Effects of Injection of Oregano Oil Submicron Emulsion on Lipopolysaccharide-induced Pneumonia in Rats

Fei HAN, Guangqiang MA, Xinli LIANG, Xiao HUANG, Hanlin XU, Huaqiang WU*

1. Jiangxi University of Traditional Chinese Medicine: a. School of Pharmacy, b. School of Life Sciences, c. Key Laboratory of Modern Preparation of TCM, Ministry of Education, Nanchang 330004, China; 2. School of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430065, China

Abstract [Objectives] To explore the effects of injection of oregano oil submicron emulsion on lipopolysaccharide-induced pneumonia in rats. [Methods] Rats induced by lipopolysaccharide were used as animal models of acute pneumonia. The experiment was divided into blank group, model group, administration group and positive drug control group. The morphology of lung tissue and the changes of cells and inflammatory factors in each group were observed, and the anti-inflammatory effects of injection of oregano oil submicron emulsion. [Results] The injection of oregano oil submicron emulsion can improve the pathological injury of rat lung tissue, inhibit the release of IL-6, IL-10, TNF-α cytokines induced by lipopolysaccharide, and significantly reduce the value of CRP. [Conclusions] Oregano oil submicron emulsion has a certain therapeutic effect on lipopolysaccharide-induced pneumonia in rats, and its mechanism may be related to reducing the release of cytoinflammatory factor IL-6, IL-10, and TNF-α and alleviating the injury to tissues and organs.

Key words Oregano oil, TCM antibiotic, Pneumonia, Cytokine, Action mechanism

1 Introduction

Oregano is the dried whole plant ofOriganumvulgareL., a perennial herb of the genusOriganumin the family Lamiaceae. It is also called small leaf mint, wild marjoram, antidiarrheal herb, Baihua Yinchen, and Tuxiangru,etc. It is mainly distributed in the Mediterranean and China’s Hunan, Hubei, Jiangxi, Henan, Anhui, Shaanxi, and Gansu provinces. It was included in theChinesePharmacopoeia(1977 version). It has the functions of clearing heat and removing toxin, regulating qi and removing dampness, diminishing swelling, sterilizing and stopping diarrhea, it is a commonly used Chinese medicine among the folks[1]. Oregano oil is a volatile oil extracted from the plantO.vulgare. It is a light yellow oily liquid. In the world, there are many kinds of health care products and veterinary products using oregano oil as a raw material. At present, our scientific research team has developed it as a submicron emulsion for injection (Patent No.ZL 201410468844.9, Publication No.CN 104224906.A), and the declaration of related new drugs is also in progress.

The anti-bacterial, antioxidant, and antiseptic effects of oregano oil have been widely recognized worldwide, but there is still no report about the anti-inflammatory effects of preparations related to oregano oil[2-5]. In view of this, taking SD rats as experimental animals, using lipopolysaccharide (LPS) to induce pneumonia in rats, and taking interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α) and C-reactive protein (CRP) as evaluation indicators, we investigated the effects of injection of oregano oil submicron emulsion on pneumonia in rats, to provide some experimental references and basis for the development of oregano oil-related preparations.

2 Materials and methods

2.1Materials

2.1.1Instruments. High speed refrigerated centrifuge (Shanghai Yuejin Medical Instrument Co., Ltd., China); ELISA-ELM3000 automatic microplate reader (DRG International In., USA); HB-2 Olympus biological microscope (Hitachi Ltd., Japan); analytical balance (Sartorius Scientific Instruments (Beijing) Co. Ltd.); electric heating thermostat (BioTek Instruments, Inc., USA).

2.1.2Reagents. Lipopolysaccharide (Beijing Solarbio Science & Technology Co., Ltd., batch No.819E037); chloral hydrate (Shanghai Zhanyun Chemical Co., Ltd., batch No.1912017); phosphate buffer solution (Xilong Chemical Co., Ltd., batch No.2033024); formaldehyde solution (analytical reagent (AR), Xilong Chemical Co., Ltd., batch No.200105); xylene solution (AR, Xilong Chemical Co., Ltd., batch No.190108); absolute ethanol (AR, Xilong Chemical Co., Ltd., batch No.200225); iodine solution (Sinopharm Chemical Reagent Co., Ltd., batch No.191223); high-efficiency sectioning paraffin (Shanghai Huashen Rehabilitation Equipment Co., Ltd., batch No.190113); cefuroxime sodium injection (Nanchang Lijian Pharmaceutical Co., Ltd. , specification 3.0 g, production batch No.F19307003); rat C-reactive protein (CRP) kit, rat tumor necrosis factor alpha (TNF-α) kit, rat interleukin-6 (IL-6), and rat interleukin-10 (IL-10) (German IBL International GmbH, batch No.20200313); oregano oil submicron emulsion (self-prepared).

