RESEARCH ARTICLE

Cholinergic anti-inflammatory pathway confers protective effects in septic shock rats

Yuchao Shen and Ying Cui^*

Department of Emergency Brain Academy District, Cangzhou Central Hospital, Cangzhou, Hebei, China

Abstract

Background: Septic shock can lead to multiple organ dysfunction. The cholinergic anti-inflammatory pathway is known to prevent organ damage from infection by modulating the inflammatory response. However, the modulatory capacity of cholinergic anti-inflammatory pathways in septic shock remains unclear.

Methods: Cecal ligation and puncture (CLP) was used to establish a rat model of septic shock. Bilateral cervical vagal nerve isolation, dual cervical vagotomy, and vagus nerve stimulation (VNS) were used to inhibit or activate cholinergic anti-inflammatory pathways. Tacrine and α-bungarotoxin groups were used to mimic the activation and shutdown of cholinergic anti-inflammatory pathways. The survival status, hemodynamic indicators, inflammatory cytokine content, and inflammatory response of liver and kidney tissues of the rats were detected.

Results: Dual cervical vagotomy significantly exacerbated the hepatic (P < 0.05) and renal (P < 0.05) impairment in septic shock rats relative to the sham-operated group. Tacrine, a cholinergic potentiator, significantly alleviated liver (P < 0.05) and kidney (P < 0.05) damage in septic shock rats. Dual cervical vagotomy significantly promoted the expression of pro-inflammatory factors caused by septic shock in rat liver and kidney tissues (P < 0.05), while Tacrine treatment inhibited the increase of inflammatory factors (P < 0.05).

Conclusions: The activation of the cholinergic anti-inflammatory pathway exerts protective effects on multiple organ dysfunction caused by septic shock. The cholinergic enhancer, Tacrine, and VNS effectively ameliorated liver and kidney damage, improved hemodynamic indicators, and enhanced survival rates in septic shock rats.

Keywords: cholinergic anti-inflammatory pathways; Septic shock; Vagotomy; organ dysfunction

 

 

Sepsis is a systemic inflammatory response syndrome caused by the invasion of pathogenic microorganisms such as bacteria or fungi (1, 2). In addition to the manifestations of extensive inflammatory reactions and primary infection lesions, patients with severe sepsis often have manifestations of organ hypoperfusion, generally including organ failure and hypotension (3, 4). Septic shock is defined as severe sepsis with uncorrectable persistent hypotension despite adequate fluid resuscitation and is also considered a special type of severe sepsis (5). The most common cause of septic shock is a bacterial infection, such as a urinary tract infection, pneumonia, or a skin infection (6, 7). Septic shock is a serious condition that can be life-threatening if left untreated. Early diagnosis and treatment can help prevent serious complications and improve the chances of a full recovery (8).

Inflammation is a normal response of the body to injury or infection (9). The correlation between septic shock and inflammation is well established (9, 10). Septic shock is caused by an overwhelming immune response to an infection (11). This response leads to an excessive release of inflammatory mediators, such as cytokines, which could cause widespread inflammation throughout the body (12). Inflammation could also lead to the release of toxins from the bacteria that cause the infection (13). Thus, septic shock or sepsis is essentially an uncontrolled self-destructive systemic inflammatory process (14). Sepsis is a systemic inflammatory response to infection, and septic shock is a severe form or stage of sepsis (15).

Traditionally, it is believed that the nervous system only regulates the inflammatory response through the neurohumoral pathway of the hypothalamus-pituitary gland (16). In recent years, it has been discovered that there is a cholinergic anti-inflammatory pathway composed of the vagus nerve and its transmitter acetylcholine between the nervous system and the immune system. Initial studies have shown that the efferent component of vagus nerve stimulation (VNS) attenuates the inflammatory response by modulating the function macrophages (17). It has also been demonstrated that electrical stimulation of the vagus nerve improves intestinal blood flow after trauma and hemorrhagic shock. Therefore, targeting the cholinergic anti-inflammatory pathway composed of the vagus nerve and acetylcholine to reveal new treatment options may be an effective way to treat septic shock. This study aimed to investigate the protective effect of cholinergic anti-inflammatory pathway on rats with septic shock. We expect to provide more treatment ideas for clinical prevention and treatment of septic shock and reduce the burden on patients.

