Protective effects of oridonin on the sepsis in mice

Abstract

This study aimed to investigate the protective effects of oridonin (ORI) on cecal ligation and puncture (CLP)‐induced sepsis in mice. Male C57BL/6 mice weighing 22–30 g and aged 8–10 weeks were randomly assigned to three groups: Sham group, CLP group, or CLP plus ORI group. In the CLP group and ORI group, CLP was induced, and intraperitoneal injection of normal saline and oridonin (100 μg/kg) was conducted, respectively. The survival rate was determined within the following 7 days. The blood, liver, and lung were collected at 24 hours after injury. Hematoxylin–eosin staining of the lung, detection of lung wet‐to‐dry ratio, and serum cytokines (tumor necrosis factor [TNF]‐α and interleukin [IL]‐6), and examination of intraperitoneal and blood bacterial clearance were conducted to evaluate the therapeutic efficacy. Results showed that ORI treatment significantly reduced the lung wet‐to‐dry ratio, decreased serum TNF‐α and IL‐6, and improved liver pathology compared with the CLP group (p < 0.05). Moreover, the intraperitoneal and blood bacterial clearance increased markedly after ORI treatment (p < 0.05). The 7‐day survival rate in the ORI group was also dramatically higher than in the CLP group (p < 0.05). Our findings indicate that ORI can attenuate liver and lung injuries and elevate bacterial clearance to increase the survival rate of sepsis mice.

Introduction

Sepsis is a syndrome, not a disease. It is an imprecise clinical diagnostic term used to describe patients who have a continuum of abnormalities in organ function [1]. The worldwide incidence of sepsis is estimated to be 18 million cases per year [2]. Sepsis remains the most common cause of death in people who have been hospitalized, and between 20% and 50% of patients with sepsis die [[3], [4]]. Furthermore, sepsis is associated with a reduced quality of life in those who survive their acute illness [5].

Unlike other major epidemic illnesses, treatment for sepsis is nonspecific and limited primarily to support organ function and consists of administration of intravenous fluids, antibiotics, and oxygen [6]. There are no approved drugs that specifically target sepsis. In recent years, increasing studies employ Traditional Chinese Medicine in the therapy of sepsis [[7], [8], [9]]. Oridonin (ORI) is a famous diterpenoid isolated from the Chinese medicinal herb Rabdosia rubescens which is also known as dong ling cao [10]. This compound has drawn attention for its remarkable apoptosis and autophagy‐inducing activity in cancer therapy [11]. However, many prominent studies have proven that ORI possesses many other therapeutic effects, such as antiinflammatory, neuroprotective, antibacterial, and antineoplastic activities [[12], [13], [14]]. In this study, the protective effects of ORI on sepsis were investigated in a mouse model. Our results show that ORI treatment attenuates lung and liver injuries and enhances the survival rate of sepsis mice, which is due to the reduction in serum proinflammatory cytokines and increase in serum and peritoneal bacterial clearance.

Materials and methods

Animals and grouping

A total of 50 male C57BL/6 mice aged 8–10 weeks and weighing 22–30 g were purchased from the Experimental Animal Center of the Second Military Medical University. Mice were randomly assigned into three groups. In the Sham group (n = 10), mice received laparotomy without manipulation of the intestine; in the CLP group, mice received cecal ligation and puncture (CLP) followed by intraperitoneal injection of normal saline (n = 10 for survival analysis; n = 10 for biochemical analysis); in the CLP+ORI group, mice received CLP followed by intraperitoneal injection of ORI at 100 μg/kg (n = 10 for survival analysis; n = 10 for biochemical analysis). Animals were housed under controlled temperatures (23 ± 2°C) with 12 hours of light/dark cycles and given ad libitum access to food and water. This study was approved by the Ethics Committee of the Second Military Medical University.

Reagents

The following reagents were used in the present study: ORI (Lot number: 28957‐04‐2; Shanghai Yuanye Biotechnology Co., Ltd. China), hematoxylin–eosin staining kit (Wuhan Boster Biotechnology Co., Ltd. China), nutrient agar medium (Shanghai Tiancheng Biotechnology Co., Ltd. China), paraformaldehyde (Sinopharm Biochemical Reagents Company, Shanghai, China), mouse tumor necrosis factor‐α (TNF‐α), Quantikin Enzyme‐Linked Immunosorbent Assay (ELISA) Kit (Cat: MTA00B), and mouse interleukin‐6 (IL‐6) Quantikin ELISA Kit (Cat:M6000B; R&D Systems Inc., MN, USA).

