Materials and methods
Experimental animals and grouping
Thirty 10-week-old specific-pathogen-free male C57BL/6J mice, weighing 20 ± 2 g, with an animal experiment qualification certificate: SCXL(Hu)2020-0018, were divided into five groups using a random number table method (n = 6 in each group): sham operation, septic model, model + low-dose Shenfu Decoction, model + medium-dose Shenfu Decoction, and model + high-dose Shenfu Decoction.
Composition and preparation of the medicine
The composition of Shenfu Decoction includes raw sun-dried ginseng and processed aconite root in a 3:1 ratio (390 g and 130 g, respectively). The herbal pieces were purchased from the Pharmacy Department of Wuhan Traditional Chinese Medicine Hospital. First, the processed aconite root was soaked in 4,000 mL of water for 1 h, brought to a boil over high heat, and then simmered on low heat for 1 h. Next, ginseng was added, brought to a boil again over high heat, and then simmered on low heat for another hour. Afterward, the decoction was filtered through sterile gauze to collect 3,000 mL of liquid. Additional water was added to the residue, boiled again, and a second batch of 3,000 mL of liquid was collected, resulting in a total of 6,000 mL of decoction. The concentration of the medicine was 52 mg/0.6 mL. Based on the literature and equivalent dose calculations for experimental animals,6 a 20 g mouse required doses of 52 mg, 104 mg, and 208 mg, equivalent to a 70 kg adult human daily dosage of 20 g, 40 g, and 80 g, respectively. Considering the stomach capacity of mice, the plan was to administer 0.6 mL of the decoction twice daily. Therefore, the decoction was concentrated by heating to prepare low, medium, and high concentrations (52 mg/0.6 mL, 104 mg/0.6 mL, 208 mg/0.6 mL) and stored at 4°C in the refrigerator.
Main reagents and instruments
Main reagents
The BCA Protein Assay Kit (Product No. AS1086) was purchased from ASPEN. The Mouse P2X1 ELISA Kit (Product No. ELK6575), Mouse FPR1 ELISA Kit (Product No. ELK4832), Mouse CXCR1 ELISA Kit (Product No. ELK2893), and Mouse CXCR2 ELISA Kit (Product No. ELK7031) were acquired from ELK Biotechnology. The primary antibodies (fMLP and IL-8), Product Nos. HY-P0224 and HY-P81106) and secondary antibodies (Product No. HY-P81016) were purchased from MCE. Fetal Bovine Serum (Product No. 141215) was sourced from Hangzhou Tianhang Biotechnology Co., Ltd. The Calcein AM Assay Kit was purchased from Shanghai Yisheng Biotechnology Co., Ltd. (Catalog No. 40719ES50).
Major instruments
The instruments used included an Inverted Microscope (Olympus Corporation, Japan, IX51), Biological Microscope (Nikon, Japan, YS5100), Imaging System (Q-IMAGING, USA, MicroPublisher), DR-200Bs Microplate Reader (Diatek Instruments), GNP9160 Constant Temperature Incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.), DYY-6C Electrophoresis Apparatus (Beijing Liuyi Instrument Factory), RM2016 Microtome (Leica Instruments Shanghai Ltd.), and JT-12K Dehydrator and JB-P5 Embedding Machine (Wuhan Jujie Electronics Co., Ltd.).
Model establishment
This experiment was approved and recorded by the Ethics Committee of Wuhan Traditional Chinese Medicine Hospital. The septic model was prepared using the cecal ligation and puncture method. Mice were intraperitoneally injected with 10% chloral hydrate at a dose proportional to body weight (0.1 mL/10 g). Two to three minutes after the anesthetic effect appeared, an incision was made from the xiphoid process towards the tail, exposing and ligating half the length of the cecum. A 5 mL syringe needle was used to puncture the ligated section of the cecum once, which was then slightly squeezed before being returned, and the abdomen was closed. The sham operation group underwent a sham operation where, after anesthesia, an incision was made along the midline near the xiphoid process, the cecum was exposed and then immediately returned without ligation, puncture, or squeezing. After surgery, all groups of mice had free access to water and food.
