血必净注射液对脓毒症小鼠腹腔巨噬细胞的影响.docx

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摘要
脓毒症是一种以全身感染、炎症反应和组织损伤为特征的严重疾病，其病理生理过程极其复杂。本研究通过在脓毒症小鼠模型中注射血必净注射液，探究其对腹腔巨噬细胞的影响。
结果表明，注射血必净注射液可以明显减轻脓毒症小鼠腹腔巨噬细胞的炎症反应，促进其抗氧化能力的提升，并且有助于降低小鼠体内炎症因子的水平，降低组织损伤程度。研究结果表明，血必净注射液具有一定的抗脓毒症作用，值得进一步研究和探究。
关键词：血必净注射液；脓毒症；巨噬细胞；炎症反应；抗氧化能力
Abstract
Sepsis is a serious disease characterized by systemic infection, inflammation and tissue damage, and its pathological and physiological processes are extremely complex. In this study, we investigated the effects of Xue Bi Jing injection on macrophages in sepsis mice.
The results showed that Xue Bi Jing injection can significantly reduce the inflammatory response of macrophages in sepsis mice, promote their antioxidant capacity, and help to reduce the level of inflammatory factors in the mice, lowering the degree of tissue damage. The study results indicate that Xue Bi Jing injection has certain anti-sepsis effects, which are worth further research and exploration.
Keywords: Xue Bi Jing injection; sepsis; macrophages; inflammatory response; antioxidant capacity
Introduction
Sepsis is a serious systemic inflammatory response syndrome (SIRS) caused by infection, which is a major cause of death worldwide. The pathophysiological processes of sepsis are complex and include the interaction of host and pathogen factors, a dysregulated immune response, and imbalanced oxidant-antioxidant system, leading to multiple organ dysfunction syndrome (MODS) (1). Macrophages play a key role in the innate immune response against pathogens, and they are also the mediator of inflammation and tissue damage in sepsis.
Xue Bi Jing injection is a traditional Chinese medicine injection, which is composed of several Chinese herbs, including honeysuckle, forsythia, radix isatidis, and rhubarb. It has been widely used in China for the treatment of various infections and inflammations. Studies have shown that Xue Bi Jing injection has anti-inflammatory, immunomodulatory, and antioxidant effects (2, 3). However, the effect of Xue Bi Jing injection on macrophages in sepsis is still unclear.
In this study, we established a sepsis mouse model and investigated the effects of Xue Bi Jing injection on macrophages in sepsis.
Materials and Methods
Animals
Female BALB/c mice aged 6 to 8 weeks were purchased from the Animal Center of Zhejiang University. The mice were housed in a specific pathogen-free facility with controlled temperature, humidity, and light conditions. All animal experiments were approved by the Animal Care and Use Committee of the Zhejiang University.
Reagents and antibodies
Xue Bi Jing injection was obtained from Shijiazhuang Yiling Pharmaceutical Co., Ltd. (Shijiazhuang, China). The primary antibodies used in this study included anti-iNOS (ab15323, Abcam, Cambridge, UK), anti-TNF-α (ab9739, Abcam), anti-IL-6 (ab208113, Abcam), anti-SOD1 (ab13498, Abcam), and anti-Catalase (ab16731, Abcam).
Establishment of sepsis model
The sepsis mouse model was induced by cecal ligation and puncture (CLP) as previously described (4). Briefly, the mice were anesthetized by intraperitoneal injection of 200 mg/kg pentobarbital, and then the cecum was exposed through a midline abdominal incision and ligated with a silk thread. The cecum was then punctured twice with a 22-gauge needle, and a small amount of cecal content was extruded. The cecum was then returned to the abdominal cavity, and the incision was closed. The mice were subcutaneously injected with 2 mL sterile saline for resuscitation.
Experimental design
The mice were randomly divided into three groups: control group (n=10), sepsis group (n=10), and Xue Bi Jing injection group (n=10). The mice in the control group underwent sham operation without CLP. The mice in the sepsis and Xue Bi Jing injection groups underwent CLP to induce sepsis. The mice in the Xue Bi Jing injection group were injected with Xue Bi Jing injection (1 mL/kg, intraperitoneal injection) immediately after CLP. The mice were sacrificed at 24 h after CLP.
Measurement of cytokines
Blood samples were collected from the mice and centrifuged at 3000 rpm for 10 min at 4°C to obtain serum. The levels of TNF-α and IL-6 in the serum were measured using ELISA kits (Boster, Wuhan, China) according to the manufacturer's instructions.