Methods

Animals

Male Sprague-Dawley rats (6–8 weeks, 220–240 g) were housed and provided with sterile feed and water in a circadian cycle. The rats were randomly divided into six groups. 1) Sham operation group (Sham): sham cecal ligation and puncture (CLP) + double cervical vagus nerve trunk isolation. 2) Model group (Vehicle): CLP surgery + double cervical vagus nerve trunk isolation. 3) Vagotomy group (Vagotomy): CLP surgery + double neck vagus nerve transection (vagotomy). 4) VNS group (18): CLP surgery + double cervical vagus nerve trunk isolation + left distal VNS. 5) Tacrine group (Tacrine) (19): CLP surgery + double cervical vagus nerve trunk isolation + iliac vein injection of tacrine (tetrahydroaminoacridine) 1.5 mg/kg. 6) α-Bungarotoxin group (VNS+α-BGT) (20): CLP surgery + double cervical vagus nerve trunk isolation + iliac vein injection of α-bungarotoxin α-bungarotoxin (α-BGT) 1.0 μg/kg + electrical stimulation of the distal left vagal trunk. The death of rats in each group within 72 h after operation was counted, and 10 rats were randomly selected in each group to extract 8 mL of rat-tail venous blood 4 h after operation. Ten rats in each group that survived 4 h after the operation were euthanized to obtain liver and kidney tissues and obtain tissue homogenate. Inflammation indexes of tissue homogenates were measured respectively. Animal studies were approved by the ethics committee of Cangzhou Central Hospital. This study was performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985).

Cecal ligation and puncture

CLP was used to construct a rat model of septic shock. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium 40 mg/kg. The limbs of anesthetized rats were fixed on homemade smooth foam boards with pressure-sensitive tape. The rat common carotid artery and vagus nerve were carefully stripped out on a sterile operating table. The common carotid artery was cannulated. Pressure transduction systems and monitors were used to continuously monitor arterial pressure. A 2–3 cm long incision was made in the middle of the anterior abdomen of the rat, and the mesentery and cecum were mobilized. A 3.0 silk thread was used for a circular ligation of the root of the cecum. A 9-gauge needle was used to puncture the blind end of the rat in two places, and the distance between the two needle holes was about 3 mm. Rats were sutured layer by layer to close the abdomen. After the operation, the rats received subcutaneous injection of normal saline (30 ml/kg) to replenish body fluids. Povidone iodine was used to disinfect and dress wounds. Animals in the sham operation group received the same procedures as above except that the cecum was not ligated and perforated. The model was considered successful when the mean arterial pressure dropped to 2/3 of the initial blood pressure. The arteriovenous catheter was removed after successful modeling.

Vagotomy

After being anesthetized, the rats were fixed in the supine position for skin preparation. The incision was made about 2.5–4 cm long along the midline of the neck and abdomen between the larynx and the sternum. Hemostats were used to separate the rat sternohyoid muscle from the sternothyroid muscle. The carotid sheath (which houses the carotid artery and cervical nerves) was shown. The common carotid artery and vagus nerve were bluntly dissected approximately 3–4 cm. 4.0 silk suture was used to ligate the vagus nerve.

Electrical stimulation of the distal left vagus nerve trunk

Platinum electrodes were used to loop around the vagus nerve in the left neck. The platinum electrodes were connected to a neurostimulator with parameters set at 5 V, 2 Hz, 1 millisecond pulse width, and 20 min. Stimulation was continued for 20 min immediately after CLP.

Liver functional assays

Aspartate transaminase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and low-density lipoprotein (LDL) were used to assess liver function in rats. Automatic biochemical analyzer (Hitachi, Tokyo, Japan) was used to analyze the above liver function indexes in rats, according to the manufacturer’s instructions.

Kidney functional assays

Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured using corresponding commercial kits (Roche).

ELISA

ELISA was used to detect the inflammatory indicators TNF-α (ab236712 for serum, ab100785 for tissue homogenates, Abcam), IL-1β (ab255730 for serum, ab100768 for tissue homogenates, Abcam), infectious markers procalcitonin (PCT, ER1235, FineTest), and C-reactive protein (CRP, C-reactive protein, Elabscience) in rats. Ten rats in each group that survived 4 h after operation were selected (21). Their liver and kidney tissues were harvested and homogenized. Inflammation indexes of tissue homogenates were measured accordingly.

