The protective effect of a short peptide derived from cold-inducible RNA-binding protein in a rat model of cardiac arrest and resuscitation

doi:10.21203/rs.3.rs-2219941/v1

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The protective effect of a short peptide derived from cold-inducible RNA-binding protein in a rat model of cardiac arrest and resuscitation

https://doi.org/10.21203/rs.3.rs-2219941/v1

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Systemic inflammation act as a contributor to neurologic deficits after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). Extracellular cold-inducible RNA-binding protein (CIRP) has been demonstrated to be responsible in part for the inflammation through binding to Toll-like receptor 4 (TLR4) after cerebral ischemia. The short peptide C23 derived from CIRP has a high affinity for TLR4, which can inhibit the downstream inflammatory response. We hypothesize that C23 reduces systemic inflammation after CA/CPR by blocking the binding of CIRP to TLR4. Adult male SD rats in experimental groups were subjected to 5 minutes of cardiac arrest followed by resuscitation. C23 peptide (8 mg/kg) or normal saline was injected intraperitoneally at the beginning of the return of spontaneous circulation (ROSC). The expressions of CIRP, TNFα, IL-6, and IL-1β in serum and brain tissues were significantly increased at 24h after ROSC (P < 0.05). C23 treatment could markedly decrease the expressions of TNFα, IL-6, and IL-1β in serum (P < 0.05). Besides, C23 can penetrate the blood-brain barrier and play an anti-inflammatory role in brain tissues. It can decrease the expressions of TLR4, TNFα, IL-6, and IL-1β in the cortex and hippocampus and inhibit the colocalization of CIRP and TLR4 (P < 0.05). In addition, C23 treatment can reduce the apoptosis of hippocampus neurons(P < 0.05). Finally, the rats in the C23 group have improved survival rate and neurological prognosis (P < 0.05). These findings suggest that C23 can reduce systemic inflammation and it has the potential to be developed into a possible therapy for post-cardiac arrest syndrome.

CIRP

C23

Cardiac arrest

Inflammation

Ischemia-reperfusion

Cardiac arrest (CA) is the leading cause of death worldwide. Timely and effective cardiopulmonary resuscitation (CPR) is very critical to the follow-up treatment of patients[1]. However, even return of spontaneous circulation (ROSC) is achieved in a proportion of patients with CA, about 50% of in-hospital and 90% of out-of-hospital CA patients still die, and nearly half of those who survive will have permanent neurological damage[2].

Post-cardiac arrest syndrome (PCAS), which is characterized by organ ischemia-reperfusion injury, systemic inflammation, post-CA brain injury, and myocardial dysfunction, can contribute to the high rate of mortality and disability after CA[3]. As a hallmark of PCAS, systemic inflammation and multi-organ failure can predict severe neurological injury and often fatal outcomes[4]. Systemic inflammation is thought to be a trigger for endothelial activation and injury, resulting in hemodynamic instability and aggravating damage to the brain, myocardium, and other organs[5]. The released cytokines like interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and IL-1β have been suggested to modulate the brain and cardiovascular dysfunction associated with PCAS. At present, hypothermia therapy is still recommended by guidelines as an important means for the early treatment of PCAS[6, 1]. However, it has been reported that hypothermia does not reduce or even increase the expression of inflammatory factors in serum after CA/CPR[7, 8], which is contrary to our commonly known. Considering the side effects of hypothermia treatment, it is necessary for us to further understand the mechanisms of CA/CPR and find new specific anti-inflammatory targets.

Cold-inducible RNA-binding protein (CIRP) is a member of the cold shock protein family, which was first discovered in 1997[9]. Decades of research have confirmed that as a low level constitutively expressed protein CIRP is highly conserved in mammalian cells. CIRP was originally described as a nuclear protein whose synthesis was induced by mild hypothermia. Increasing evidences have shown that CIRP can be synthesized in large quantities under stress conditions, such as hypothermia, hypoxia, and UV irradiation, and transferred from the nucleus to the cytoplasm even released out of the cells[10–12]. Previous studies have demonstrated that as a novel damage-associated molecular pattern (DAMP), released CIRP can increase the level of proinflammatory cytokines, such as TNF-α and IL-6 through the Toll-like receptor 4 (TLR4)-mediated pathway[13]. In a rat model of cerebral ischemia, CIRP has been proven to be a detrimental factor in stimulating inflammation to cause brain damage[14]. In addition, CIRP knockout rats had reduced central nervous system inflammation and microglial activation compared with wild-type rats [15]. However, the relationship between PCAS and CIRP-related inflammation has rarely been studied.

