Effect of Shenfu injection on lipopolysaccharide (LPS)-induced septic shock in rabbits

Journal of Ethnopharmacology 234 (2019) 36–43

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Effect of Shenfu injection on lipopolysaccharide (LPS)-induced septic shock in rabbits

T

Xi Liua,b, Runzhe Liua,b, Zhenfeng Daia,b, Hao Wua,b, Ming Lina,b, Fang Tiana,b, Zeyu Gaoa,b, ⁎ Xin Zhaoa,b, Yi Suna,b, Xiaoping Pua,b, a b

National Key Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China

ARTICLE INFO

ABSTRACT

Keywords: Shenfu injection LPS Septic shock MALDI-TOF-MSI Energy metabolism

Ethnopharmacological relevance: Shenfu injection is a popular Chinese herbal formula that has been widely used in the treatment of shock in China. Aim of the study: To investigate the effect of Shenfu injection on lipopolysaccharide (LPS)-induced septic shock in rabbits. Materials and methods: We established a septic shock model in rabbits by administering an intravenous injection of 0.6 mg/kg LPS to anesthetized rabbit, and 15 min after LPS challenge, the rabbits were intravenously administered the Shenfu injection. In these in vivo experiments, the jugular vein of the rabbits was cannulated for LPS and drug administration, and the right common carotid artery was cannulated to record the mean arterial pressure (MAP) over a 6-h period. In addition, various serum biochemical parameters, including lactate dehydrogenase (LDH), aspartate aminotransferase (AST), glutamate transaminase (ALT), creatinine (Cre), and urea nitrogen (Urea), were measured at 0, 3, and 6 h. Serum LPS levels at 6 h were determined by the test kit. And histological changes in the heart, liver and kidney tissues were observed by HE staining. Furthermore, some related small molecules in the heart tissues were detected by MALDI-TOF-MSI. Results: We found that Shenfu injection can increase the MAP, decrease the serum LPS, LDH and AST levels, and improve the tissue morphology of the heart, liver and kidney in rabbits with LPS-induced septic shock. In addition, Shenfu injection can increase the contents of ATP and taurine while reducing the content of AMP in the heart tissue during septic shock. Conclusions: These results indicate that Shenfu injection exerts a protective effect on LPS-induced septic shock in rabbits.

1. Introduction Sepsis is a systemic inflammatory response syndrome that is generally caused by burns, trauma, and surgery and might develop into septic shock (Dellinger et al., 2013), which is often accompanied by microcirculatory disorders, tissue cell damage, and multiple organ dysfunction. Septic shock is the most common type of shock seen in the clinic, and its mortality can reach 40–80% (Delano and Ward, 2016;

Gaieski et al., 2013). The most common cause of septic shock is lipopolysaccharide (LPS), a component in the cell wall of Gram-negative bacteria that can cause shock, disseminated intravascular coagulation (DIC), and multiple organ failure (Bone, 1996). Cardiac insufficiency is a typical characteristic of septic shock. The current treatment for septic shock in patients is still mainly based on adjusting the hemodynamics, reducing the cardiac load and enhancing myocardial contractility through the administration of vasopressor and

Abbreviations: LPS, lipopolysaccharide; MAP, mean arterial pressure; LDH, lactate dehydrogenase; AST, aspartate aminotransferase; ALT, glutamate transaminase; Cre, creatinine; Urea, urea nitrogen; MALDI-TOF-MSI, matrix-assisted laser desorption ionization time-of-flight mass spectrometry imaging; SFI, Shenfu injection; DA, dopamine; DIC, disseminated intravascular coagulation; ITO, indium tin oxide; AMPK, adenosine 5′-monophosphate (AMP)-activated protein kinase; NF-κB, nuclear factor kappa B; TLR4, Toll-like receptor 4; ROS, reactive oxygen species ⁎ Correspondence to: Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Rd, Haidian District, Beijing 100191, PR China. E-mail addresses: [email protected] (X. Liu), [email protected] (R. Liu), [email protected] (Z. Dai), [email protected] (H. Wu), [email protected] (M. Lin), [email protected] (F. Tian), [email protected] (Z. Gao), [email protected] (X. Zhao), [email protected] (Y. Sun), [email protected] (X. Pu). https://doi.org/10.1016/j.jep.2019.01.008 Received 28 September 2018; Received in revised form 19 December 2018; Accepted 9 January 2019 Available online 11 January 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.