Statistical analysis

SPSS 19.0 statistical software package was used for statistical analysis. The measurement data were described by means ± standard deviation. Data were analyzed by Log-rank (Mantel-Cox) test or one-way analysis of variance with a post hoc test. P < 0.05 was considered statistically significant.

Results

Overall survival of rats during 72 h recording post-CLP

We first investigated the impact of cholinergic anti-inflammatory pathways on the survival of rats with septic shock. As shown in Fig. 1, the sham operation group showed normal survival rates. Dual cervical vagotomy exacerbated the death of rats with septic shock, reaching statistical significance (P < 0.001). Tacrine, a cholinergic enhancer drug, improved the survival of rats within 72 h, but without significant difference. Distal electrical stimulation of the left vagus nerve trunk (VNS) reduced the death of septic shock rats within 72 h, reaching statistical significance (P < 0.05). However, α-bungarotoxin counteracted the effect of VNS (P < 0.05).

Fig. 1. Overall survival of rats during 72 h recording post-CLP from each group. n = 20 for each group. * P < 0.05, *** P < 0.001 compared to vehicle. ^ P < 0.05 between the indicated groups. Log-rank (Mantel-Cox) test.

Effects of cholinergic anti-inflammatory pathway on physiological indicators in rats with septic shock

We further analyzed the effects of cholinergic anti-inflammatory pathways on heart rate (HR) (Fig. 2a), blood pressure (Fig. 2b), and hemodynamic indicators (Fig. 2c, d) in septic shock rats. The HR (beats/min) of septic shock rats in Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 481.08 ± 24.68, 266.91 ± 20.75, 187.47 ± 24.34, 355.04 ± 33.75, 377.82 ± 30.21, and 286.86 ± 31.22, respectively. The MAP (mmHg) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 97.76 ± 4.55, 61.85 ± 7.98, 52.22 ± 6.61, 77.98 ± 8.53, 85.62 ± 8.79, and 72.85 ± 6.69, respectively. The +dp/dtmax (mmHg/ms) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 3.58 ± 0.31, 1.36 ± 0.28, 0.82 ± 0.24, 2.37 ± 0.38, 2.73 ± 0.43, and 1.96 ± 0.37, respectively. The -dp/dtmax (mmHg/ms) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 2.88 ± 0.29, 1.01 ± 0.17, 0.69 ± 0.18, 1.84 ± 0.38, 2.17 ± 0.26, and 1.33 ± 0.28, respectively. Dual cervical vagotomy exacerbated the changes of HR (187.47 ± 24.34 vs. 481.08 ± 24.68, P < 0.001), blood pressure (52.22 ± 6.61 vs. 97.76 ± 4.55, P < 0.001), and hemodynamic indexes (0.82 ± 0.24 vs. 3.58 ± 0.31/0.69 ± 0.18 vs. 2.88 ± 0.29, P < 0.001) of septic shock rats compared with the Sham group, reaching statistical significance. As a cholinergic enhancer, Tacrine could improve the HR (355.04 ± 33.75 vs. 481.08 ± 24.68, P < 0.001), blood pressure (77.98 ± 8.53 vs. 97.76 ± 4.55, P < 0.01), and hemodynamic indicators (2.37 ± 0.38 vs. 3.58 ± 0.31/1.84 ± 0.38 vs. 2.88 ± 0.29, P < 0.001) of rats in septic shock, reaching statistical significance. VNS improved HR (P < 0.001), blood pressure (P < 0.001), and hemodynamic indicators (P < 0.001) in rats with septic shock statistically compared with the Vagotomy group. However, α-bungarotoxin partially counteracts the effect of VNS (P < 0.05).

Fig. 2. Comparisons of HR (a), MAP (b), +dp/dtmax (c), and -dp/dtmax (d) of rats from each group at 4 h post-CLP. n = 10 for each group.