In recent years, a 15-mer peptide (GRGFSRGGGDRGYGG) derived from human CIRP called C23 has been forward. It has a high affinity for the TLR4-myeloid differentiation factor 2 (MD2) complex. Peptide C23 can competitively bind to the TLR4/MD2 complex receptor with CIRP, and attenuate the downstream proinflammatory effects of CIRP. Some studies showed that C23 could play a protective role in renal injury, lung injury, and intestinal ischemia reperfusion injury[16–18]. However, whether C23 has a protective role in PCAS remains unclear.

In previous studies, our laboratory has demonstrated that CA/CPR can increase CIRP expression in brain tissues[19]. In this study, we hypothesize that systemic inflammatory response after CA is caused, at least in part, by released CIRP. C23 could attenuate peripheral and central inflammatory damage, protect neurons, and improve prognosis by inhibiting the binding of CIRP with TLR4 in a rat model of CA/CPR.

Experimental animals

Adult male Sprague-Dawley rats, weighing 280-320g, were provided by Animal Central of Southeast University Medical College. The temperature of the feeding room was kept at 21–25℃, the humidity is maintained at 45–50%, and there was a 12-hour light and dark cycle. The animal experimental ethical inspection form of Southeast University has been signed.

Animal Model Of Cardiac Arrest And Resuscitation

According to previous reports, we modified the preparation of cardiac arrest models[20]. Pentobarbital sodium (50mg/kg) was injected intraperitoneally for anesthesia. A 14-gauge cannula was used to perform tracheal intubation. Two 24G intravenous indwelling needles were inserted into the left femoral artery and vein (Fig. 1A), which can be used to monitor blood pressure and give fluids intravenously. Ventricular Fibrillation (VF) or pulseless electrical activity (PEA) was induced by transoesophageal cardiac pacing. Continuous cardiac pacing (frequency 25Hz; intensity 25V; stimulus duration width 10ms) was performed and maintained for at least 60 seconds to prevent spontaneous defibrillation. Then pause the pacing for 1–2 seconds to observe the changes in the electrocardiogram (ECG). Once the VF or PEA recovered automatically, additional 15 seconds of burst pacing was performed immediately until the VF of PEA persisted. The flow chart of the establishment of the CA model was shown in Fig. 1B, C. 5 minutes after the onset of VF or PEA, advanced cardiac life support was initiated with pure oxygen mechanical ventilation (70 breaths/min, 6 ml/kg). The same investigator performed manual chest compressions at a rate of 180 compressions/min, with equal compression-relaxation duration. At the beginning of CPR, adrenalin (5ug/100g) and 0.2 ml sodium bicarbonate (5%) were administrated. Repeat the dosing at 2 minutes intervals as needed. If the return of spontaneous circulation (ROSC) did not resume within 10 minutes, the CPR failed. ROSC was defined as recovery of supraventricular rhythm with mean aortic pressure (MAP) ≥ 50 mmHg for at least 30 minutes. We used a heating pad to maintain rectal temperature around 37 ℃ during resuscitation. All catheters, including the endotracheal tube, were removed after the rats had regained consciousness. Then they were placed back in cages, and all the rats were closely monitored for 72 hours.

Experiment Design

All the rats were randomly assigned to these groups: sham group (S), ischemia reperfusion group (IR), C23 group (C23), and ischemia reperfusion normal saline control group (IR-control). According to different time points of reperfusion, the IR group was divided into IR6h, IR12h, and IR24h subgroups. The rats in the sham group only received endotracheal intubation and arteriovenous puncture. The rats in group IR, C23, and IR-control underwent CA for 5 minutes followed by CPR. According to previous studies, C23 (8mg/kg) was administrated at the beginning of reperfusion in group C23. As a control, rats in group IR-control were administered the same dose of normal saline at the same time points. Synthetic C23 (GRGFSRGGGDRGYGG) was obtained from Synpeptide Co., Ltd (Nanjing, China; >98% purity). The quality test report is attached in the supplementary material.