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(Beijing, China) with the license number SCXK (Beijing) 2015–0001. The animals were independently maintained with a temperature of 22–24 °C, 50–60% humidity, and a 12-h light/12-h dark cycle for a 1week acclimatization period before the experiments. All the experiments were conducted in accordance with the requirements of the Peking University Biomedical Ethics Committee in Beijing, China (approval number: LA2017282). The experimenters are approved by the Peking University Health Science Center.

inotropic agents, such as dopamine (Dellinger et al., 2013). However, this type of treatment might be a risk factor contributing to the poor prognosis of these patients (Walley, 2018). Some studies have suggested that energy-related therapies, such as β-receptor blockers, can improve the prognosis of patients with heart failure (Landesberg et al., 2012; Neubauer, 2007). Therefore, drugs that can improve myocardial energy metabolism might be helpful in the treatment of septic shock. Shenfu injection is derived from traditional Chinese medicine, and its main active ingredients are ginsenosides and aconitine alkaloids. Compared to chemical drugs, traditional Chinese medicine is cheap, available, and considered safe because they have been used for thousands of years (Unschuld, 1999). In China, Shenfu injection has been widely used in the treatment of cardiovascular diseases, such as shock, congestive heart failure, chronic arrhythmia, coronary heart disease, acute myocardial infarction, viral myocarditis, and others, with remarkable curative effect (Guo and Li, 2013). Although a previous study showed that Shenfu injection can exert anti-inflammatory effects and improve lung and liver damage in rats with systemic inflammatory response syndrome (Wang et al., 2008), the role of Shenfu injection in animal septic shock models has not been reported thus far. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry imaging (MALDI-TOF-MSI) allows the acquisition of spatial distribution maps of lipids, proteins, peptides and small molecules in situ (Lefcoski et al., 2018). Therefore, MALDI-TOF-MSI can be used as an efficient tool to evaluate the effect of Shenfu injection on myocardial energy metabolism in LPS-induced shock because it can detect the content and distribution of small molecules, such as ATP and taurine, in situ. Here, we evaluated the effect of Shenfu injection on a rabbit model of LPS-induced shock and clarified its mechanism of action.

2.3. Grouping and model establishment The rabbits were randomly assigned into one of six groups: control group, model group, dopamine group, and doses of 4.5, 6, and 8 ml/kg Shenfu injection groups. Each group contained four to six rabbits. The rabbits were fasted for 12 h before the experiment and anesthetized via an injection of sodium pentobarbital (30 mg/kg for induction and 6 mg/kg for maintenance) into the ear vein, and their body temperature was maintained with a heating pad throughout the experiment. A catheter with an outer diameter of 1.2 mm was inserted into the right external jugular vein for LPS and drug administration, and a catheter with an outer diameter of 1.8 mm was inserted into the right common carotid artery for monitoring the mean arterial pressure (MAP) using the MedLab Biofunctional Experimental System (Beijing Zhishu Duobao Biotechnology Co., Ltd., China) and for blood sample collection. Sodium heparin solution (2.5 mg/kg) was slowly injected through the intravenous catheter to protect against the formation of blood clots, and 30 min later, 0.6 mg/kg LPS was injected into all the rabbits except those in the control group using a syringe pump (Rivend Life Technologies Co., Ltd.) at an injection speed of 20 μl/min. The control group was administered an equal amount of normal saline instead of LPS.