Animal model of sepsis

CLP was employed to induce sepsis in mice. Briefly, following isoflurane anesthesia, the cecum was exposed after a 1‐cm‐long midline incision was made. The cecum was ligated ∼15 mm proximal to the cecal pole with a 5/0 Prolene thread without stricture of the ileocecal valve. The ligated cecum was then punctured once with a 22‐gauge needle. The cecum was slightly compressed until a small drop of stool appeared. The abdominal wall was closed, and fluid resuscitation was conducted with subcutaneous injection of 1 mL of normal saline after surgery. In the CLP group, mice received intraperitoneal injection of normal saline immediately after surgery; in the CLP+ORI group, mice received intraperitoneal injection of oridonin at 100 μg/kg immediately after surgery.

Detection of serum cytokines

At 24 hours after surgery, mice were deeply anesthetized with 10% chloral hydrate at 0.3 mL/100 g and sacrificed. Blood was collected from the heart, and serum was collected after centrifugation. Serum contents of TNF‐α and IL‐6 were detected with corresponding ELISA kits according to the manufacturer's instructions.

Pathological examination

At 24 hours after surgery, mice were anesthetized, and the liver and lung were collected, fixed in 4% paraformaldehyde, and embedded in paraffin and sectioned. Hematoxylin–eosin staining was performed to evaluate the liver and lung injuries.

Detection of lung wet‐to‐dry lung weight ratio

At 24 hours after surgery, animals were sacrificed and the left lungs were collected and weighed after removing water on the lung (wet weight). The lung was then heated at 60°C for 72 hours until the lung weight remained stable. The lung was weighed (dry weight). The wet to dry (W/D) weight ratio was calculated.

Detection of bacterial clearance

At 24 hours after CLP, mice were anesthetized with 2.5% isoflurane. Mice were fixed on a table and anesthesia was maintained with 2.5% isoflurane. The skin of the abdomen was cut open (0.5 cm) in the midline after thorough disinfection. Blood (200 μL) was collected by sterile puncture of the left cardiac ventricle with a syringe, transferred into a sterile tube and stored at 4°C. Then, laparotomy was performed and the peritoneal cavity was exposed. Sterile 1× phosphate buffer solution (Sinopharm Co., Ltd, China) (2 mL) was injected into and aspirated out of the peritoneal cavity five times. The peritoneal lavage fluid (PLF) was stored at 4°C. Bacteria were grown, and identification tests were conducted in accordance with routine bacteriological methods. Bacterial counts are given as number of milliliter of peritoneal lavage or blood.

Survival rate

The survival status was observed within 7 days, and the survival rate was calculated, followed by delineation of survival curve.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Quantitative data are expressed as mean ± standard deviation. Significant differences among groups were assessed with one way analysis of variance, followed by Student–Newman–Keuls‐q test. The survival was analyzed with Kaplan–Meier survival curves and log‐rank test. A p value < 0.05 was considered statistically significant.

Results

Survival analysis

Survival curves were analyzed in three groups. None died in the Sham group. The survival rate was 20% in the CLP group and 70% in the CLP+ORI group at 7 days after surgery, showing a significant difference between them (Figure 1). This suggests that postoperative oridonin treatment improves the survival of CLP mice.

Figure 1.

Seven‐day survival rate in three groups. The survival rate was 20% in the cecal ligation and puncture (CLP) group and 70% in the CLP+oridonin (ORI) group at 7 days after surgery. The survival rate in the CLP group was significantly lower than in the CLP+ORI group.

Microscopic analysis

In the Sham group, the liver structure was nearly normal and there were no hepatocyte necrosis, no hemorrhage, and no infiltration of inflammatory cells. In the CLP group, severe hepatocyte swelling was present, the hepatic cord was irregularly arranged, and the hepatic sinus was compressed and narrowed. After ORI treatment, the liver injury was significantly attenuated: the hepatocytes were nearly normal, hepatocyte swelling was alleviated, and hepatic sinus compression was mitigated (Figure 2). In the Sham group, the lung structure was intact: no alveolar hemorrhage, normal alveolar septum, normal pulmonary capillaries, and no inflammatory cells. In the CLP group, the lung structure was significantly disrupted, and alveolar hemorrhage, pulmonary interstitial edema, alveolar collapse, and infiltration of a large amount of inflammatory cells were observed in the lung. After ORI treatment, the lung injury was markedly attenuated: alveolar septum was widened slightly, there was no obvious alveolar hemorrhage, and only a few inflammatory cells infiltrated in the lung (Figure 2).