Effects of cholinergic anti-inflammatory pathway on liver function in rats with septic shock

To study the protective effect of cholinergic anti-inflammatory pathway on organ damage caused by septic shock, we analyzed the liver function of postoperative rats. We compared serum levels of LDH (Fig. 3a), LDL (Fig. 3b), ALT (Fig. 3c), and AST (Fig. 3d) of rats of each group at 4 h post-CLP. The serum LDH (U/L) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 75.84 ± 14.25, 438.91 ± 83.29, 612.89 ± 117.73, 312.39 ± 67.82, 243.77 ± 55.84, and 321.44 ± 61.24, respectively. The serum LDL (mmol/L) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 1.85 ± 0.38, 5.37 ± 1.36, 7.11 ± 1.07, 3.26 ± 0.63, 2.89 ± 0.65, and 3.86 ± 1.11, respectively. The serum ALT (U/L) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 9.23 ± 4.39, 475.76 ± 68.93, 601.42 ± 69.81, 278.85 ± 53.29, 243.78 ± 57.93, and 360.67 ± 76.55, respectively. The serum AST (U/L) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 9.98 ± 3.39, 604.86 ± 97.03, 786.79 ± 104.11, 436.83 ± 82.94, 375.84 ± 68.81, and 458.68 ± 91.63, respectively. Dual cervical vagotomy upregulated the levels of LDH (P < 0.001), LDL (P < 0.001), ALT (P < 0.001), and AST (P < 0.001) in the serum of rats. Tacrine reduced the levels of LDH (P < 0.01), LDL (P < 0.01), ALT (P < 0.001), and AST (P < 0.01) in the serum of rats with septic shock. VNS at the distal end of the left vagus nerve trunk also reduced the levels of LDH (P < 0.001), LDL (P < 0.001), ALT (P < 0.001), and AST (P < 0.001) in the serum of rats with septic shock. However, α-bungarotoxin partially offset the effect of VNS (P < 0.05).

Fig. 3. Comparisons of serum levels of LDH (a), LDL (b), ALT (c), and AST (d) of rats from each group at 4 h post-CLP. n = 10 for each group.

Effects of cholinergic anti-inflammatory pathway on renal function in rats with septic shock

In order to study the protective effect of cholinergic anti-inflammatory pathway on organ damage caused by septic shock, we analyzed the renal function of postoperative rats. We compared serum levels of BUN (Fig. 4a) and creatinine (Fig. 4b) of rats from each group at 4 h post-CLP. Serum BUN (mg/dL) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 15.47 ± 3.85, 76.57 ± 16.85, 98.84 ± 14.14, 51.65 ± 12.96, 42.84 ± 11.71, and 62.87 ± 16.45, respectively. Serum creatinine (mg/dL) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 0.48 ± 0.08, 1.87 ± 0.35, 2.43 ± 0.39, 1.34 ± 0.28, 1.18 ± 0.28, and 1.96 ± 0.37, respectively. Bilateral cervical vagotomy upregulated the BUN (P < 0.001) and creatinine (P < 0.001) levels in septic shock rats. Tacrine reduced the BUN (P < 0.01) and creatinine (P < 0.01) levels of rats with septic shock. VNS also reduced the BUN (P < 0.001) and creatinine (P < 0.001) levels of rats with septic shock. However, α-bungarotoxin partially counteracted the effect of VNS (P < 0.05).

Fig. 4. Comparisons of serum levels of BUN (a) and creatinine (b) of rats from each group at 4 h post-CLP. n = 10 for each group.

Effect of cholinergic anti-inflammatory pathway on serum inflammation level and infectious index in rats with septic shock