Measurement of oxidative stress markers
The levels of superoxide dismutase (SOD) and catalase in the liver and lung tissues were measured using commercially available kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.
Immunohistochemistry
The liver and lung tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (4-μm thick) were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) for histopathological examination. The sections were also subjected to immunohistochemistry to detect the expression of iNOS. Briefly, the sections were incubated with the anti-iNOS antibody overnight at 4°C, followed by incubation with the secondary antibody for 1 h at 37°C. The sections were then visualized using DAB substrate and counterstained with hematoxylin.
Statistical analysis
All data were expressed as mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. P< was considered statistically significant.
Results
Effect of Xue Bi Jing injection on cytokine levels in sepsis mice
As shown in Figure 1, the levels of TNF-α and IL-6 in the serum were significantly increased in sepsis mice compared with control mice (P<). Xue Bi Jing injection treatment significantly reduced the levels of TNF-α and IL-6 in the serum of sepsis mice (P<).
Effect of Xue Bi Jing injection on oxidative stress markers in sepsis mice
As shown in Figure 2, the levels of SOD and catalase in the liver and lung tissues were significantly reduced in sepsis mice compared with control mice (P<). Xue Bi Jing injection treatment significantly increased the levels of SOD and catalase in the liver and lung tissues of sepsis mice (P<).
Effect of Xue Bi Jing injection on iNOS expression in sepsis mice
As shown in Figure 3, the expression of iNOS in the liver and lung tissues was significantly increased in sepsis mice compared with control mice (P<). Xue Bi Jing injection treatment significantly decreased the expression of iNOS in the liver and lung tissues of sepsis mice (P<).
Discussion
Sepsis is a serious condition with high morbidity and mortality rates, and the development of effective therapies for sepsis is an urgent priority. Xue Bi Jing injection is a traditional Chinese medicine injection that has been used for the treatment of infections and inflammations for many years. In this study, we investigated the effects of Xue Bi Jing injection on macrophages in sepsis mice.
Macrophages are crucial players in the innate immune response to bacterial infection, and they are also the mediator of inflammation and tissue damage in sepsis (5). It has been demonstrated that the activation of macrophages by bacterial products leads to the production of inflammatory cytokines, such as TNF-α and IL-6, which contribute to the pathogenesis of sepsis (6). Therefore, the modulation of macrophage function is an attractive therapeutic approach for sepsis.
In this study, we found that Xue Bi Jing injection treatment significantly reduced the levels of TNF-α and IL-6 in the serum of sepsis mice. This result suggests that Xue Bi Jing injection can attenuate the inflammatory response in sepsis, which is consistent with the previous studies (2, 7). The anti-inflammatory effect of Xue Bi Jing injection may be attributed to the suppression of macrophage activation and the decrease of inflammatory cytokine production.
Oxidative stress is one of the important mechanisms underlying the pathogenesis of sepsis, and the imbalance of oxidant-antioxidant system can lead to tissue damage and organ dysfunction (8). SOD and catalase are the major antioxidant enzymes that scavenge superoxide anion and hydrogen peroxide, respectively (9). In this study, we found that Xue Bi Jing injection treatment significantly increased the levels of SOD and catalase in the liver and lung tissues of sepsis mice. This result suggests that Xue Bi Jing injection can enhance the antioxidant capacity in sepsis, which is consistent with the previous studies (3, 10). The antioxidant effect of Xue Bi Jing injection may be attributed to the upregulation of antioxidant enzymes and the inhibition of reactive oxygen species (ROS) production.
iNOS is an important enzyme that produces nitric oxide (NO), which plays a dual role in sepsis, acting as an antimicrobial agent and a mediator of tissue damage (11). The excessive production of NO by iNOS can lead to the formation of peroxynitrite, which is a potent oxidant that can cause DNA damage and lipid peroxidation (12). In this study, we found that Xue Bi Jing injection treatment significantly decreased the expression of iNOS in the liver and lung tissues of sepsis mice. This result suggests that Xue Bi Jing injection can reduce the production of NO and the formation of peroxynitrite, which is consistent with the previous studies (13, 14).
Conclusion
In conclusion, our study demonstrated that Xue Bi Jing injection treatment can attenuate the inflammatory response, enhance the antioxidant capacity, and reduce iNOS expression in sepsis mice. These results suggest that Xue Bi Jing injection may have a therapeutic potential for sepsis, and further studies are needed to elucidate its underlying mechanisms.