Collection Of Blood And Brain Tissues

About 4ml blood was drawn from the heart, then it was centrifuged at 3,000g for 5 minutes. Taking the upper layer of serum stored at -80°C until assayed. A section of brain tissues was preserved in 4% paraformaldehyde for pathological analysis. The remaining brain tissues were snap-frozen in liquid nitrogen and stored at -80°C until assayed.

Western Blot Analysis

The level of TNFα, IL-6, IL-1β, cysteinyl aspartate specific proteinase 3(Caspase3), Bcl2-Associated X (Bax), B-cell lymphoma 2 (Bcl2) and TLR4 in hippocampus and cortex region were separately detected by western blot at 24 hours after ROSC. 15% SDS-PAGE was used to separate samples, and then transferred to polyvinylidene difluoride (PVDF) membranes. Nonspecific binding sites were blocked by 3% bovine serum albumin (BSA), the PVDF membranes were incubated with primary anti-CIRP antibody (1:100, Affinity Biosciences), TNFα (1:1000, Cell Signaling Technology), IL-6 (1:1000, Cell Signaling Technology), IL-1β (1:1000, Cell Signaling Technology), Caspase3 (1:1000, Affinity Biosciences), Bax (1:1500, Affinity Biosciences), Bcl2 (1:1500, Affinity Biosciences) and TLR4 (1:1200, Affinity Biosciences) at 4℃ for one night. The PVDF were then incubated with second antibody for 1 hour. Immunoreactive bands were identified by chemiluminescence method, and the gray values of immunoreactive bands were calculated by ImageJ software.

Immunofluorescence Staining

The brain was removed and coronal tissue from 1 to 4mm posterior to the optic chiasm was removed. Nonspecific binding sites were blocked with 3% BSA. Then, the following primary antibodies were used to incubate overnight: anti-TLR4 (1:500, Affinity Biosciences), anti-CIRP antibody (1:300, Affinity Biosciences). The secondary antibodies with fluorescence were incubated for 1 hour. Then, the slices labeled with DAPI. Nikon ECLIPSE Ti microscope was used to observe the images (Nikon, Tokyo, Japan).

Enzyme-linked Immunosorbent Assay

We used Enzyme-linked immunosorbent assay (ELISA) to measure the levels of TNFα, IL-6, IL-1β, CIRP and LDH in serum and tissues. The levels of these proteins were detected by ELISA kits (Jingmei Biotech. Co., China) according to the manufacture.

Tunel Assay And He Staining

Sections were perfused with xylene, sequentially rinsed with various concentrations of ethanol, and blocked in 3% H₂O₂. These sections were incubated in 5% BSA for 30 minutes, followed by labeling of DNA fragments with terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay (TUNEL) reaction solution for 1 hour at 37˚C.Converter peroxidase were added at 37˚C. DAB staining solution was added 30 minutes earlier to show TUNEL-positive nuclei. For HE, these sections were cut and stained with hematoxylin and eosin. Survival neuronal in CA1 region of hippocampus were counted by 2 investigators blinded to treatment assignment.

Neurological Function

Neurological function was assessed by the neurological deficit scoring system (NDS), which has been described in previous studies[21]. The contents of NDS related to consciousness, nervous reflex, motor, sensory and coordination. Scores range from 0 to 500 (0 brain dead, 500 normal).

Statistical analysis

Data conforming to normal distribution were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and Bonferroni correction were used for pairwise comparisons between multiple groups. The survival rate was calculated by Kaplan–Meier analysis and the log-rank test. The NDS was analyzed by Kruskal-Wallis analysis, as the data did not conform to the normal distribution. The Mann-Whitney U analysis was used for comparison between multiple experimental groups. A value of P ≤ 0.05 was considered statistically significant.