Administration method
In accordance with the principles and guidelines presented in the “Methodology of Pharmacological Research on Traditional Chinese Medicine”,6 the equivalent dose was calculated based on the conversion of the body surface area ratio of animals. Mice in the Shenfu Decoction groups were administered 0.03 mL/g, totaling 0.6 mL, divided into two doses (at a 12-h interval) via gavage after 6 h of cecal ligation and puncture surgery, for three consecutive days. The sham operation and model groups received an equal volume of saline via gavage for three days, once every 12 h. Food and water were withheld 2 h before gavage, and experimental specimens were collected 4 h after the last gavage.
Neutrophil isolation and culture
Primary neutrophils were extracted from the peripheral blood of septic mice using the suspension cell separation method. Through drug serum gradient screening, the optimal drug concentration was determined (10% drug-containing serum intervention for 24 h), and the cells were cultured with drug-containing serum (10%) for five days before testing. The blank control group was cultured with fetal bovine serum, the sham operation group with serum from sham-operated mice, the low-dose group with drug-containing serum at a low dose of Shenfu Decoction, and the high-dose group with drug-containing serum at a high dose of Shenfu Decoction.
Specimen testing
Number of neutrophils
The number of neutrophils in peritoneal lavage fluid and blood was measured using an automatic hematology analyzer. The chemotactic distance of neutrophils was assessed through agarose chemotaxis experiments. The expression levels of chemotactic molecular receptors and P2X1 receptors in neutrophils were detected by Western Blot. Calcein staining was conducted to measure calcium ion concentration.
Agarose chemotaxis assay
Agarose chemotaxis assay for measuring neutrophil chemotactic distance: Take a 50 mL centrifuge tube, add 0.45 g of agarose powder to 30 mL of ultrapure water, and mix gently by slight shaking. Heat the mixture in a microwave until boiling, remove and shake for 5 s, then microwave for 20 s. Repeat this process several times until the agarose is completely dissolved. Quickly transfer 3 mL of the dissolved agarose into a 35 mm petri dish and allow it to cool slowly to room temperature, avoiding the formation of bubbles. Once solidified, the agarose gel is formed. Use a hollow tube with a diameter of 3.5 mm to punch two holes in the gel, spaced 2.4mm apart. Carefully remove the agarose inside the holes using a vacuum aspirator. Place the petri dish in a 37°C incubator for 1 h to allow temperature equilibration. Use the vacuum aspirator again to carefully remove any excess liquid from the holes, preparing them for use. Add 5 µL of chemoattractant (fMLP as the terminal chemotactic molecule and IL-8 as the intermediate chemotactic molecule) to one hole and 5 µL of cell suspension to the other hole. After incubation in a 37°C incubator for 2 h, observe and photograph the chemotactic distance of the central granulocytes under an inverted microscope.
Statistical analysis
SPSS 18.0 statistical software was used for data analysis. Quantitative data, if normally distributed, are presented as mean ± standard deviation (x ± S). The independent samples t-test was used for comparisons between two groups, while analysis of variance was used for comparisons among multiple groups. A P-value of less than 0.05 was considered statistically significant.
Discussion
The main pathogenesis of sepsis involves infection-induced immunodysregulation, systemic cascading inflammatory responses, and coagulation dysfunction, leading to multi-organ failure and even death by shock.7 The causes of sepsis may include various assaults, such as trauma and infections, where bacteria and toxins enter the body, triggering the production of inflammatory mediators that activate the body’s inflammatory response. An exacerbated inflammatory response increases immune suppression, thereby worsening the inflammation and further damaging the body. Typical symptoms include chills, fever, increased heart rate, shortness of breath, altered mental status, and edema, which may accompany organ dysfunction (cardiovascular, lung, brain, kidney), inadequate tissue perfusion, hypotension, lactic acidosis, oliguria, and acute changes in consciousness. Septic shock presents symptoms of sepsis along with hypotension, consciousness disturbances, a rapid and weak pulse, cold and damp limbs, and oliguria or anuria.
