Yang Huang, Guo-Bao Wu, Yan-Jun Zhong

Department of critical medicine, Xiangya Second Hospital, Central South University, Changsha 410011

Keywords:

ABSTRACT

1. Introduction

Sepsis is an uncontrollable inflammatory reaction in the body. There are many causes of sepsis, and the number of patients is increasing. Patients may have infection at any part of the body, and organ dysfunction in severe cases. It is a common complication of shock, infection and other surgical operations [1]. According to a large number of literatures, the lung is the most vulnerable organ after sepsis, which can cause acute lung injury (ALI). Ali is caused by interstitial or direct causes of lung cell damage, leading to alveolar edema, clinical manifestations of patients with pulmonary insufficiency, ventilation / blood flow imbalance, leading to the emergence of respiratory distress syndrome [2].

At present, the treatment of chemical drugs for the prevention and treatment of sepsis lung injury is not ideal. Traditional Chinese medicine has a wide range of drug sources, long-lasting effect, high security and other characteristics. Curcumin is the extract of traditional Chinese medicine. According to previous studies, curcumin has a wide range of activities and has the effect of resisting bacteria, viruses and inflammation. It is commonly used in clinical research to inhibit a variety of malignant tumors [3]. Strand RNA dependent protein kinase (PKR) is a kind of protein kinase, which plays a significant role in immune response and myocardial cells [4]. The immune function of sepsis patients is abnormal, but there are few literatures about PKR signal and immune function of sepsis infected lung damage rats. So this paper discusses the effect of curcumin on PKR signal and immune function of sepsis infected lung damage rats.

2. Materials and methods

2.1 Main animals, reagents and instruments

SPF SD rats (Shanghai kilton Biology); curcumin and DMSO (Shanghai Beinuo biology, China); PKR (USA, Abcam); biochemical experiment electrophoresis protein instrument (Shanghai Zhixin, China); hematoxylin eosin Kit (Shanghai jianglai, China); f-800 blood cell counter (Shanghai Yonggui, China).

2.2 Animal grouping and Administration

Fifteen SPF grade SD rats, male, were randomly divided into three groups (5 rats in each group): sham group (sham group), acute lung injury group (ALI group) and curcumin group. In Ali group and curcumin group, 0.1 DMSO (1ml / 1kg) and 200mg curcumin were injected intraperitoneally. 2 times a day for 3 consecutive days.

2.3 animal model making

Model establishment of sepsis rats, reference [5]: 18 experimental rats were anesthetized with ketamine at a dose of 2-4mg, sterilized and kept sterile. A 1-2cm incision was made in the abdomen, the cecum was taken out, and the cecum was punctured with a medical special surgical needle at a distance of 4cm, three times to form a wound. Finally, the end of the cecum was sutured, returned to the original position, and the skin group was sutured Weaving. In sham group, the distal cecum was separated from the abdomen without any other treatment. Use normal saline to maintain life, eat and drink normally.

2.4 Tissue extraction

Two rats in each group were killed by bleeding from the right heart after anesthesia. The right lung lobe was extracted and stored in liquid nitrogen for test.

2.5 HE staining to observe lung tissue pathology

The right lungs of rats in each group were cryopreserved. After paraffin embedding, the sections were taken out for 4 μ m and sealed with goat serum. The first staining time was 6-8s and the second staining time was 10s. After the completion, the remaining lung tissues were stored at - 80 ° C and sealed for use in subsequent experiments.

2.6 Inflammatory factors in serum

After the drug intervention of the remaining rats, the tail vein blood was taken and the expression of TNF - α, IL-1 β, IL-6 in the supernatant was detected by ELISA. The process was in accordance with the instructions. The experiment was completed within 20 minutes and the average value was taken

2.7 Detection of immune function index

In addition, 2ml of the whole blood of each group of rats was taken and the level of leukocytes, lymphocytes, monocytes and ZTE granulocytes were counted on the f-800 blood cell counter.