2. Materials and methods 2.1. Chemical

2.4. Administration

Sodium pentobarbital and lipopolysaccharide (LPS, Escherichia coli serotype O111: B4) were purchased from Sigma-Aldrich (MO, USA). Sodium heparin (150 U/mg) was acquired from Coolaber (Beijing, China), and Dopamine hydrochloride injection was purchased from Hefeng Pharmaceutical Co., Ltd. (Shanghai, China). The test kit for the detection of Gram negative bacteria LPS was purchased from Zhanjiang A & C Biological Ltd. (Guangdong, China). Shenfu injection (batch number: 170902050) was procured from Huarun Sanjiu Pharmaceutical Co., Ltd. (Ya’an, Sichuan, China). Shenfu injection contains 2 herbal materials, as described in Table 1. Its quality was strictly controlled in compliance with the standard of China Food and Drug Administration (approval No: WS3-B-3427-98-2013) in Table 2 and was ensured by using fingerprint technology during production. The work flow of the Shenfu injection preparation is presented in Fig. 1. In brief, the two crude Chinese herbs were respectively soaked and concentrated into 1 mg/ml Panax ginseng C. A. Mey. and 2 mg/ml Aconitum carmichaelii Debeaux, by which Shenfu injection was mixed.

Fifteen minutes after LPS injection, the rabbits belonging to the Shenfu injection groups were administered Shenfu injection via an intravenous catheter; the rabbits in the dopamine group were administered 3.2 mg/kg dopamine hydrochloride injection; and the rabbits in the control and model groups were given the same amount of normal saline. The injection speed of the syringe pump was set to 250 μl/min. The workflow of the model establishment and administration is illustrated in Fig. 2. 2.5. Serum LPS levels detection Blood (1.5 ml) was withdrawn through the carotid artery catheter at three time points, namely, 0, 3, and 6 h, and the same volume of saline was injected to replace the withdrawn blood. The blood samples were then centrifuged at 3000 rpm for 15 min, and the serum was stored at −20 °C until use in various assays. Serum LPS levels at 6 h were determined at Hospital 302 of the People's Liberation Army (Beijing, China), measured by the photometric assay using a LKM-02-32 Kinetic Tube Reader (Zhanjiang A & C Biological Ltd., China). And the operations were carried out in strict accordance with the instructions.

2.2. Animals Male Japanese white rabbits weighing 2.2–2.5 kg were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Table 1 Information of raw herbs in Shenfu injection. Latin scientific name

Plant parts

English name

Pinyin name

Voucher no

Voucher specimens no

Ratio

Panax ginseng C. A. Mey. Aconitum carmichaelii Debeaux

Root Tuber

Red Ginseng Root Aconite Tuber

Hongshen Fuzi

13010560 13010423

Hongshen: 1305001 Fuzi: 1305041

33.3% 66.6%

37

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Table 2 The quality standard of Shenfu injection (batch number: 170902050). Compound

Chemical structure

Content

Quality control

Benzoylmesaconine

1.58 μg/ml

0.50–4.50 μg/ml

Ginsenoside Rg1

0.12 mg/ml

> 0.04 mg/ml

Ginsenoside Re

0.11 mg/ml

> 0.02 mg/ml

Ginsenoside Rb1

1.0 mg/ml

0.6–1.8 mg/ml

were determined at Peking University Third Hospital (Beijing, China). 2.7. Histopathological analyses Six hours after LPS administration, three rabbits from each group were sacrificed and perfused with normal saline, and the heart, liver and kidney were rapidly obtained and immersed in 10% paraformaldehyde solution overnight. The tissues were prepared into 4-μmthick paraffin sections and stained with hematoxylin-eosin (HE). Pathological changes in the heart, liver and kidney were observed under an optical microscope. 2.8. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry imaging (MALDI-TOF-MSI) Six hours after LPS administration, three rabbits from the 8 ml/kg Shenfu injection, control and model groups were sacrificed, and their hearts were collected, immediately snap-frozen in liquid nitrogen and stored at −80 °C for the MALDI-TOF-MSI experiment. Frozen slices of heart tissue were prepared using a microtome (Scotsman Jencons, Germany) at −18 °C. The slices had a thickness of 10 µm at a cross-section in the middle of the heart. The slices were transferred to ITO-coated slides (Bruker Daltonics, Germany), dried in a vacuum pump for 20 min and subjected to matrix spraying and MALDITOF-MSI according to the methods described by Liu et al. (2014).