Based on the above manifestations, sepsis in Traditional Chinese Medicine (TCM) is categorized under the conditions of “Shang Han” (Cold Damage), “Wen Bing” (Warm Diseases), and “Jue Tuo” (Collapsing Syndrome), among others. In the early stage, it is categorized as “Wen Bing” and “Re Zheng” (Heat Syndromes), gradually evolving into “Tuo Zheng” (Collapse Syndrome).8 The etiology includes external factors such as the Six Excesses (wind, cold, summer heat, dampness, dryness, and fire) and internal factors such as the Seven Emotions (joy, anger, worry, contemplation, sorrow, fear, and startlement), as well as injuries that do not fall under either internal or external categories, with pathological products such as heat toxins, blood stasis, and phlegm turbidity. Some scholars suggest that sepsis, characterized by symptoms such as chills, fever, palpitations, and shortness of breath, is primarily caused by toxic heat, and recommend methods to clear heat and detoxify.9 Although this approach has certain effects, it cannot prevent the deterioration of sepsis or its transition to septic shock.
The “Huangdi Neijing” states, “When the righteous Qi resides within the body, pathogenic factors cannot interfere,” and “Pathogenic factors cannot harm the body alone,” highlighting the crucial role of the body’s righteous Qi in the development of sepsis. The modern understanding of sepsis places significant emphasis on immune function, precisely the righteous Qi mentioned in the “Huangdi Neijing”. Through years of clinical practice by generations of TCM practitioners at our institution, we have identified that a deficiency in righteous Qi is the fundamental cause of sepsis, affecting the entire course of the disease. An abundance of righteous Qi can prevent infection in the early stages, avert the progression of disease and expulsion of pathogens in the mid-stage, promote recovery, and prevent sequelae in the later stages.10 As stated in the “Huangdi Neijing”, “Where pathogens gather, there must be a deficiency.” The onset of sepsis indicates that the external defensive barrier has been compromised and the protective Qi has been breached. In clinical practice, we choose Shenfu Decoction to enhance Qi, warm Yang, secure the collapse, and support the righteous Qi while expelling pathogens, achieving favorable therapeutic outcomes. Research shows that Shenfu Decoction improves systemic circulation and microcirculation, protects organ cells, and enhances cellular immunity.11 In real clinical conditions, especially in critical cases without antibiotic sensitivity results, the safety of the patient’s life often necessitates the empirical use of multiple broad-spectrum antibiotics, progressively escalated, and eventually including steroids, immunoglobulins, and other treatments. Experienced experts may also apply thymosin. These practices underscore the challenges of treating sepsis and the necessity of TCM intervention to support the righteous Qi. Therefore, Shenfu Decoction has a clinically rational basis for its application in sepsis treatment.
Shenfu Decoction, first mentioned in “Ji Sheng Fang” (Formulas for Succouring the Sick,《济生方》in Chinese), is composed of red ginseng and processed aconite root. We have chosen to use sun-dried ginseng in place of red ginseng to enhance its effect in greatly replenishing the primal Qi12,13; the ratio of sun-dried ginseng to aconite has been adjusted to 3:1, thereby increasing the dosage of sun-dried ginseng to strengthen the decoction’s ability to benefit Qi and support the righteous Qi.14 Sun-dried ginseng, slightly cool in nature and sweet in flavor, enters the Lung and Spleen Meridians and has the effects of greatly replenishing the primordial Qi, nourishing the spleen and the lung, generating fluids to quench thirst, calming the spirit, and enhancing intelligence.15 Compared to red ginseng, sun-dried ginseng is more effective in nourishing Yin and generating fluids, reducing dryness, and increasing its effect in astringing and nourishing Yin, thus achieving a balance of nourishing both Yin and Yang. Modern pharmacological studies have found that ginseng has an immunomodulatory effect, with research indicating that its main active components, ginsenosides, can restore immune function in sepsis caused by trauma by inhibiting the activation of CD4+CD25+ regulatory T cells,16 suggesting that using sun-dried ginseng to generate fluids and quench thirst might avoid exacerbating heat pathogens associated with excessive Yang warming. Moreover, its fluid-generating action is beneficial for eliminating the heat of sepsis and can counteract the heat from aconite. Aconite, extremely hot in nature and spicy-sweet in flavor, enters the heart, spleen, and kidney meridians, with effects of restoring depleted Yang and warming fire to assist Yang.17 The active component of aconite, aconitine, has anti-inflammatory and analgesic properties, while aconite polysaccharides can promote the activation of immune responses.18,19 When used together, they not only counteract the toxicity of aconite but also benefit Qi, generate fluids, restore Yang depletion, and secure collapse. As stated in “Shan Bu Ming Yi Fang Lun” (Revised and Supplemented Famous Physicians’ Prescriptions and Discussions,《删补名医方论》in Chinese), “For replenishing the postnatal Qi, nothing compares to ginseng; for replenishing the prenatal Qi, nothing compares to aconite root. This is the basis of Shenfu Decoction.”