Physiological and animal model parameters

166 rats were enrolled in this experiment (Fig. 2). 22 rats were assigned to the sham group and the other 144 rats underwent CA and CPR. Of these, 114 rats were successfully resuscitated. The success rate of resuscitation was close to 80%. The baseline values of physiological variables, such as weight, heart rate (HR), mean arterial pressure (MAP), temperature, pH, PaO₂, PaCO₂, Na⁺, K⁺, and lactate in the four groups were not statistically significant (Table 1). In addition, the variables of CA/CPR model were similar among the four groups (Table 2). These data demonstrated that we successfully produced a rat model of CA/CPR and peptide C23 did not affect the related parameters of CA/CPR model.

Table 1

Baseline characteristics of all groups
	Sham	IR	C23	IR-control	P
Weight, g	299.3 ± 4.5	302.6 ± 6.4	303.8 ± 7.2	300.3 ± 7.9	0.83
HR, bpm	425.2 ± 18.7	420.3 ± 20.3	419 ± 13.1	421.3 ± 22.2	0.87
MAP, mmHg	116.1 ± 6.6	117.6 ± 7.6	118.9 ± 8.6	120 ± 9.4	0.79
Temperature, ℃	37.5 ± 0.5	37.2 ± 0.3	37.2 ± 0.2	37.3 ± 0.5	0.51
pH	7.41 ± 0.02	7.39 ± 0.02	7.39 ± 0.02	7.39 ± 0.03	0.87
PaO₂, mmHg	99.2 ± 7.9	100.9 ± 8.5	98.3 ± 11.6	101.7 ± 9.6	0.78
PaCO₂, mmHg	40.1 ± 2.8	40.2 ± 2.9	42.1 ± 2.6	40.1 ± 2.6	0.79
Na⁺, mM	141.8 ± 2.4	140.3 ± 2.2	140.6 ± 2.5	141.6 ± 2.1	0.75
K⁺, mM	3.83 ± 0.14	3.89 ± 0.11	3.91 ± 0.09	3.90 ± 0.11	0.85
Lactate, mM	0.89 ± 0.22	0.81 ± 0.13	0.91 ± 0.11	0.85 ± 0.13	0.47
Data are expressed as mean ± SD.
pH: potential of hydrogen; HR, heart rate; bpm, beats per minute; MAP, mean arterial pressure; PaO₂, arterial oxygen pressure; PaCO₂, arterial carbon dioxide pressure.

Table 2

Variables of cardiac arrest and resuscitation model
	IR	C23	IR-control	P
Preparation time, minutes	30.6 ± 3.1	31.8 ± 4.1	33.8 ± 6.1	0.38
Time to ROSC, seconds	98.3 ± 12.1	102.6 ± 15.5	92.8 ± 8.7	0.61
Total dose of adrenalin, µg	20.3 ± 2.3	22.7 ± 2.7	22.7 ± 2.7	0.69
Maximal systolic BP after ROSC, mmHg	162.5 ± 13.4	167.9 ± 14.7	175 ± 10.7	0.71
Successful resuscitation	20/24	20/25	20/25	0.93
Temperature at the end of experiment, ℃	37.7 ± 0.4	37.7 ± 0.4	37.1 ± 0.5	0.85
Data are expressed as mean ± SD.
ROSC, return of spontaneous circulation; BP, arterial blood pressure.

The Expressions Of Cirp Was Increased In Brain Tissues And Serum After Ca/cpr

We used Elisa to find that CIRP expression increased at 6 hours after ROSC and peaked at 24 hours in the cortex (P = 0.002 versus sham; P = 0.017 versus 6h; P = 0.333 versus 12h) and hippocampus (P < 0.001 versus sham; P < 0.001 versus 6h; P = 0.471 versus 12h), which were consistent with our previous studies[19]. In addition, the expression of CIRP in serum also increased at 6 hours after ROSC and peaked at 24 hours after ROSC (P = 0.003 versus sham; P = 0.002 versus 6h; P = 0.232 versus 12h; Fig. 3). The results ensured that our CA/CPR model has done sufficient damage to generate extracellular CIRP. Therefore, this model was appropriate to test whether C23 could antagonize the CIRP-TLR4 complex.