Fig. 1. Work flow of the Shenfu injection preparation.

2.9. Statistical analysis Fig. 2. Workflow of the model establishment and administration.

The data of MAP, biochemical parameters and intensity ratio of small molecules obtained by MALDI-TOF-MSI are presented as the means ± SDs. One-way ANOVA was used to analyze the differences among groups, and statistical significance was set to α = 0.05 (twosided). The statistical analysis of the MALDI-TOF-MSI data was performed using SCiLS Lab software, and this software was also used for image generation.

2.6. Biochemical analyses Various serum biochemical parameters at 0, 3, and 6 h, including lactate dehydrogenase (LDH), aspartate aminotransferase (AST), glutamate transaminase (ALT), creatinine (Cre), and urea nitrogen (Urea), 38

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Table 3 The effect of Shenfu injection on MAP of LPS-induced shock in rabbits (means ± SD, mmHg). time (h)

Control

0 1 2 3 4 5 6

106.8 105.2 106.5 106.6 105.6 104.1 104.3

± ± ± ± ± ± ±

4.6 8.8 9.2 7.2 9.9 9.5 7.0

Model

SF 4.5 ml/kg

SF 6 ml/kg

SF 8 ml/kg

DA

108.8 ± 4.6 91.9 ± 14.6 86.5 ± 11.4### 82.2 ± 6.0### 69.6 ± 8.5### 69.3 ± 3.9### 70.1 ± 3.6###

106.7 ± 5.4 92.7 ± 12.0 82.2 ± 8.9 75.0 ± 10.5 68.2 ± 9.7 67.3 ± 6.4 70.2 ± 4.6

114.0 ± 6.0 83.8 ± 15.6 86.9 ± 6.1 83.8 ± 7.5 83.4 ± 5.0* 86.2 ± 5.0*** 84.2 ± 8.2***

107.1 ± 6.7 89.5 ± 9.0 86.0 ± 8.0 84.6 ± 9.2 79.2 ± 8.5* 80.3 ± 7.8** 85.3 ± 7.5***

106.8 ± 9.7 87.7 ± 4.6 86.3 ± 10.4 79.2 ± 8.0 79.1 ± 5.1 85.5 ± 7.3** 88.9 ± 3.9***

Control: normal group; Model: model group; SF 4.5 ml/kg: 4.5 ml/kg Shenfu injection group; SF 6 ml/kg: 6 ml/kg Shenfu injection group; SF 8 ml/kg: 8 ml/kg Shenfu injection group; DA: dopamine group. The data are presented as the means ± SDs; n = 4–6. ### p < 0.001 vs. the control group. *** p < 0.001 vs. the model group. ** p < 0.01 vs. the model group. * p < 0.05 vs. the model group.

3. Results

3.3. Shenfu injection affected the levels of serum biochemical indices

3.1. Shenfu injection increased the mean arterial pressure (MAP) in rabbits with LPS-induced septic shock

The serum biochemical indices were analyzed as the ratios of the values after treatment to the initial values (Fig. 4). LPS administration resulted in abnormal increases in the LDH, AST, ALT, Cre and Urea levels at 6 h. The administration of Shenfu injection at the dose of 8 ml/ kg yielded significant improvements in the LDH and AST levels at 6 h (p < 0.01 and p < 0.05 vs. the model group, respectively), and dopamine also improved the levels of LDH, AST and ALT at 6 h (all p < 0.05 vs. the model group).