Figure 2.

Liver and lung morphology in different groups. In the Sham group, the liver structure was nearly normal. In the cecal ligation and puncture (CLP) group, severe hepatocyte swelling, irregularly arranged hepatic cord, and compressed and narrowed hepatic sinus were observed. After oridonin (ORI) treatment, the morphology of hepatocytes was nearly normal, hepatocyte swelling was alleviated, and hepatic sinus compression was mitigated. In the Sham group, the lung structure was complete. In the CLP group, the lung structure was significantly disrupted, and alveolar hemorrhage, pulmonary interstitial edema, alveolar collapse, and infiltration of a large amount of inflammatory cells were observed. After ORI treatment, the alveolar septum was widened slightly, there was no obvious alveolar hemorrhage and only a few inflammatory cells infiltrated in the lung. Scale bar: 50 μm.

Lung W/D ratio

In the sham group, the lung W/D ratio was 3.85 ± 0.28. At 24 hours after CLP, the lung W/D ratio was 4.23 ± 0.19, which was significantly higher than in the Sham group (p < 0.05). However, ORI treatment dramatically attenuated lung edema, characterized by a reduction in lung W/D ratio (3.99 ± 0.15, p < 0.05 vs. CLP group; Figure 3).

Figure 3.

Lung wet‐to‐dry (W/D) ratio in different groups. The lung W/D ratio was 3.85 ± 0.28 in the Sham group, 4.23 ± 0.19 in the cecal ligation and puncture (CLP) group and 3.99 ± 0.15 in the CLP+oridonin (ORI) group. ORI treatment dramatically reduced lung W/D ratio in mice after CLP. * p < 0.05 versus Sham group. ** p < 0.05 versus CLP group.

Serum contents of TNF‐α and IL‐6

The serum contents of TNF‐α and IL‐6 were 5.8 ± 5.4 pg/mL and 50.2 ± 11.2 pg/mL in the Sham group. At 24 hours after CLP, the serum contents of TNF‐α and IL‐6 increased markedly (49.8 ± 17.1 pg/mL and 539 ± 133.7 pg/mL, respectively, p < 0.05). However, ORI treatment after CLP significantly reduced the serum TNF‐α and IL‐6 contents (22.5 ± 4.1 pg/mL and 240.8 ± 67.1 pg/mL, respectively, p < 0.05 vs. CLP group; Figure 4).

Figure 4.

Serum contents of tumor necrosis factor (TNF)‐α and interleukin‐6 (IL‐6) in different groups. The serum contents of TNF‐α and IL‐6 were 5.8 ± 5.4 pg/mL and 50.2 ± 11.2 pg/mL, respectively, in the Sham group, 49.8 ± 17.1 pg/mL and 539 ± 133.7 pg/mL, respectively, in the cecal ligation and puncture (CLP) group, and 22.5 ± 4.1 pg/mL and 240.8 ± 67.1 pg/mL, respectively, in the CLP+oridonin (ORI) group. ORI treatment significantly reduced the serum contents of TNF‐α and IL‐6. * p < 0.05 versus Sham group. ** p < 0.05 versus CLP group.

Blood and peritoneal bacterial clearance

Bacterial culture showed there were no bacteria in both blood and PLF in the sham group. At 24 hours after CLP, results showed the bacterial counts of the blood and PLF increased remarkably (71.7 ± 25.4 and 6.9 ± 1.0 × 10⁴, respectively, p < 0.05 vs. sham group). However, ORI treatment after CLP significantly reduced the bacterial counts of the blood and PLF (15.3 ± 7.5 and 0.14 ± 0.07 × 10⁴, respectively, p < 0.05 vs. CLP group; Figure 5).

Figure 5.

Bacterial counts in different groups. The bacterial counts of the blood and peritoneal fluid increased significantly in the cecal ligation and puncture (CLP) group compared with the Sham group. However, oridonin (ORI) treatment significantly reduced the bacterial counts of the blood and peritoneal fluid. * p < 0.05 versus Sham group. **p < 0.05 versus CLP group. CFU = colony forming units; PLF = peritoneal lavage fluid.