The Expressions Of Inflammatory Cytokines Were Increased In Brain Tissues And Serum

We detected the expressions of inflammatory cytokines in brain tissues and serum by Elisa. The expressions of TNF- α, IL-1β, IL-6 increased at 6 hours after ROSC and peaked at 24 hours in cortex (P < 0.001, P < 0.001, P < 0.001 versus sham; P = 0.017, P = 0.006, P = 0.004 versus 6h; P = 0.403, P = 0.749, P = 0.037 versus 12h; Fig. 4A-C). The expressions of TNFα, IL-1β, and IL-6 also increased at 6 hours after ROSC and peaked at 24 hours in the hippocampus (P < 0.001, P < 0.001, P = 0.002 versus sham; P = 0.002, P < 0.001, P = 0.001 versus 6h; P < 0.001, P = 0.155, P = 0.012 versus 12h; Fig. 4D-E). Likewise, the level of TNF-α, IL-1β, and IL-6 in serum also increased at 6 hours after ROSC and peaked at 24 hours compared with other groups (P < 0.001, P < 0.001, P = 0.037 versus sham; P = 0.018, P < 0.001, P = 0.246 versus 6h; P = 0.017, P = 0.001, P = 0.553 versus 12h; Fig. 5A-C). Besides, we detected the expression of lactate dehydrogenase (LDH) in serum, which was released mainly by the liver, but also by the myocardium and skeletal muscle, kidney, and brain. The expression of LDH also peaked at 24 hours after ROSC (P < 0.001 versus sham; P = 0.003 versus 6h; P = 0.013 versus 12h; Fig. 5D). The above results showed that severe systemic inflammatory response and organ damage occur after CA/CPR. In addition, we found that the changing trend of CIRP was consistent with that of inflammatory factors in the first 24 hours after ROSC. It is reasonable to infer that at least part of the inflammation after PCAS is caused by CIRP-TLR4 pathway.

C23 Decreased The Expressions Of Inflammatory Cytokines And Ldh In Serum

Based on the previous results, 24 hours after ROSC was selected as the time point for studying C23. The level of TNF-α was significantly increased in the rats of IR and IR-control groups compared with the sham group(292 ± 20 versus 163 ± 22pg/ml, P < 0.001; 300 ± 30 versus163 ± 22pg/ml, P < 0.001), which was markedly reduced by C23 compared with IR-control group (193 ± 16 versus 300 ± 30pg/ml, P = 0.001; Fig. 6A). The level of IL-1β was markedly increased in the rats of the IR and IR-control groups compared with the sham group (515 ± 28 versus 234 ± 29pg/ml, P < 0.001; 499 ± 46 versus 234 ± 29pg/ml, P < 0.001), while C23 treatment could significantly decrease it compared with IR-control group (418 ± 45 versus 499 ± 46pg/ml, P < 0.001), but still higher than sham group (418 ± 45 versus 234 ± 29pg/ml, P < 0.001; Fig. 6B). Likewise, the level of IL-6 was markedly increased in the rats of the IR and IR-control groups compared with the sham group (114 ± 19 versus 89 ± 10 pg/ml, P = 0.037; 119 ± 15 versus 89 ± 10 pg/ml, P = 0.031), which was completely reversed by C23 treatment (72 ± 16 versus 119 ± 15 pg/ml, P = 0.001; Fig. 6C). The level of LDH was markedly increased in the rats of the IR and IR-control groups compared with the sham group (73 ± 9 versus 37 ± 8 ng/ml, P < 0.001; 77 ± 7 versus 37 ± 8 ng/ml, P < 0.001), which was completely reversed by C23 treatment (45 ± 10 versus 77 ± 7 ng/ml, P = 0.001; Fig. 6D). The above results indicated that C23 had an obvious anti-inflammatory effect in the blood.