As shown in Table 3, the MAP of the rabbits in the control group remained almost unchanged over the 6-h experimental period, whereas that of the rabbits belonging to the model group decreased to 70.1 mmHg after LPS injection (p < 0.001), indicating successful establishment of the model. The administration of Shenfu injection at a dose of 4.5 ml/kg did not change the MAP of rabbits with septic shock, but higher doses of 6 and 8 ml/kg significantly increased the MAP during the period from 4 to 6 h (p < 0.05, p < 0.001, p < 0.001 vs. the model group; p < 0.05, p < 0.01, p < 0.001 vs. the model group). Dopamine, which was used as a positive control, also showed an effect from 5 to 6 h (p < 0.01, p < 0.001 vs. the model group).

3.4. Shenfu injection protected heart, liver and kidney tissues from LPSinduced injury As shown in Fig. 5, impaired myocardial cells (red arrow) and inflammatory cell infiltration (dark blue arrow) were observed in the heart of the animals belonging to the model group. The degrees of myocardial cell lysis, edema and vacuolar lesions were reduced in the rabbits treated with Shenfu injection or dopamine after LPS administration. The liver of the rabbits from the model group showed pathological abnormalities, including hepatic edema, large area of hepatocyte waterlike degeneration, round fat vacuoles in the cytoplasm, cytoplasmic loose vacuoles (yellow arrow), inflammatory cell aggregation in the hepatic sinus and central venous stasis (purple arrow). However, significant improvements in the extents of water-like degeneration, round fat vacuoles and edema were observed in the Shenfu injection and dopamine groups. The kidney tissue from the model group showed glomerular shrinkage (light blue arrow), water-like and vacuolar degeneration in renal tubular epithelial cells (green arrow), and lumen dilatation and tubular formation in the renal tubules. Less pathological damage was detected in the Shenfu injection and dopamine groups.

3.2. Shenfu injection decreased the levels of serum LPS in rabbits with LPSinduced septic shock Fig. 3 shows the LPS levels in the serum of rabbits at 6 h. The increase of the LPS level in the model group was significantly depressed by 6 and 8 ml/kg Shenfu injection (p < 0.05, p < 0.01 vs. the model group, respectively). In contrast, no significant difference was noted in the LPS level of the dopamine group, compared with the model group.

3.5. Shenfu injection improved cardiac energy metabolism, as detected by MALDI-TOF-MSI MALDI-TOF-MSI showed that the contents of ATP and taurine were decreased in the heart of the rabbits in the model group (p < 0.01, 0.001), whereas the 8 ml/kg Shenfu injection increased the contents of ATP and taurine (p < 0.05). Additionally, an abnormal increase in AMP was observed in the model group (p < 0.01), and this increase was inhibited by 8 ml/kg Shenfu injection (p < 0.05). The contents of glucose, glutamate, hypoxanthine and creatine were reduced in the model group, and Shenfu injection did not exert a significant improvement in these changes (Fig. 6).

Fig. 3. Effects of Shenfu injection on serum LPS levels at 6 h of rabbits with LPSinduced septic shock (means ± SD). Control: normal group; Model: model group; SF 4.5 ml/kg: 4.5 ml/kg Shenfu injection group; SF 6 ml/kg: 6 ml/kg Shenfu injection group; SF 8 ml/kg: 8 ml/kg Shenfu injection group; DA: dopamine group. The data are presented as the means ± SDs; n = 3–5. ### p < 0.001 vs. the control group, ** p < 0.01 vs. the model group, * p < 0.05 vs. the model group. 39

