Xiao-Yun Wei, Na Wei

Zhangjiagang Hospital Affiliated to Nanjing University of Chinese Medicine Pediatrics, Zhangjiagang, Jiangsu 215600

Keywords:

ABSTRACT

1. Introduction

The Human Respiratory Syncytial Virus (RSV) is a common respiratory infection disease [1-2] frequently occurring in infants and children with immune system defects, it is highly contagious and can cause patients' lung function to be affected. It is an important cause of asthma occurrence and development. About 80% of children were infected with RSV during the age of 2 years, and the younger the infant was when he became sick, the higher the RSV load and the heavier the disease [3]. Approximately 160,000 infants and young children die each year from RSV-associated lower respiratory tract infections, which seriously threatens patients' lives [4]. At present, the pathogenesis of RSV is not completely clear. Studies have found that RSV is closely related to the inflammatory response. Patients' immune function is often affected after the onset, and various cytokine imbalances appear. Interleukin-6 (Interleukin 6, IL- 6) and Interleukin-8 (IL-8) are often over-expressed, aggravating local airway inflammation, causing bronchitis, pneumonia and other diseases [5-6]. There is still a lack of safe and effective treatments in clinical practice. RSV is currently used to meet the standard treatment, mainly to control the corresponding symptoms of the disease, many adverse drug reactions, poor results, and prone to drug resistance [7-8].

It is important to find an antiviral drug that is both safe and effective for the prevention and / or treatment of RSV infection. At present, the epidemic trend of RSV infection is still severe, but there are still no safe and effective vaccines and specific control methods. In recent years, studies have found that Kekeling mixture has certain efficacies in the treatment of respiratory infections, significantly improve the respiratory function of patients and reduce the recurrence of wheezing [9]. In order to further clarify the effect of Kekeling Mixture in the treatment of RSV, this article investigates the effect of Kekeling Mixture on the levels of IL-6 and IL-8 in RSV infected mice, and then provides a theoretical basis for the treatment of RSA. The experiment is reported as follows.

2. Materials andmethods

2.1 Materials

2.1.1About the subjects

The experimental animals in this study were 60 healthy BALB / c mice, half male and half male, weighing 120-200g, clean grade, provided by Beijing Experimental Animal Center, and kept in a clean, constant temperature and dark and alternating clean grade In the environment (12h each for light and dark), drink freely, drink water, and adaptively breed for 7d to adapt to the environment and avoid external stimuli.

2.1.2 Cells and reagents

The human laryngeal cancer epithelial cells (Hep-2) were purchased from Guangzhou Bote Biological Engineering Co., Ltd .; respiratory syncytial virus strains were purchased from Nanjing Jitian Biological Co., Ltd.

Reagents: Kekeling Mixture (Ephedra sinensis 3g, Scutellariabaicalensis 6g, Bitter Almond 10g, Honeysuckle 10g, Dai Clam Powder 6g, Wild Chrysanthemum 6g, Gypsum 20g, Melon Skin 6g, Dilong 6g, Mulberry Skin 6g, Raw Licorice 3g ) Purchased from Zhangjiagang Traditional Chinese Medicine Hospital, Nanjing University of Traditional Chinese Medicine; mouse IL-6, IL-8 enzyme-linked immunosorbent assay (EL ISA) kits were purchased from Shanghai Enzyme Biological Co., Ltd .; fetal bovine serum (FBS) was purchased from Beijing Yami Biological Technology Co., Ltd .; Ether was purchased from Wuhan Hengwo Technology Co., Ltd .; Ribavirin was purchased from Beijing Huarui Biological Pharmaceutical Co., Ltd .; Pancreatin was purchased from Hebei Lihua Biotechnology Co., Ltd .; Saline was purchased from Tianjin Damao Reagent Co., Ltd .; Paraformaldehyde was purchased from Zhejiang Lianshuo Biological Technology Co., Ltd .; Paraffin was purchased from JiyuanHengshun New Material Co., Ltd.

2.2 Methods

2.2.1 Preparation and evaluation of RSV infection model

Diethyl ether inhalation was usedfor anesthesia.When the consciousness of the mouse was lost, the limbs were paralyzed, and the breathing increased, the anesthesia is considered successful. Use a pipette to suck 50 μL of 100TCID50 RSV suspension, and slowly instill along the nostril of the mouse until the virus solution is completely inhaled. They were then returned to the cage and inoculated once. The normal group was given a nasal drip of DMEM culture medium. After successful modeling, the mice mainly showed increased body temperature, decreased food intake, decreased activity, and atrophy.

2.2.2 Groups and administration of drugs

According to the digital random grouping method, 60 mice were randomly divided into normal group, model group, Kekeling mixture high, medium and low dose group, ribavirin group, 10 mice in each group. On the second day, the normal group and the RSV infection model group were given the same amount of physiological saline 0.2mL / 10g, twice a day; the Kekeling mixture high, medium and low dose groups were given 88.4g / (kg.d) , 44.2g / (kg.d), 22.1g / (kg.d) were treated, and ribavirin was treated with 27.6mg / (kg · d) for 6 days.