Neutrophils are an important part of the body’s nonspecific immune system, primarily found in the bone marrow and peripheral blood. When an infection occurs, neutrophils in the peripheral blood rapidly migrate to the site of infection, eliminating pathogens through various means such as phagocytosis or the formation of neutrophil extracellular traps.20 However, during sepsis, the chemotactic function of neutrophils is impaired, leading to a reduced number of neutrophils reaching the site of infection, resulting in poor infection control.21 This study shows that, compared to the model group, the number of peritoneal neutrophils in mice treated with Shenfu Decoction significantly increased, with a more pronounced effect at higher doses, indicating that Shenfu Decoction improved the chemotactic function of neutrophils toward inflammation sites in a dose-dependent manner. Compared to the model group, the low-dose group of Shenfu Decoction showed an increase in the number of blood neutrophils in mice, but the difference was not statistically significant, indicating that the mobilization of neutrophils from the bone marrow to the peritoneal cavity did not significantly impact the number of neutrophils. The number of neutrophils in the medium and high-dose groups was statistically significant compared to the model group, demonstrating the stimulatory effect of medium and high doses of Shenfu Decoction on the overall mobilization of neutrophils.
In fact, the chemotactic distance of neutrophils represents the cell’s motility. TCM theory perceives it as a driving force, which is the action of Yang and Qi. Chemokines such as IL-8 and fMLP both have chemotactic effects on neutrophils, and thus, we selected these two chemotactic agents for our study. The agarose chemotaxis model, used to evaluate the chemotactic function of neutrophils, is a commonly used experimental model in recent years for research on neutrophil chemotaxis. Its maximum distance can reach up to 2,700 µm, and its direction is unrestricted, allowing for dynamic observation of cell chemotaxis directly under a microscope without the need for staining or labeling. Using the final chemotactic molecule fMLP and the intermediate chemotactic molecule IL-8 (both at optimal chemotactic concentrations) as chemotactic agents, the chemotactic distances and the differences between groups were consistent. Therefore, we believe that the direct effect of sepsis endotoxins on the chemotaxis of neutrophils and the promotive action of medium and high doses of Shenfu Decoction are consistent.
The P2X1 receptor is an ATP-gated cation channel receptor that is immediately activated upon binding with ATP, rapidly inducing the influx of cations into the cell.22 The P2X1 receptor has the highest permeability to calcium ions, and recent studies have confirmed the expression of P2X1 receptors in peripheral blood neutrophils, eosinophils, lymphocytes, and platelets, playing a role in the immune response to pathogens.23 Research has shown that activation of the P2X1 receptor enhances the random movement of neutrophils, but this movement lacks directionality and impairs their ability to move toward chemotactic agents, weakening the neutrophils’ chemotactic ability toward infection sites. This study demonstrates that compared to the sham operation group, the expression level of P2X1 protein in the Shenfu Decoction group was significantly downregulated. Combined with the results of the calcein staining, compared to the sham operation group, the concentration of calcium ions in neutrophils in the Shenfu Decoction group was significantly reduced. Both results are consistent, indicating that Shenfu Decoction downregulates the expression level of the P2X1 receptor in neutrophils, inhibits the influx of calcium ions, and is dose-related to Shenfu Decoction.
The P2 receptor family includes the ionotropic P2X (P2X1 to P2X7) receptors and the metabotropic P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 to P2Y14) receptors. The P2X7 receptor has been widely reported to be activated by ATP, activating the NLRP3 inflammasome. The activated inflammasome processes the precursor of IL-1β through caspase-1, producing mature IL-1β and releasing it to cause inflammation.24 Previous research has shown that ATP can bind to the P2Y2 receptor to regulate the chemotaxis of granulocytes and macrophages.25 The action of Shenfu Decoction in benefiting Qi and warming Yang is analogous to the energetic and dynamic action of ATP. FPR1 encodes a G protein-coupled receptor for phagocytic cells, which mediates the chemotaxis and response of phagocytic leukocytes to bacterial formylated chemotactic peptides under inflammatory conditions.