We further studied the effect of cholinergic anti-inflammatory pathway on serum inflammation level and infectious index in rats with septic shock. We compared serum levels of PCT (Fig. 5a), CRP (Fig. 5b), TNF-α (Fig. 5c), and IL-1β (Fig. 5d) of rats from each group at 4 h post-CLP. Serum PCT (ng/mL) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 1.76 ± 0.31, 25.86 ± 4.75, 34.05 ± 3.84, 14.53 ± 3.85, 11.46 ± 3.72, and 19.94 ± 4.21, respectively. Serum CRP (mg/L) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 5.03 ± 2.18, 25.34 ± 3.79, 32.42 ± 6.51, 16.64 ± 3.32, 14.36 ± 3.27, and 22.84 ± 4.74, respectively. Serum TNF-α (pg/mL) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 23.35 ± 5.97, 154.82 ± 30.94, 200.78 ± 37.62, 107.12 ± 22.18, 88.96 ± 19.59, and 134.79 ± 28.99, respectively. Serum IL-1β (pg/mL) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 44.85 ± 8.98, 188.02 ± 40.53, 273.54 ± 45.31, 136.69 ± 34.42, 122.63 ± 31.17, and 196.57 ± 42.64, respectively. Double cervical vagotomy significantly upregulated the levels of inflammation factors in septic shock rats (P < 0.05). Tacrine treatment suppressed the levels of inflammation factors in septic shock rats (P < 0.05). Similarly, VNS also suppressed the levels of inflammation factors in septic shock rats. α-Bungarotoxin partially counteracted the effect of VNS (P < 0.05).

Fig. 5. Comparisons of serum levels of PCT (a), CRP (b), TNF-α (c), and IL-1β (d) of rats from each group at 4 h post-CLP. n = 10 for each group.

Effect of cholinergic anti-inflammatory pathway on liver and kidney inflammation in septic shock rats

In order to study the protective effect of cholinergic anti-inflammatory pathway on organ inflammation caused by septic shock, we analyzed the inflammatory indicators of postoperative rat liver and kidney. We compared the concentrations of TNF-α (Fig. 6a) and IL-1β (Fig. 6b) in the liver, as well as TNF-α (Fig. 6c) and IL-1β (Fig. 6d) in the kidney of rats from each group at 4 h post-CLP. Liver IL-1β (pg/mg tissue) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 11.26 ± 2.25, 79.96 ± 19.95, 108.50 ± 22.13, 55.46 ± 14.27, 38.75 ± 12.14, and 67.97 ± 19.84, respectively. Renal IL-1β (pg/mg tissue) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 12.34 ± 3.98, 112.77 ± 23.58, 145.71 ± 24.06, 47.85 ± 18.47, 38.74 ± 14.39, and 81.74 ± 20.08, respectively. Liver TNF-α (pg/mg tissue) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 9.35 ± 2.73, 78.75 ± 14.15, 98.46 ± 10.62, 42.46 ± 10.61, 40.36 ± 9.21, and 52.29 ± 11.18, respectively. Renal TNF-α (pg/mg tissue) of septic shock rats in the Sham group, Vehicle group, Vagotomy group, Tacrine group, VNS group, and VNS+α-BGT group was 12.47 ± 3.54, 85.76 ± 13.22, 105.19 ± 13.88, 56.46 ± 12.15, 47.68 ± 11.16, and 68.49 ± 14.78, respectively. Double cervical vagotomy significantly upregulated the levels of inflammation factors in the liver and kidney tissues of septic shock rats (P < 0.05). Tacrine treatment suppressed the levels of inflammation factors in the liver and kidney tissues of septic shock rats (P < 0.05). Similarly, VNS also suppressed the levels of inflammation factors in the liver and kidney tissues of septic shock rats (P < 0.05). α-Bungarotoxin partially counteracted the effect of VNS (P < 0.05).

Fig. 6. Comparisons of concentrations of TNF-α (a) and IL-1β (b) in the liver, as well as TNF-α (c) and IL-1β (d) in the kidney of rats from each group at 4 h post-CLP. n = 10 for each group.

Discussion

Sepsis is a severe inflammatory response syndrome that can result in multiple organ dysfunction and high mortality rates (22). It is a type of shock that is caused by a severe infection, usually bacterial (23). The incidence of septic shock is increasing. It is estimated that there are more than 750,000 cases of septic shock in the United States each year (24). Certain populations, such as the elderly, with weakened immune systems and with chronic illnesses are at a higher risk of developing septic shock (25). Treatment for septic shock includes antibiotics, fluids, and medications to support blood pressure and organ function. In some cases, surgery may be necessary to remove the source of the infection (26). The goal of treatment is to reduce the infection and restore blood pressure and organ function. The cure rate of septic shock depends on several factors, including the severity of the infection, the patient’s age and overall health, and the speed of diagnosis and treatment (27). Early diagnosis and treatment are essential for improving the cure rate of septic shock (28). Despite advances in medical technology, septic shock remains a major cause of death in critically ill patients (27). Therefore, it is necessary to explore new ways to treat septic shock.