2.8 Oxidative stress level of lung tissue

Take 100 μ g of lung tissue, use PMSF to homogenize the lung tissue, centrifugate and retain the supernatant, detect the expression of MDA, SOD, GSH PX and cat oxidative stress activity through each index kit, and operate in strict accordance with the instructions

2.9 Immunoblotting for PKR

The lung tissue was centrifuged with protein lysate, and then the solution was added to the sample pore. After electrophoresis, take off PVDF membrane TBS and soak it for 10min, seal it, wash PBS repeatedly, add PKR (1:500), hybridize at 40 ℃ for 1D, repeat the above cleaning steps, add second antibody (1:2000), hybridize for 2h. The membrane was immersed in ECL working solution, and then detected to obtain electrophoretic image.

2.10 Statistical analysis

Spss22.0 software was used to analyze the content of this paper. The pathology of lung tissue, serum inflammatory factors, immune function, PKR positive and protein in lung tissue of each group were detected by t-test (±s), P ＜ 0.05.

3. Results

3.1 Pathological examination of lung tissue in each group

In the sham group, the structure of pulmonary bubbles was clear, and there was no inflammatory infiltration in the interstitium; in the Ali group, the structure of pulmonary bubbles was disorder, intraluminal hemorrhage, a lot of inflammation and interstitial changes occurred; in the curcumin group, compared with the Ali group, the inflammatory infiltration was reduced, the interstitial width was improved, and the lung tissue injury was improved. See figure 1.

Fig.1 lung histopathology of rats in each group (200 times)

3.2 Expression of serum inflammation in rats of each group

The levels of TNF - α, IL-1 β and IL-6 in Ali group were (750.24 ± 21.03), (487.21 ± 60.33) and (2632.10 ± 175.06) respectively, which were significantly lower than those in sham group (65.85 ± 19.25), (66.38 ± 10.58) and (404.6565.23) (P ＜ 0.05), while those in curcumin group were (340.29 ± 31.26), (201.14 ± 30.57) and (985.14 ± 116.38), respectively (P ＜ 0.05) )See Table 1

Table 1 Serum inflammatory expression of rats in each group

Table 2 Comparison of immune function indexes of rats in each group

3.3 Comparison of immune function indexes of rats in each group

The levels of leukocytes, lymphocytes and neutrophils in Ali group were (5.30 ± 0.85), (4.23 ± 0.60), (0.12 ± 0.05) and (1.23 ± 0.16) respectively, which were significantly different from those in sham group (8.51 ± 1.02), (6.90 ± 0.78), (0.32 ± 0.14) and (0.75 ± 0.09) (P ＜ 0.05), while those in curcumin group were (7.16 ± 0.66), (5.78 ± 0.09) 42), (0.24 ± 0.06) and (0.96 ± 0.35) were significantly different from those in Ali group (P ＜ 0.05), as shown in Table 2

3.4 Comparison of oxidative stress indexes in lung tissue of each group

MDA, SOD, GSH in Ali group- PX and cat levels were (34.65 ± 5.36), (25.57 ± 4.30), (13.25 ± 1.89) and (15.87 ± 2.50) respectively, which were significantly different from those of sham group (12.45 ± 1.80), (53.16 ± 7.80), (27.56 ± 5.41) and (36.44 ± 6.03) (P ＜ 0.05). Curcumin group (22.40 ± 3.54), (38.69 ± 5.21), (20.33 ± 2.90) and (27.08 ± 3.26) respectively )Compared with ALI group, there was significant difference (P ＜ 0.05), as shown in Table 3

Table 3 Comparison of oxidative stress indexes in lung tissue of each group

3.5 Comparison of PKR protein levels in lung tissues of each group

The PKR protein level in Ali group was lower than that in sham group (P ＜ 0.05), and the PKR protein level in curcumin group was higher than that in Ali group (P ＜ 0.05), as shown in Figure 2