2.1.3Animals. Eighty healthy SD rats, male, weight (200 ± 20) g, reared in a barrier environment with temperature of 18-26 ℃ and relative humidity of 40%-70%, provided by Hunan SJA Laboratory Animal Co., Ltd. with license number of SCXK (Hunan) 2009-0004.

2.2Methods

2.2.1Pneumonia modeling. (i) Modeling of inhalation of different doses of lipopolysaccharide. As a chemical component in the cell wall of gram-negative bacteria, lipopolysaccharide is composed of core polysaccharide, O-polysaccharide side chain and lipid A. It is the main component of endotoxin and can stimulate the synthesis and release of different inflammatory mediators such as neutrophils and other inflammatory cells. It can cause damage to various tissues and cells, lead to inflammation, and particularly cause a higher incidence of lung tissue. Extensive studies have indicated that[6-8], the lipopolysaccharide-induced pneumonia model of rats is a mature animal model with a very high success rate.

Before modeling, healthy SD rats were fasted for 12 h but provided with water, and were randomly divided into six groups, each with 6 rats. The rats were anesthetized intraperitoneally with 10% chloral hydrate (0.5 g/kg). When the rat entered an anesthesia state and the limbs were stimulated without response, the rat head was tilted back vertically, the neck rat hair was removed, the skin was exposed, and the rat was placed in supine position on a self-made plate at 45°. Cut the neck skin using the surgical scissors to open the neck muscles and expose the upper trachea. Used a disposable syringe (specification 0.5 mL) to extract the lipopolysaccharide solution (sterilized water for injection was prepared into a 10 mg/mL solution), inserted into the trachea and injected into the lungs at one time. Each group of rats was injected with 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg lipopolysaccharide in sequence. The rats were kept in the supine position on the plate for 2-3 min to ensure that the lipopolysaccharide can smoothly enter their lungs. Then the wound was sutured and disinfected with iodine solution to prevent infection. The rats in anesthetized state were put back to the cage to wake up naturally, and provided with sufficient water and food. They were sacrificed three days later, and the alveolar tissue was collected for experiment and observed for inflammation.

(ii) Building of pneumonia model. After the above experiment determined the appropriate dose of lipopolysaccharide, the remaining rats were randomly divided into 6 groups, and lipopolysaccharide was injected in accordance with the above method. No treatment was carried out for the normal group. Three days later, administered drugs and observed the physiological changes of the rats.

2.2.2Administration method. From the fourth day of modeling, intraperitoneal injection was administered. With reference to theLD50value of oregano oil[6], took the oregano oil submicron emulsion 0.2 g/kg as the low dose group, 0.4 g/kg as the medium dose group, 0.6 g/kg as the high dose group, and cefuroxime sodium as the positive drug group. Combined with the actual clinical administration situation[7], according to the 0.4 g/kg group, the blank group and the model group were given equal amount of normal saline, intraperitoneally injected once a day for 10 consecutive days. During this period, the physiological changes of the rats were observed.

2.2.3Bronchoalveolar lavage fluid (BALF) sample retention and cell count. The rats were killed by cervical dislocation, the thoracic cavity was opened, the left hilum was clamped, and 1 mL of PBS buffer was injected into the tracheal intubation. The recovery rate was about 85%, 4 000 rpm, centrifuged for 10 min, and took the supernatant and stored at -80 ℃. The BALF residue was resuspended in 1 mL of PBS solution, and the sample was dropped on a glass slide as a smear. After the BALF residue smear was dried naturally, HE stained, and the cells were classified and counted under a high-power microscope.

2.2.4Tissue specimen processing and morphological observation. After the rats were killed, the thoracic cavity was cut along the abdomen to the neck. Took out the lung tissue, washed the blood stains on the surface with normal saline, and absorbed to dry with filter paper. Took the upper part of the left lung large lobe into the microporous embedded box and immersed it in 10% formaldehyde solution for 48 h; took it out and washed with pure water for 10 min, soaked in 75% and 85% ethanol for 1.5 h, and soaked in 95% and absolute ethanol for 2.0 h; soaked twice in xylene solution, and the time was 15 and 10 min, respectively; finally, placed into liquid paraffin at 65 ℃ to impregnate and do it three times, and each time of impregnation was 1.0 h; after completion of the impregnation, used the embedded box to make a paraffin block, which was sectioned with a rotary microtome, and the section thickness was 3.5 μm. After the sections were deparaffinized and fixed with various concentration gradients of ethanol and xylene, stained with eosin and hematoxylin, and then treated with different concentrations of ethanol and xylene solutions, the sections were kept dry and mounted with balsam mounting medium. The pathological changes of lung tissues were observed under a microscope.