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Fig. 4. Effects of Shenfu injection on serum biochemical indices of LPS-induced shock in rabbits (means ± SD). (a-c): Relative levels of LDH, AST and ALT at 6 h. Control: normal group; Model: model group; SF 4.5 ml/kg: 4.5 ml/kg Shenfu injection group; SF 6 ml/kg: 6 ml/kg Shenfu injection group; SF 8 ml/kg: 8 ml/kg Shenfu injection group; DA: dopamine group. The data are presented as the means ± SDs; n = 3–5. # p < 0.05 vs. the control group, ** p < 0.01 vs. the model group, * p < 0.05 vs. the model group.

shock in rabbits. The HE staining results also showed that Shenfu injection exerts obvious protective effects on major organs, including the heart, liver and kidney. Cardiac function damage, characterized by contractile and diastolic dysfunction in the heart, is a typical clinical feature of septic shock and is also closely related to the patient's prognosis (Ronco et al., 1993). Previous studies have shown that mitochondrial dysfunction, rather than myocardial hypoxia, is the main cause of energy metabolism disorder during septic shock (Chen et al., 2003). LPS can directly impair myocardial mitochondrial function, inhibit electron transfer, and reduce ATP production (An et al., 2012). The MALDI-TOF-MSI results obtained in this study indicate that Shenfu injection can improve cardiac energy metabolism. Compared with the control group, the ATP and AMP contents were significantly decreased and significantly increased in the model group, which is consistent with the pathological features of LPS-induced shock (Supinski and Callahan, 2006), and Shenfu injection at a dose of 8 ml/ kg can reverse the changes in the ATP and AMP contents. In addition, previous studies revealed that adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) activity is inhibited when the AMP/ATP ratio is increased (Jiang et al., 2014; Meares et al., 2013; Tadie et al., 2012; Xing et al., 2013). Although AMPK plays an important negative regulatory role in energy metabolism, the activation of AMPK can also inhibit TLR4/NF-κB-related signaling and thereby inhibit inflammatory responses (Liu et al., 2015). In our study, Shenfu injection reversed the increase in the AMP/ATP ratio observed after LPS administration, and thus, we hypothesize that Shenfu injection might inhibit the inflammatory response by stimulating the activation of AMPK. However, further studies are needed to confirm this hypothesis. In addition, Shenfu injection might also improve oxidative stress damage caused by LPS. The MALDI-TOF-MSI results showed that Shenfu injection can reverse the reduction in taurine content caused by LPS. Taurine, as an antioxidant, is mainly found in skeletal muscle and myocardial tissue, and many studies have shown that taurine can protect major organs from LPS (Jeon et al., 2009; Kim and Kim, 2002; Liu et al., 2017). In addition, taurine can inhibit the increase in LPS-induced reactive oxygen species (ROS) accumulation and enhance the activity of superoxide enzymes to exert antioxidant effects (Jeon et al., 2009). Traditional methods for detecting small molecules, such as ATP and taurine, include chromatography, electrophoresis and optical analysis, but these methods have some drawbacks, such as low extraction efficiency and solvent contamination (Chida and Kido, 2014; Fonseca et al., 2018). MALDI-TOF-MSI is a promising, powerful, highthroughput technology for measuring hundreds of different molecules directly on tissues and can be applied for molecule detection, structural