2.2.3 Detection method

The six groups of mice were fasted and water-free after the drug intervention. Samples were collected at 8:00 the next morning. Ten mice from each group were randomly selected, and five were randomly intubated, and 0.8 mL of PBS was injected into the lung 3 times. , 0.5mL, 0.5mL, then collect about 1mL of alveolar lavage fluid, use ELISA to detect the levels of IL-6, IL-8 in the alveolar lavage fluid; the remaining 5 were killed directly, first remove the posterior heart leaf, put A 4% paraformaldehyde solution was used for internal fixation, embedded in paraffin, and pathological examination of mouse lung tissue using HE staining. The remaining lung tissue was removed and stored in a -80 ° C ultra-low temperature refrigerator. The levels of IL-6 and IL-8 in the alveolar lavage fluid were measured by ELISA. All operations were performed strictly according to the instructions.

2.2.4 Statistical method

This study used SPSS 20.0 statistical software to process experimental data. Counting data uses the "mean ± standard deviation" meter, represented by (±s), the independent sample t test is used between normal distribution data groups, the counting data is expressed as%, and the Chi-square test P ＜0.05 was considered statistically significant.

3. Results

3.1 General condition

One-dayafter the model was built, the general livingconditions of the mice in each group was analyzed. The normal group had smooth, shiny hair and good mental status, while the RSV-infected mice had standing hair, slow movement, reduced food intake, and mental status.

3.2 Lung-tissue lesion condition

The analysis and comparison of lung lesions of mice in each group, normal alveolar epithelial cells are relatively complete, alveolar spaces are thin, bronchial and pulmonary spaces are intact, as shown in Figure 1A; mouse alveolar epithelial cells in RSV infection model group Broken, alveolar wall is significantly thickened, and the blood vessels are swollen and congested. Monocytes, lymphocytes and neutrophils infiltrate the connective tissue of the lungs, as shown in Figure 1B. After treatment with ribavirin, the lungs of mice Tissue lesions were significantly improved, bronchial skin cells in the lungs were arranged neatly, and no obvious inflammatory exudates in the cavity were close to normal levels, as shown in Figure 1C. After treatment with high-dose Kekeling mixture, lung tissue lesions were repaired to a certain extent. The number of mouse alveolar epithelial cell damage was significantly reduced, and vascular congestion was improved, but the treatment effect was lower than that of the ribavirin group; see Figure 1D. After treatment with the mediumdose Kekeling mixture, the number of mouse alveolar epithelial cell damage was reduced. Significantly reduced, alveolar interval decreased, lung tissue inflammation and infiltration weakened, the effect was lower than that in the high-dose group, see Figure 1E; after treatment with low-dose Kekeling mixture, Tissue damage were improved, but the effect is limited, the overall effect is less than in the high dose group, see Figure 1F.

The HE staining method was used to detect the effect of Kekeling Mixture on lung tissue lesions in RSV infected mice, and the lung tissue lesions in mice were significantly improved after treatment. A: Lung tissue of normal group mice. B: Lung tissue of model group. C: Lung tissue of ribavirin group. D: Lung tissue of high dose group. E: Lung tissue of medium dose group. F: lung tissue of mice in low dose group.

Figure 1: Pathological pictures of each group

3.3 Levels of IL-6 and IL-8 in alveolar lavage fluid within each group

The levels of IL-6 and IL-8 in the alveolar lavage fluid of each group of mice were measured. Compared with the normal group, the IL in the model group, Kekeling mixture high, medium and low dose groups, and the ribavirin group -6, the level of IL-8 was significantly increased, and the difference was statistically significant (P ＜0.05). Compared with the model group, IL- in the high, medium and low dose Kekeling mixture group and the ribavirin group,the level of IL-8 decreased significantly; the levels of IL-6 and IL-8 in the alveolar lavage fluid of the mice in the high-low and middle-dose groups were significantly higher than those in the Ribavirin group, and the difference was statistically significant (P ＜ 0.05). The expression levels of IL-6 and IL-8 in alveolar lavage fluid of each group were ranked as follows: high-dose group ＜medium-dose group ＜low-dose group, and the differences were statistically significant (P ＜0.05). See Table 1 for specific results.

Table 1 levels of IL-6 and IL-8 in BALF within each group(±s ; ng/L)

Table 1 levels of IL-6 and IL-8 in BALF within each group(±s ; ng/L)

Compared with the normal group, * P ＜0.05; compared with the model group, ** P ＜0.05; compared with the Ribavirin group, *** P ＜0.05

GroupsnIL-6IL-8Ft Normal group1056.18±7.29407.28±21.15 35.18 3.872 Model group10 288.96±36.45*1461.27±32.37* 69.76 4.879 Ribavirin group10 98.28±21.56**439.51±23.84** 41.82 3.076 Kekeling highdose group10 109.32±27.33**/*** 469.27±31.19**/*** 45.38 3.689 Kekelingmediumdose group10 131.26±23.45**/*** 538.58±34.29**/*** 50.98 3.825 Kekelinglowdose group10 152.56±30.15**/*** 784.29±30.22**/*** 45.56 4.292