The cholinergic anti-inflammatory pathway has been reported to play a crucial role in regulating the inflammatory response and reducing mortality in septic shock (29). In the present study, we investigated the impact of cholinergic anti-inflammatory pathways on survival and physiological indicators of rats with septic shock. The results of this study showed that dual cervical vagotomy exacerbated the death of rats with septic shock, while distal electrical stimulation of the left vagus nerve trunk reduced the death of septic shock rats within 72 h, reaching statistical significance. These findings are consistent with previous studies that have shown that the cholinergic anti-inflammatory pathway plays a crucial role in the regulation of the inflammatory response during sepsis (30–32). In addition, the present study also analyzed the effects of cholinergic anti-inflammatory pathways on HR, blood pressure, and hemodynamic indicators in septic shock rats. Dual cervical vagotomy exacerbated the changes of these indicators in septic shock rats, while Tacrine, a cholinergic enhancer drug, and distal electrical stimulation of the left VNS significantly improved these indicators. These findings are consistent with previous studies that have shown that the cholinergic anti-inflammatory pathway can regulate cardiovascular function during sepsis (33, 34).

Furthermore, the present study analyzed the protective effect of cholinergic anti-inflammatory pathways on organ damage caused by septic shock. Double cervical vagal trunk dissection exacerbated liver and renal injury in septic shock rats, while Tacrine and distal electrical stimulation of the left VNS significantly improved liver and renal functions. These findings are consistent with previous studies that have shown that the cholinergic anti-inflammatory pathway can protect against organ dysfunction during sepsis (35). Finally, we analyzed the effect of cholinergic anti-inflammatory pathways on serum inflammation level and infectious index in rats with septic shock. Dual cervical vagotomy significantly exacerbated the levels of infection and inflammation in septic shock rats, while Tacrine and distal electrical stimulation of the left VNS suppressed these levels. These results suggest that the cholinergic anti-inflammatory pathway can regulate the inflammatory response in septic shock and protect against infection. These findings are consistent with previous studies that have shown that the cholinergic anti-inflammatory pathway can regulate systemic inflammation during sepsis (36, 37).

However, one aspect that remains unaddressed in the current manuscript is the underlying mechanistic attributes of how the cholinergic anti-inflammatory pathway exerts its protective effects. Although the study demonstrated the pathway’s modulatory capacity and its impact on various physiological and biochemical markers, the specific molecular mechanisms involved were not thoroughly investigated. Understanding the detailed cellular and molecular pathways through which the cholinergic anti-inflammatory pathway regulates inflammation and protects against organ damage would provide valuable insights and could pave the way for targeted therapeutic interventions in septic shock.

Future studies could explore the downstream signaling pathways activated by cholinergic stimulation in immune cells, such as macrophages and lymphocytes, as these cells play a central role in the immune response during septic shock. Investigating the interaction between the vagus nerve and immune cells in various organs would provide a more comprehensive understanding of the pathway’s protective effects on multiple organ dysfunction.

Moreover, investigating the role of specific acetylcholine receptors, such as the alpha 7 nicotinic acetylcholine receptor (α7nAChR), in mediating the anti-inflammatory effects of the cholinergic pathway could shed light on the precise targets for therapeutic interventions. Activating α7nAChR has been shown to suppress inflammation and improve survival in various inflammatory conditions, and its involvement in the cholinergic anti-inflammatory pathway could provide a mechanistic basis for its protective effects in septic shock.

Conclusions

In conclusion, the manuscript presented compelling evidence that the activation of the cholinergic anti-inflammatory pathway exerts protective effects in septic shock rats. The study’s comprehensive experimental design and results provide valuable insights into the potential therapeutic benefits of targeting this pathway. However, further investigations into the underlying mechanistic attributes of the pathway’s protective effects would enhance our understanding and potentially lead to novel treatment strategies for septic shock. By elucidating the cellular and molecular mechanisms involved, future studies can contribute to the development of targeted therapies that may improve outcomes and reduce the burden of septic shock in clinical settings.

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