4. Discussion Several methods have been developed for preparing animal models of septic shock: an LPS-induced shock model, an LPS-combined-withsensitizer-induced shock model, and a cecal-ligation-and-perforation (CLP)-induced shock model (Fink and Heard, 1990). Among these methods, the LPS-induced shock model has been widely used in the laboratory because it requires a single pathogenic factor, is easy to perform and produces easily analyzed data. In 2016, ESICM published the Sepsis 3.0 consensus, which lists hypotension and organ dysfunction as the most important clinical features of septic shock (Feng et al., 2012). Therefore, the development of septic shock in our experiment was monitored by the MAP, biochemical parameters and histopathological staining, and these parameters are also important indicators of drug-related effects. In this study, a rabbit model of LPS-induced septic shock was established through a slow intravenous injection of 0.6 mg/kg LPS. The MAP of the rabbits in the model group decreased to less than 75% from 2 to 6 h. Six hours after LPS injection, the levels of LPS, LDH, AST, ALT, Cre, and Urea in the rabbit serum were significantly increased in the model group, and HE staining showed that the heart, liver and kidney were injured. These characteristics are typical of LPS-induced septic shock, as detailed in the literature, and indicate that the model was successfully established in this study (Liu et al., 2018; Shao et al., 2011). The active ingredients of Shenfu injection include ginsenosides and aconitine alkaloids. Ginsenosides Rg1, Re, and Rb1 are reported to have anti-inflammatory activity (Li et al., 2016), and higenamine has cardiotonic function (Feng et al., 2012). In addition, clinical studies have shown that Shenfu injection can increase the MAP in patients with septic shock (Mou et al., 2015). Similarly, we found that 6 or 8 ml/kg Shenfu injection can improve the MAP in septic shock, consistent with the results detailed in a clinical report (Mou et al., 2015). In our study, Shenfu injection at a dose of 6 and 8 ml/kg can protect LPS-induced septic shock of rabbits by lowering serum LPS levels, indicating that Shenfu injection can enhance rabbits' ability to eliminate LPS, but whether Shenfu injection can directly bind and neutralize LPS still needs to be validated by experiments. The serum LDH and AST levels reflect the functions of the heart and liver, respectively. Shenfu injection at a dose of 8 ml/kg significantly reduced the levels of LDH and AST in the serum of LPS-treated rabbits, which suggests that Shenfu injection can improve LPS-induced damage in the heart and liver. However, 8 ml/kg Shenfu injection failed to reverse the serum levels of ALT. Combined with the serum levels of AST and the histological changes observed by HE staining, 8 ml/kg Shenfu injection had protective effect on hepatic damage of LPS-induced septic 40

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Fig. 5. Microphotographs of morphological changes in heart, liver and kidney tissues (×200). C: normal group; M: model group; SF 4.5: 4.5 ml/ kg Shenfu injection group; SF 6: 6 ml/ kg Shenfu injection group; SF 8: 8 ml/ kg Shenfu injection group; DA: dopamine group. Heart: the red arrow indicates myocardial cell lysis, and the dark blue arrow indicates inflammatory cell infiltration. Liver: the yellow arrow indicates hepatocyte water-like degeneration and fat vacuoles, and the purple arrow indicates inflammatory cell infiltration. Kidney: the green arrow indicates renal tubule epithelial cell water-like degeneration and vacuolar degeneration, and the light blue arrow indicates glomerular shrinking. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

observations of histopathology and drug screening (Mainini et al., 2015). Several limitations of this study should be noted. First, we didn’t measure the active ingredients of Shenfu injection in the serum. Second,

our research couldn’t cover all types of septic shock. The present study supports the benefits of the Shenfu injection to only LPS-induced sepsis. In addition, some previous study (Xing et al., 2015) revealed that Shenfu injection can significantly alleviate intestinal epithelial damage 41

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Fig. 6. MALDI-TOF-MSI analysis of the effect of Shenfu injection on the distribution and content of small molecules in heart tissues of rabbits with LPS-induced shock. The intensity ratio of the small molecules was analyzed using SCiLS Lab software. The thickness of the heart slice was 10 µm, and the spatial resolution was set to 200 µm. Scale bar = 5 mm. Control: control group; Model: model group; SF 8 ml/kg: 8 ml/kg Shenfu injection group. The data are presented as the means ± SDs; n = 3. ### p < 0.001 vs. the control group; ## p < 0.01 vs. the control group; # p < 0.05 vs. the control group; * p < 0.05 vs. the model group.

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in CLP (cecal ligation and puncture) models of rats, which was a typical septic shock model with mixed bacterial infection. The positive effect of the medicine should be attributed to the only tested conditions so the usage be confined to the defined/tested conditions.