2.2.5Detection of IL-6, IL-10, TNF-α, and CRP levels by enzyme-linked immunosorbent assay (ELISA). After the rats were fixed, pressed the rat head to make the eyeballs protrude, used ophthalmic tweezers to clamp the rat eyeballs and then remove the eyeballs. Collected the rat blood in a 10 mL centrifuge tube, did a mark, and stored at 4 ℃. After the blood of each group was collected, centrifuged at 4 000 rpm for 5 min. After centrifugation, took the upper serum and stored at -80 ℃. Used ELISA kit to detect the content of IL-6, IL-10, TNF-α and CRP in accordance with the instructions.

3 Results and analysis

3.1InvestigationandhistopathologicalsectionofdifferentdosesoflipopolysaccharideDuring the screening process of establishing the rat pneumonia model with lipopolysaccharide, two rats died in the 1.0 mg lipopolysaccharide injection group and one rat died in the 0.8 mg injection group. The remaining rat models did not die within 3 d. After intratracheal injection of lipopolysaccharide solution, rats in each group experienced shortness of breath, did not like to move, and had the drowsiness,etc. Two days later, the rat hair lost luster, the color became yellow and the hair standing appeared, such situations were severe in 0.6, 0.8, and 1.0 mg injection groups. Rats showed anorexia, drink increased, and occasionally trembling, coughing and other symptoms, body weight loss, slow movement, and rats in 0.8 and 1.0 mg injection group showed symptoms of curled body and drowsiness, and these symptoms were more serious. Through observing the pathological section, it was found that the alveolar walls of the 0.1, 0.2, and 0.4 mg injection groups were relatively intact without obvious thickening, and a small number of neutrophils and lymphocytes appeared in the alveolar interstitium. In the 0.8 and 1.0 mg injection groups, the alveolar wall was severely damaged, a large number of plasma cells and neutrophils appeared in the alveolar cavity and interstitium, and the congestion was severe. In the 0.6 mg injection group, the alveolar wall of was slightly damaged, the alveolar wall thickened moderately to severely, and the alveolar interstitium was infiltrated by a large number of lymphocytes and neutrophils, and local congestion appeared, and the symptoms were slightly milder than the 0.8 and 1.0 mg injection groups. Through comprehensive consideration, we took 0.6 mg lipopolysaccharide to induce inflammation in rats as the experimental model standard, as shown in Fig.1.

Note: The above pictures are the modeling of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg lipopolysaccharide.Fig.1 HE stained pathological sections of different concentrations of lipopolysaccharide (×200)

3.2StatusandhistopathologicalsectionsineachgroupofexperimentalanimalsDuring the administration period, the rats in the normal group had normal physiological activities, active movement, shiny hair, strong limbs, and normal breathing, and there were no symptoms such as shortness of breath or cough, and the diet and mental states were normal. Compared with the normal group, the rats in model group showed severe dull hair, drowsiness, weak limbs, not like to move, reduced food intake, increased water intake, weight loss, and occasional chill shivering. Compared with the model group, the rats in low dose oregano oil group showed improved hair gloss, increased food and water intake, and increased the number of activities, which were much different from the normal group. Compared with the model group, the rats in medium and high dose oregano oil groups and the positive drug group showed recovered hair luster, strong limbs, normal food intake, gradually normal body, and improved drowsiness. In the normal group, the alveolar wall of the pathological section was intact, the thickness of the alveolar wall was normal, and there were no large numbers of inflammatory cells in the lung interstitium. In the model group, the alveolar walls were damaged, a few alveolar cavities were congested, the alveolar walls were moderately thickened, and a large number of inflammatory cells infiltrated in the lung interstitium. In the low dose oregano oil emulsion group, alveolar walls were moderately thickened, and a large number of neutrophils and plasma cells infiltrated in the lung interstitium. In the oregano oil medium and high dose groups, the alveolar wall of the positive drug group was relatively intact, the alveolar wall was slightly thickened, and a small number of neutrophils infiltrated in the lung interstitium (Fig.2).

3.3ClassificationandcountingofBALFsmearsIn the BALF of the blank group, mononuclear macrophages dominated. Compared with the blank group, the number of neutrophils in BALF of the model group was significantly higher, and the difference was statistically significant (P<0.01); compared with the model group, the number of neutrophils in the BALF of the oregano oil group and the positive drug group was significantly reduced, and the number of mononuclear macrophages was significantly increased, and the difference was statistically significant (P<0.01). The results are shown in Fig.3 and Table 1.

3.4Effectsoforeganooilsubmicronemulsiononthedetectionindicatorsofpneumonia

3.4.1Effects on the levels of IL-6 and IL-10 in serum. Compared with the blank control group, the levels of IL-6 and IL-10 in the model group were significantly increased, while the levels of IL-6 and IL-10 in the oregano oil submicron emulsion group and the positive drug group decreased (Fig.3).