C23 Decreased The Expressions Of Inflammatory Cytokines And Apoptotic Proteins In Brain Tissues

Peptides have been shown to cross the blood-brain barrier (BBB) [22]. In addition, the establishment of the CA model will lead to the destruction of the BBB, which also facilitates the passage of C23 through the BBB. To further observe C23's ability to penetrate BBB, we engineered C23 with autofluorescence FITC. As shown in Fig. 7, we scanned faint green fluorescence in the cortex and hippocampus at 24 hours after ROSC. Then we detected the effects of C23 on the inflammatory factors and apoptotic proteins in the brain tissues by western blot. As shown in Fig. 8A and B, the expressions of TLR4 in the cortex and hippocampus were increased in the rats of IR group and IR-control group (P < 0.001), which were totally abolished by C23 treatment in the cortex (P = 0.001) and partly reduced by C23 treatment (P = 0.008) in the hippocampus. In addition, immunofluorescence showed that C23 could decrease TLR4 expression and reduce the colocalization of TLR4 and CIRP (Fig. 8C and D). Compared with sham group the expressions of TNFα, IL-6, IL-1β, Caspase3, and Bax in cortex were significantly increased in the rats of IR group (P = 0.003, P < 0.001, P = 0.001, P = 0.002, P = 0.003) and IR-control group (P = 0.001, P < 0.001, P < 0.001, P = 0.001, P = 0.001), which were abolished by C23 treatment compared with IR-control group (P = 0.007, P = 0.001, P < 0.001, P = 0.007, P = 0.006). Compared with the IR-control group, C23 treatment could markedly increase the expression of Bcl2 in the cortex (P = 0.001; Fig. 9). Likewise, compared with the sham group the expressions of TNFα, IL-6, IL-1β, Caspase3, and Bax in the hippocampus were markedly elevated in the rats of the IR group (P < 0.001) and IR-control group (P < 0.001). In contrast, these levels were partly reduced in the C23-treated rats compared with group IR-control (P = 0.001, P = 0.001, P = 0.005, P = 0.003, P = 0.001). Compared with the IR-control group, C23 treatment could markedly increase the expression of Bcl2 in the hippocampus (P = 0.001; Fig. 10). The above results indicated that C23 exerted an anti-inflammatory and anti-apoptotic role in the cortex and hippocampus.

C23 Attenuated Hippocampus Neuronal Injury And Reduced Neuron Apoptosis

In the sham group, there was no injury, and the morphology of neurons was intact and arranged regularly. In contrast, at 24 hours after ROSC, there was severe histopathological damage in the CA1 region in IR and IR-control groups. The number of surviving neurons was reduced and their arrangement was disordered. At the same time, vacuolization, tissue edema, and pyknotic nuclei were also observed in the IR group and IR-control group. The number of surviving neurons in the IR group and IR-control groups were less than that in the sham group (23 ± 10 versus 106 ± 10, P < 0.001; 25 ± 8 versus 106 ± 10, P < 0.001). C23 treatment alleviate the abnormalities of the hippocampus CA1 region, and the surviving neurons were much more than IR-control group (61 ± 10 versus 25 ± 8, P = 0.012), however, still less than that in the sham group (61 ± 10 versus 106 ± 10, P = 0.023; Fig. 11A and B). The TUNEL-positive cells of hippocampus CA1 region were increased in the IR group and IR-control group compared with the sham group (78 ± 14 versus 4 ± 2%, P < 0.001; 68 ± 18 versus 4 ± 2%, P < 0.001), which were decreased by C23 treatment compared with the IR-control group (33 ± 15 versus 68 ± 18%, P = 0.013), but still more than that in the sham group (33 ± 15 versus 4 ± 2%, P = 0.029; Fig. 11C and D). The above results showed that C23 could play a protective role in hippocampus neurons.

Survival Rate And Neurological Deficit Scores

As shown in Fig. 12, the 7-day survival rate of rats in the IR group and IR-control group were approximately 35%-30% (P = 0.002, P = 0.001versus 100% in the sham group), which was significantly increased to 65% by C23 treatment (P = 0.05 versus IR-control group). Neurological function was evaluated at 24 hours and 48 hours after ROSC. Rats in the IR group and IR-control group showed neurological deficit at 24 hours (P < 0.001, P < 0.001 versus sham group) and 48 hours (P < 0.001, P < 0.001 versus sham group) after ROSC. C23 treatment could attenuate neurological deficit (P = 0.009, P = 0.045 versus IR-control group), but not completely reverse it (P = 0.032, P = 0.006versus sham group). These results indicated that C23 administered at the beginning of ROSC significantly improved survival rate and neurological outcome after CA/CPR.