3.4 Comparison of IL-6 and IL-8 levels in lung tissue of each group

The levels of IL-6 and IL-8 in the lung tissue of the mice in each group were analyzed and compared. The levels of IL-6 and IL-8 in the serum of RSV-infected mice were higher than those in the normal group, and the differences were statistically significant ( P ＜0.05); Compared with the model group, the levels of IL-6 and IL-8 in the serum of the mice decreased significantly after treatment, and the difference was statistically significant (P ＜0.05); The levels of IL-6 and IL-8 in lung tissue of Chinese mice were significantly higher than those of Ribavirin group, and the difference was statistically significant (P ＜0.05). The contents of IL-6 and IL-8 in the lung tissue of each group of mice were high-dose group ＜medium-dose group ＜low-dose group, and the differences were statistically significant (P ＜0.05). The specific results are shown in Table 2.

Table 2 levels of IL-6 and IL-8 within each group(±s; ng/L )

Table 2 levels of IL-6 and IL-8 within each group(±s; ng/L )

Note: Compared with the normal group, * P ＜0.05; compared with the model group, ** P ＜0.05; compared with the Ribavirin group, *** P ＜0.05

GroupsnIL-6IL-8Ft Normal group10 27.23±3.16*307.22±17.25* 39.17 3.79 Model group10 100.06±7.12** 1358.28±19.37** 58.76 4.68 Ribavirin group10 63.58±4.53** 355.42±21.32** 38.29 5.34 Kekeling highdose group10 63.32±9.46**/*** 419.33±23.45**/*** 40.23 3.19 Kekelingmediumdose group10 79.41±8.18**/*** 512.26±25.37**/*** 51.27 4.26 Kekeling high lowdose group10 93.37±9.23**/*** 865.43±20.72**/*** 59.28 3.68

4. Discussion

The Human RSV infection is an important cause of airway epithelial cell destruction and inflammatory response. The pathogenesis is so far not yet clear and is one of the important causes of respiratory infection among children [9]. Clinical studies have shown that cytokines play an important role in the occurrence and development of diseases and can be used as important indicators for clinical diagnosis and treatment. RSV infection can stimulate the release of IL-8 and L-6, which is one of the important causes of lung inflammation and lung injury [11].

TheIL-6 is one of the important immune cytokines in the human body. It can be produced by T, B cells, monocyte macrophages and other immune cells. It participates in many biological activities in the body's immunity, and is closely related to inflammatory reactions and tumor immunity. Related [12]. When the body is infected with RSV, the cytokine IL-6 in the body will rise significantly. Liu Yi, Fan Ruyan, et al. [13] conducted research and analysis on the IL-6 content in the serum of children with RSV infection, and the results showed that the serum IL-6 in children with RSV infection was significantly higher than normal levels. Zhu Zhumei et al. [14] measured the levels of cytokines in the serum of children with respiratory syncytial virus infection, and the results showed that IL-6 was significantly increased in RSV infected patients. IL-6 is an important inflammatory marker, which can reflect the patient's condition to a certain extent and provide a reference for drug treatment. In this study, compared with the normal group, the IL-6 level in the serum of the RSV model group mice was significantly increased, and the IL-6 level was significantly decreased after treatment with Kekeling mixture. IL-8 is also one of the members of interleukins. It is mainly produced by monocytes and has a wide range of biological functions. It has certain links with inflammatory reactions and tumorigenesis. A small dose of IL-8 can resist infection and promote The body's immune function, but high doses of IL-8 can induce inflammation and cause tissue damage. Song Shufan, Xin Pingping [15] conducted research and analysis on the IL-8 content in the serum of patients with respiratory syncytial virus infection, and the results showed that the serum IL-8 level in children with RSV infection was significantly higher than normal levels, and the IL-8 level could be to a certain extent It reflects the degree of patient's body infection and has important guiding significance for the diagnosis and treatment of the disease. Zhou Hailan [16] analyzed the relationship between interleukin-8 and the occurrence and development of children with acute bronchitis induced by respiratory syncytial virus. 70 children with acute bronchitis treated in the hospital were studied. The expression of IL-8 in this group of patients was analyzed and studied. The results show that IL-8 is closely related to the development of the pathogenesis of RSV, and can be used as one of the important indicators for assessing the condition and indicating the prognosis.

In this study, the levels of IL-6 and IL-8 in alveolar lavage fluid and lung tissue of RSV-infected mice were measured, and the effect of Kekeling mixture on RSV infection was investigated. After treatment with Kekeling mixture, mice The levels of IL-6 and IL-8 in alveolar lavage fluid and lung tissues were significantly reduced, and the treatment effect of Kekeling high-dose group was better than that of medium-dose group than that of low-dose group, but Kekeling mixture was high, medium and low dose The effect of the groups on the levels of IL-6 and IL-8 was lower than that of ribavirin. In summary, IL-6 and IL-8 can be used as potential targets for the treatment of RSV infection. Kekeling mixture can effectively reduce the levels of IL-6 and IL-8 in the serum of RSV infected mice, thereby inhibiting the inflammatory response and improving clinical symptoms. TheKekelingMixture is one of the effective drugs for treating RSV, and its specific mechanism is to be further explored.