Discussion

Sepsis remains a major cause of morbidity and mortality in hospitalized patients. Although numerous studies have been conducted to investigate the pathogenesis and therapy of sepsis, treatment for sepsis is still nonspecific and there are no approved drugs that specifically target sepsis [6]. In recent years, Traditional Chinese Medicine is employed for the prevention and therapy of sepsis. In this study, we investigated the protective effects of ORI on sepsis in a mouse model. Our results showed that ORI significantly reduced the lung W/D ratio, decreased serum proinflammatory cytokines and improved liver and lung pathology, as well as increased the intraperitoneal and blood bacterial clearance and 7‐day survival rate.

ORI is a famous diterpenoid isolated from the Chinese medicinal herb R. rubescens [10]. Increasing studies have been conducted to investigate the biological activities of ORI and its protective effects in many disease models [15]. In recent years, increasing attention has been paid to its antineoplastic activity [11] although studies have reported its antiproliferative, neuroprotective, antibacterial, and antiinflammatory effects as well as effects on the immune system [[12], [13], [14], [16]]. In this study, we investigated the protective effects of ORI on sepsis in a mouse model.

Sepsis is more properly defined as “a failure of homeostasis,” i.e., “an inability for the organism or cell to maintain internal equilibrium by adjusting its physiological processes under fluctuating environmental conditions in response to infection or injury” [3]. Inflammation and immune dysfunction are closely related to the pathogenesis of sepsis. Events developing early in sepsis suggest that a hyperinflammatory state exists, and development of immunosuppression and degraded innate and adaptive immune responses are well‐established complications of sepsis. Studies have revealed a marked suppression of cell‐mediated immunity that appears to contribute to the morbidity and mortality in sepsis [17] and there is a shift from a Th1 to a Th2 response as well as the induction of an immune suppressive macrophage phenotype in sepsis [18]. Thus, investigators have attempted to treat sepsis with immunomodulatory strategies in clinical practice [19]. The immune dysfunction may induce the hyperinflammatory response, resulting in the production of proinflammatory cytokines (cytokine storm) [20]. Hu et al [16] found that ORI had a distinct effect on promoting CD4+/CD25+ Treg differentiation and modulating Th1/Th2 balance, which was achieved via inducing the antiinflammatory target HO‐1. Ku and Lin [21] also reported that ORI had a relative Th1‐inclination property. There is evidence showing that ORI is able to inhibit the secretion of IL‐2, interferon‐γ, IL‐12p40, and TNF‐α in murine splenic lymphocytes [22]. However, ORI may also facilitate phagocytic activity against apoptotic cells through TNF‐α and IL‐1β release, thereby contributing to its antitumor activity [23]. Our results also showed that ORI was effective to reduce serum proinflammatory cytokines, and also increased the blood and peritoneal bacterial clearance in sepsis mice.

Of note, ORI does not easily dissolve in water, and thus its blood concentration cannot easily match its need. To overcome this, some groups have prepared ORI nanosuspension and nanoparticles [[24], [25]] as well as ORI analogs [26].

There were still limitations in this study. The dose‐response curve of ORI was not investigated in our study, and thus we can not confirm the optimal dose of ORI for the treatment of sepsis. In our pilot study, sepsis mice were initially treated with ORI at 10 mg/kg which is the most common dose used in available studies [27]. However, the alanine aminotransferase further increased in sepsis mice after ORI treatment. Then, the dose was reduced, and ORI at 100 μg/kg and 1 mg/kg was used. The alanine aminotransferase slightly increased after treatment with ORI at 1 mg/kg in sepsis mice. Considering that there is pre‐existing liver injury in sepsis, ORI at 100 μg/kg was used in this study. In addition, only short‐term treatment with ORI was performed in mice. Whether long‐term ORI treatment has a better therapeutic efficacy and whether repeated treatment with ORI increases the risk for adverse effects in case of sepsis are still unclear. Moreover, the specific mechanism underlying the therapeutic effects of ORI was not further studied. Studies have identified several proteins targeted by ORI and transcription factors and signaling pathways regulated by ORI [15]. Thus, more studies with elegant design are required to elucidate the potential mechanisms underlying the protective effects of ORI on sepsis.

Taken together, our results indicate that ORI reduces proinflammatory cytokines and increase blood and peritoneal bacterial clearance to exert protective effects on sepsis in mice. More studies are required to investigate the optimal dose of ORI in the treatment of sepsis, explore the specific mechanism underlying its protective effects on sepsis, and improve the bioavailability and safety of ORI in the therapy of diseases.