In this study, we found severe systemic inflammation and elevated expression of CIRP in rats after CA/CPR. The CIRP derived peptide C23 not only reduced the expressions of TNFα, IL-1β, IL-6, and LDH in serum but also reduced the expressions of proinflammatory mediators in brain tissues. In particular, C23-treated rats exhibited significant decreases in apoptosis proteins and TUNEL-positive neurons in the hippocampus CA1 region, as well as improved survival rate and neurological function. These data demonstrated for the first time that C23 can inhibit peripheral and central inflammation and thus exert neuronal protective effects in a CA model.

Transoesophageal cardiac pacing-induced cardiac arrest is a typical model in that we can explore the systemic pathophysiological changes after cardiopulmonary resuscitation. Exogenous pathogen-associated molecular pattern molecules (PAMPs) during infection can trigger systemic inflammation, which also can be induced by endogenous damage-associated molecular pattern molecules (DAMPs) that are released by host cells during tissue injury[23]. Although the related pathological mechanisms PAMPs are well studied, the role of endogenous molecules in inducing inflammation after CA has not been clearly illustrated.

It is currently known that CIRP stabilizes specific mRNA and promotes the translation of cell survival advantages under stress. Previous studies by us and other researchers have shown that cold-induced CIRP has a protective effect[19, 24, 25]. In 2013, an article published in Nature Medicine confirmed for the first time that extracellular CIRP was a newly discovered DAMP. They demonstrated that the released CIRP could bind to the TLR4 receptor and trigger a downstream inflammatory response based on hemorrhagic shock and sepsis models[13]. The direct pro-inflammatory role of CIRP has been proved in different models, where it could induce the production of inflammatory cytokines to further exaggerate inflammation[14, 26]. The knockdown of CIRP decreased inflammatory cytokine expression[27]. The post-resuscitation phase after cardiac arrest is characterized by a systemic inflammatory response similar to other systemic inflammatory conditions such as severe sepsis. As a “sepsis-like” syndrome, there are many similarities in pathophysiology and even treatment strategies[28, 29]. In clinical research, elevated expression of DAMPs (HMGB-1) after CA increased the levels of inflammatory factors and may be associated with a poor neurological outcome[30]. Based on the above evidence, we speculated that CIRP expression would also increase after CA and activate downstream inflammatory factors.

In the current study, we found CIRP increased in both the cortex and hippocampus at 24 hours after ROSC, which was consistent with our previous studies. Besides, the levels of TNF-α, IL-1β, ans IL-6 were also elevated from 6 hours to peak at 12 or 24 hours after ROSC. These results were consistent with previous studies[31, 32]. In addition, we found for the first time that the expression of CIRP was increased in rat serum after CA/CPR. This also indicates that PCAS-related peripheral inflammation has a great correlation with the massive expression of extracellular CIRP. To further demonstrate that the systemic inflammatory response and organ damage after PCAS are caused by CIRP, here we used a short peptide C23 to test the effectiveness of CA/CPR in terms of reducing pro-inflammatory response and brain injury.