Note: Normal: normal group; Model: model group; L: low dose group; M: medium dose group; H: high dose group; P: positive drug group. Compared with the normal group, a: P<0.05, b: P<0.001; compared with the model group, A: P<0.05, B: P<0.001.Fig.3 Comparison of the levels of IL-6 and IL-10 in each group(n=6)

3.4.2Effects of oregano oil submicron emulsion on the levels of CRP and TNF-α in serum. Compared with the normal group, the levels of TNF-α and CRP in the model group were significantly increased, and the levels of TNF-α and CRP in the oregano oil submicron emulsion group and the positive drug group were significantly decreased (Fig.4).

4 Conclusions and discussion

4.1LipopolysaccharidemodelingThrough observation of multiple pathological sections, lipopolysaccharide induced interstitial pneumonia in SD rats. However, since the dosage of lipopolysaccharide cannot be determined, the initial stage of this experiment is mainly to screen the appropriate dosage of lipopolysaccharide. According to the fatality rate of intratracheal injection of lipopolysaccharide and the physiological activity characteristics of rats, combined with the pathological sections of lung tissue, 0.6 mg lipopolysaccharide can effectively induce pneumonia in rats, the mortality is low, lung inflammation is obvious, and external characteristics are clear, the model is stable.

Note: Normal: normal group; Model: model group; L: low dose group; M: medium dose group; H: high dose group; P: positive drug group. Compared with the normal group, a: P<0.05, b: P<0.001; compared with the model group, A: P<0.05, B: P<0.001, n=6.Fig.4 Comparison of CRP and TNF-α levels in each group

4.2ObservationofvitalsignsofeachgroupandselectionofpositivedrugsafteradministrationIn the early stage of intratracheal injection of lipopolysaccharide, the rats showed shortness of breath, hair standing and losing luster, curling body, weak limbs, and reduced food intake. After intraperitoneal injection of oregano oil submicron emulsion, the curling of rats was improved. Five days after drug administration, the hair of rats in the medium and high dose oregano oil submicron emulsion groups gradually recovered their luster and the number of activities increased. Eight days after administration, the external characteristics of the rats returned to normal.

In this experiment, we selected the clinically common chemical antibiotic, cefuroxime sodium, as the positive drug. After its administration, the symptoms of rats in the positive drug group were improved significantly, the time for returning to normal signs was shorter, and the number of activities was increased. Combined with the results of pathological tissue sections and various drug efficacy indicators, we found that the alveoli of the normal group were intact and had no damage or thickening. Compared with the normal group, the alveolar cavity of the model group was severely damaged and a large number of inflammatory cells infiltrated in the lung interstitium. In the medium and high dose groups of oregano oil submicron emulsion and the cefuroxime sodium administration groups, the lung interstitium was infiltrated with a small number of inflammatory cells, the alveolar wall was moderately thickened, and the lung tissue inflammation was significantly reduced compared with the model group, indicating that western medicine antibiotics are more effective in treating pneumonia than oregano oil submicron emulsion. These are consistent with the actual situation.

4.3ResultsofindicatorsIL-6, IL-10, TNF-α and CRP are all routine examination indicators of pneumonia and can directly reflect the inflammatory characteristics of pneumonia. In this experiment, the contents of four indicators in the model group were higher than those in the normal group, indicating that the symptoms of the rat pneumonia model were severer. The levels of IL-6, IL-10, TNF-α and CRP in the oregano oil submicron emulsion low dose group were lower than those of the model group, and the pneumonia symptoms were slightly improved, but not obvious compared with the model group. The levels of IL-6, IL-10, TNF-α and CRP in the medium and high dose groups of oregano oil submicron emulsion were significantly lower than those of the model group, indicating that oregano oil submicron emulsion can effectively reduce the secretion of various cytokines and thus effectively improve the pneumonia symptoms.

Experimental results suggest that the mechanism of oregano oil affecting the inflammatory response may be related to inhibiting the activation of neutrophils and promoting the differentiation of monocytes and macrophages, controlling the production of inflammatory mediators, and then playing the role of anti-inflammation and immune regulation[9-10]. However, the specific mechanism is still unclear and needs further investigation and research. The levels of IL-6, IL-10, TNF-α, and CRP in the cefuroxime sodium group were significantly lower than those in the model group, and the improvement of pneumonia symptoms was the most obvious, indicating that chemical antibiotics have a significant effect in the treatment of pneumonia, but considering that the long-term application of traditional antibiotics may produce resistance, oregano oil-related preparations have certain advantages and characteristics[11]. In summary, oregano oil submicron emulsion has a certain therapeutic effect on lipopolysaccharide-induced pneumonia, and has a strong anti-inflammatory effect. Thus, it can be developed as a substitute for traditional chemical medicine antibiotics, the market potential is large and it is worthy of further research and development and promotion.