to further determine whether liensinine preconditioning could inhibit LPS-induced apoptosis. Two key factors in apoptosis are Bcl-2 and Bax, which are regulatory genes that inhibit and promote apoptosis, respectively. The balance of the ratio between the two is the key factor in determining the inhibitory effect on apoptosis [9]. In addition, the caspase cascade plays a central role in most of the modulation factors in the complex process of apoptosis. LPS treatment boosted the production of Bax (Bcl2-associated X)/Bcl-2 (B-cell lymphoma-2), Cytochrome C, Caspase 3, and Caspase 9 ( Figure 2C–H). However, these phenomena were significantly down-regulated after treatment with liensinine, thus protecting the heart tissue from LPS-induced apoptosis.

Figure 2 .

Liensinine protects cardiac tissue from apoptosis in septic mice and increases autophagy in cardiac tissue

(A) Transmission electron microscopy was used to observe the cell morphology in the heart tissue. (B) TUNEL detection of apoptotic cells in cardiac tissue. (C–E) Protein extracts from heart tissues were used to examine the expressions of the apoptosis-related proteins Bax, Bcl-2 and Cytochrome C, and greyscale value analysis was performed. (F–H) Expressions of the apoptosis-related proteins Caspase 3 and Caspase 9 were detected, and greyscale value analysis was performed. (I) Quantitative analysis of the results of B. (J–M) Extracted proteins from heart tissues were examined by western blot analysis for the autophagy-related proteins LC3, P62, and ATG5, and greyscale value analysis was performed. * P<0.05, ** P<0.01, *** P<0.001 vs control group; # P<0.05, ## P<0.01, ### P<0.001 vs LPS group.

Autophagy is a vital self-protection system, and autophagy imbalance has been shown to have serious consequences for the heart [10]. Autophagy activation has been observed in a number of heart disorders, including myocardial hypertrophy and ischemia/reperfusion injury, implying that autophagy may play a role in myocardial dysfunction, so we detected key proteins related to autophagy. According to our results, compared with the control group, the LC3 II/I value increased after LPS stimulation, and LC3 II/I also showed an increasing trend after liensinine administration. Similarly, the value of P62 was highest in the control group and decreased gradually with LPS administration and liensinine preconditioning. The trend of autophagy protein 5 (ATG5) was the same as that of LC3 II/I ( Figure 2J–M). Therefore, autophagy is enhanced after LPS stimulation, and liensinine further enhances LPS-induced autophagy.

In summary, this study revealed that liensinine, an alkaloid extracted from lotus seeds, exhibits an excellent prophylactic protective effect against LPS-induced septic heart injury. Liensinine protects against septic heart injury by alleviating abnormal CK and LDH elevation and cardiac tissue damage, mainly involving the processes of targeting NF-κB to inhibit inflammation, targeting Nrf2 to inhibit oxidative stress and apoptosis, and enhancing autophagy. Therefore, liensinine treatment may become an effective approach to prevent sepsis-related cardiac injury ( Supplementary Figure S2).

Supplementary data

Supplementary data is available at Acta Biochimica et Biophysica Sinica online.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported by the grants from the Basic Science (Natural Science) Research Project of Higher Education of Jiangsu Province (No. 21KJB230001), the Open-end Funds of Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening (No. HY202101), the National Natural Science Foundation of China (No. 81773968), the Postgraduate Research & Practice Innovation Program of Jiangsu Ocean University (No. KYCX2022-34), and the Priority Academic Program Development of Jiangsu Higher Education Institutions of China for financial support.