C23 is a 15-amino-acid peptide derived from the human CIRP protein (Ser110-Glu125). It has been shown to be protective and reduce inflammation and injury in models of bleeding, renal ischemia reperfusion, and sepsis[16, 13, 17, 18]. C23 and TLR4 have a strong affinity, thus inhibiting CIRP binding to TLR4/MD2 complex[13]. Toll-like receptors are widely expressed in brain tissues, myocardial tissues, and serum in CA/CPR model[33–36]. Both the PAMPs and DAMPs can activate TLR4 receptors on the surface of cells. And the activated TLR4 induces an inflammatory response characterized by the release of chemokines and cytokines that attract innate immune cells[37]. In this study, we demonstrated that C23 can reduce the levels of inflammatory cytokines in serum, which were consistent with other studies[38, 16]. Although we did not directly address the role of C23 in binding and antagonizing TLR4/MD2 in serum, our study did demonstrate the effectiveness of C23 in inhibiting CIRP-induced proinflammatory responses in CA/CPR models, possibly by interfering with CIRP binding to its receptor. Then, we sought to illustrate the anti-inflammatory effect of C23 in brain tissues. As we know, CA/CPR caused a breakdown of the BBB in the first 24 hours after ROSC, which facilitates the entry of the molecule into brain tissues[39, 40]. C23, a small peptide, was also theoretically capable of, or at least partially, crossing the BBB[22, 41, 42]. Western blot and immunofluorescence results showed that C23 could inhibit CIRP binding to TLR4 and inhibit TLR4 activation in the cortex and hippocampus. As expected C23 treatment could significantly reduce the downstream expressions of TNFα, IL-1β, IL-6, Caspase3, and Bax and increase the expression of Bcl2 in the cortex and hippocampus. Furthermore, we engineered C23 with autofluorescence FITC in hope to observe it in brain tissues. And we scanned very faint green fluorescence in the cortex and hippocampus tissues 24 hours after ROSC. These results directly illustrated that C23 can competitively bind to the TLR4/MD2 complex receptor with CIRP and antagonize CIRP-TLR4/MD2 inflammatory pathways. Besides, we also noted that C23 almost completely abolished the expression of inflammatory factors and apoptotic proteins in the cortex, which may be related to the more C23 reached the cortex. In addition, histological findings revealed less tissue damage and TUNLE-positive cells of hippocampus CA1 region in C23 group. And the peripheral and central anti-inflammatory effects of C23 improved the survival rate and neurological functions of rats after CA/CPR.

There are also some limitations in this study. Firstly, the dose and administration of C23 were decided based on previous studies, but it is not yet clear whether higher doses or a more prolonged continuous infusion bring better results. Besides, we only observed the changes in inflammatory factors and CIRP in the first 24 hours after ROSC, but the subsequent changes are not clear. In addition, due to the existence of the blood-brain barrier, a portion of C23 may not reach the brain parenchyma, intrathecal administration may be a good option. Finally, we did not isolate intracellular or extracellular proteins in either the cortex or hippocampus. As we know CIRP plays different roles depending on its location, so subsequent cell experiments are needed to further clarify the problem.

In summary, we demonstrated the effectiveness of C23 with significantly increased survival rate and improved neurological functions. These advantages were closely related to the decreases in peripheral and central inflammation, brain tissue damage, and neuron apoptosis seen in the C23 treatment group. Although hypothermia is still the first choice for the treatment of CA patients, seeking a combination therapy is also a good clinical treatment strategy. Therefore, we recommend the development of this novel therapy. The therapeutic window, the optimal route of administration, and the timing of C23 would be detected by preclinical work. Eventually, the hope is to translate this approach to CA/CPR treatment in humans.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 82102309 and 82071196)

Funding

This work was supported by the National Natural Science Foundation of China (No. 82102309 and 82071196).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Disclosure of Interest

The authors declare no conflicts of interest.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection were performed by Yu Gao and Haoxin Liu. Jiejie Zhou had done English editing. Min Guo and Jie Sun carried out literature research, data acquisition, and data analysis. Manlin Duan did article editing and article revision. The first draft of the manuscript was written by Yu Gao and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval

The experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China. The animal experimental ethical inspection form of Southeast University has been signed (NO.20210301033).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

Not applicable

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 82102309 and 82071196)

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The protective effect of a short peptide derived from cold-inducible RNA-binding protein in a rat model of cardiac arrest and resuscitation

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Abstract

Figures

Introduction

Methods

Experimental animals

Animal Model Of Cardiac Arrest And Resuscitation

Experiment Design

Collection Of Blood And Brain Tissues

Western Blot Analysis

Immunofluorescence Staining

Enzyme-linked Immunosorbent Assay

Tunel Assay And He Staining

Neurological Function

Statistical analysis

Results

Physiological and animal model parameters

The Expressions Of Cirp Was Increased In Brain Tissues And Serum After Ca/cpr

The Expressions Of Inflammatory Cytokines Were Increased In Brain Tissues And Serum

C23 Decreased The Expressions Of Inflammatory Cytokines And Ldh In Serum

C23 Decreased The Expressions Of Inflammatory Cytokines And Apoptotic Proteins In Brain Tissues

C23 Attenuated Hippocampus Neuronal Injury And Reduced Neuron Apoptosis

Survival Rate And Neurological Deficit Scores

Discussion

Declarations

References

Supplementary Files

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