Fig.2 Comparison of PKR protein levels in lung tissues of each group (a represents per protein electrophoretogram in lung tissues of each group; B represents per protein expression in lung tissues of each group; * represents P ＜ 0.05 compared with sham group, Chen represents P ＜ 0.05 compared with ALI group;)

4. Discussion

Ali in sepsis is one of the organ damages in sepsis patients. The pathological manifestations are disorder of pulmonary bubble structure, intracavitary hemorrhage, massive inflammation, widening of lung interstitium. The clinical manifestations are dyspnea, resulting in respiratory distress and other symptoms, and poor survival state [6-7]. In recent years, sepsis has been the focus of many scholars, but its research mechanism needs further elaboration.

In the Ali of sepsis, the imbalance of inflammatory response and oxidative stress are the main causes of multiple organ damage, and multiple cytokines participate in multiple links of lung damage [8]. TNF - α is the main factor of inflammatory reaction in Ali with sepsis. It can activate NF-kB signal and has many biological activities, which will accelerate the occurrence and development of Ali with sepsis [9]. IL-6 is an important cytokine involved in systemic inflammatory response. IL-1 β is mainly produced by macrophages and belongs to the inflammatory promoter. When Ali occurs in sepsis, IL-1 β damages endothelial cells and induces lung injury [10]. This study indicated that the level of serum cytokines in ALI rats with sepsis was significantly increased, and the level of inflammatory factors decreased after curcumin treatment. Curcumin is a kind of traditional Chinese medicine material which has been extracted from Curcuma block for thousands of years. It has been recognized that curcumin has a wide range of biological functions. A large number of studies have shown that curcumin has a significant anti-inflammatory effect [11-12]. According to Xiao Xuefei [13], curcumin can inhibit the expression of nuclear factors, reduce the release of cytokines from macrophages, and improve Ali in sepsis. The results of this study are similar to those of others.

There is a serious immune imbalance in Ali with sepsis. According to the research, the increase of neutrophils in Ali with sepsis can accelerate cell damage, lung organ dysfunction and the occurrence and development of Ali with sepsis [14]. In this study, curcumin can improve the immune function of ALI rats with sepsis, increase the level of leukocytes and decrease the level of neutrophils. The literature shows that curcumin can increase leukocyte activity by activating natural killer cells, promote cell surface binding of a variety of bioactive factors, inhibit the level of neutrophils, and improve the immune function [15]. The results of this study are similar to those of others.

Antioxidants can reduce inflammatory response by reducing the level of oxidative stress index in Ali. MDA can directly reflect the oxidation level of the body. The increase of MDA level in Ali lung tissue homogenate indicates that there is oxidative stress in the lung tissue [16]. SOD, contrary to MDA, can inhibit tissue oxidation. GSH - PX and cat are important components of the body's antioxidant system, which can reduce oxidized substances and relieve their toxicity [17]. In this study, the level of MDA in Ali group increased, the level of SOD, GSH PX and cat decreased, the level of MDA decreased after curcumin intervention, and the other levels increased. Subin Varghese Thomas [18] study shows that curcumin can restore the antioxidant function of lung tissue, eliminate free radicals, and improve the level of oxidative stress indicators. The results of this study are similar to those of this study.

PKR is a kind of double stranded RNA protease. In previous studies, PKR expression decreased in patients with lung injury [19]. In addition, Lei Chu [20] found that PKR mRNA level was significantly lower than that of the control group in the lung tissue injury caused by radiotherapy. PKR expression decreased in the lung tissue injury rats, indirectly promoting the increase of Caspase-3 level. In this study, curcumin can improve the PKR protein level in lung tissue of ALI rats with sepsis. But what kind of signal pathway curcumin can enhance PKR protein level needs further study.

In conclusion, curcumin can improve the immune function of ALI rats infected with sepsis and reduce the expression of serum inflammation, which may be related to the improvement of PKR protein level.