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5. Conclusion In conclusion, Shenfu injection exerts good therapeutic effect in rabbits with LPS-induced septic shock, as demonstrated by increases in the MAP, reductions in major organ damage and improvements in heart function. According to our MALDI-TOF-MSI results, Shenfu injection eases the abnormal changes in the levels of ATP, AMP and taurine in the heart tissue observed after LPS administration, indicating that Shenfu injection plays a role in improving energy metabolism and antioxidation. Therefore, Shenfu injection plays a protective role in LPS-induced shock in rabbits. CRediT authorship contribution statement Xi Liu: Data curation, Formal analysis, Methodology, Writing original draft. Runzhe Liu: Conceptualization, Formal analysis, Writing - review & editing. Zhenfeng Dai: Methodology. Hao Wu: Formal analysis. Ming Lin: Writing - review & editing. Fang Tian: Formal analysis. Zeyu Gao: Resources. Xin Zhao: Writing - review & editing. Yi Sun: Writing - review & editing. Xiaoping Pu: Conceptualization, Funding acquisition, Supervision, Writing - review & editing. Acknowledgements Project 2017JZ0036 was supported by the Key Project of Sichuan Province Science and Technology Support Program. Conflict of interest The authors declare that there are no conflicts of interest. References An, J., Du, J., Wei, N., Guan, T., Camara, A.K., Shi, Y., 2012. Differential sensitivity to LPS-induced myocardial dysfunction in the isolated brown Norway and Dahl S rat hearts: roles of mitochondrial function, NF-kappaB activation, and TNF-alpha production. Shock (Augusta, Ga) 37 (3), 325–332. Bone, R.C., 1996. Immunologic dissonance: a continuing evolution in our understanding of the systemic inflammatory response syndrome (SIRS) and the multiple organ dysfunction syndrome (MODS). Ann. Intern. Med. 125 (8), 680–687. Chen, H.W., Hsu, C., Lu, T.S., Wang, S.J., Yang, R.C., 2003. Heat shock pretreatment prevents cardiac mitochondrial dysfunction during sepsis. Shock (Augusta Ga) 20 (3), 274–279. Chida, J., Kido, H., 2014. Extraction and quantification of adenosine triphosphate in mammalian tissues and cells. Methods Mol. Biol. (Clifton N. J.) 1098, 21–32. Delano, M.J., Ward, P.A., 2016. The immune system's role in sepsis progression, resolution, and long-term outcome. Immunol. Rev. 274 (1), 330–353. Dellinger, R.P., Levy, M.M., Rhodes, A., Annane, D., Gerlach, H., Opal, S.M., Sevransky, J.E., Sprung, C.L., Douglas, I.S., Jaeschke, R., Osborn, T.M., Nunnally, M.E., Townsend, S.R., Reinhart, K., Kleinpell, R.M., Angus, D.C., Deutschman, C.S., Machado, F.R., Rubenfeld, G.D., Webb, S.A., Beale, R.J., Vincent, J.L., Moreno, R., 2013. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit. Care Med. 41 (2), 580–637. Feng, S., Jiang, J., Hu, P., Zhang, J.Y., Liu, T., Zhao, Q., Li, B.L., 2012. A phase I study on pharmacokinetics and pharmacodynamics of higenamine in healthy Chinese subjects. Acta Pharmacol. Sin. 33 (11), 1353–1358. Fink, M.P., Heard, S.O., 1990. Laboratory models of sepsis and septic shock. J. Surg. Res. 49 (2), 186–196. Fonseca, B.M., Cristovao, A.C., Alves, G., 2018. An easy-to-use liquid chromatography method with fluorescence detection for the simultaneous determination of five neuroactive amino acids in different regions of rat brain. J. Pharmacol. Toxicol. Methods 91, 72–79. Gaieski, D.F., Edwards, J.M., Kallan, M.J., Carr, B.G., 2013. Benchmarking the incidence

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