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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 4 | Page : 399-403
Anti-inflammatory effect of Dendrobium huoshanense polysaccharides on carrageenan-induced air pouch synovitis in mice
Hai-Jun Xu1, Yang Yang2, Ya-Qi Dong2, Kai-Min Niu3, Tian-Yu Li2, Hui Deng2, Jing-Wen Hao2, Wassie Teketay4
1 Teaching and Research Section of Animal Medicine, College of Biological and Pharmaceutical Engineering, West Anhui University; Engineering Technology Research Center of Plant Cell Engineering; Anhui Engineering Laboratory for Conservation and Sustainable Utilization of Traditional Chinese Medicine Resources, Anhui Province, Luan, China
2 Teaching and Research Section of Animal Medicine, College of Biological and Pharmaceutical Engineering, West Anhui University, Luan, China
3 Institute of Biological Resources, Jiangxi Academy of Sciences, Jiangxi Province, Nanchang, China
4 Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Hunan Province, Changsha, China; Department of Animal Science, Assosa University, Assosa, Ethiopia
Correspondence Address:
Associate Prof. Hai-Jun Xu
College of Biological and Pharmaceutical Engineering, West Anhui University, Anhui Province, Luan 237012
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.372728
Objective: The purpose was to observe the anti-inflammatory effects of Dendrobium huoshanense polysaccharides (DHPs) on carrageenan-induced sterile air pouch synovitis. Materials and Methods: A total of 30 Institute of Cancer Research (ICR) mice were randomly and equally assigned to the control, carrageenan-induced air pouch synovitis model (model), and carrageenan-induced air pouch synovitis model + DHP (200 mg/kg of body weight) (model + DHP) groups. Mice in the model + DHP group were intragastrically administered 200 mg/kg BW of DHP solution daily for 10 days. Mice in the control and model groups were intragastrically administered the same amount of distilled water. Two hours after intragastric administration on day 10, 1 mL of a 1% carrageenan solution in a sterile 0.9% saline solution was injected into the air pouch of mice in the model and model + DHP groups. Six hours later, the mice were sacrificed and 4 mL of ice-cold sterile 0.9% saline solution was injected into the air pouch to fully wash its inner wall. The lavage fluid was collected to observe the color and turbidity of the lavage fluid, as well as the appearance of the backside of the air pouch. The exudate volume, total number of leukocytes, protein content, levels of malondialdehyde (MDA), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α), and total superoxide dismutase (T-SOD) activity of the lavage fluid were analyzed. Results: The results showed that pretreatment with DHPs reduced the carrageenan-induced exudate volume (P < 0.01), total leukocytes (P < 0.05), and protein content (P < 0.01) in the air pouch lavage fluid. Furthermore, mice in the model + DHP group had significantly higher (P < 0.01) T-SOD activity and lower MDA content (P < 0.05), IL-1β (P < 0.05), and TNF-α (P < 0.01) levels in the air pouch lavage fluid compared with the model group. Conclusion: It is concluded that DHPs partially alleviated carrageenan-induced sterile inflammation, and its mechanism may be related to reducing exudation and scavenging oxygen-free radicals, inhibiting lipid peroxidation, and reducing the level of the proinflammatory factors, such as IL-1β and TNF-α.
Keywords: Anti-inflammation, carrageenan, Dendrobium huoshanense, polysaccharides, sterile inflammation
Dendrobium huoshanense is a precious Chinese herbal medicine in Huoshan County, Anhui Province, China. It is also an important raw material for many medicinal meals and health products. According to the Compendium of Materia Medica which is a pharmaceutical compiled by Li Shizhen, a Chinese medical scientist in the Ming Dynasty, D. huoshanense has the effects of strengthening Yin, as well as benefiting essence, gastrointestinal function, muscle growth, and intelligence.[1] It has been well documented that polysaccharides which have the functions of immune regulation, anti-inflammatory, antioxidation, liver protection, and regulating intestinal flora among others are the main active ingredients of D. huoshanense.[2],[3] The available literature on the immunomodulatory function of D. huoshanense polysaccharides (DHPs) has mainly focused on its enhancement of immune function.[4],[5],[6] However, DHPs can also downregulate hyperimmunity such as hyperactivity induced by Bacillus Calmette–Guerin (BCG) vaccine,[7] suggesting its anti-inflammatory activity. Because lipopolysaccharide (LPS), the main component of the BCG vaccine, mainly comes from the bacterial wall of Gram-negative bacteria, it is related to infectious inflammation. However, the effect of DHPs on sterile inflammation has not been extensively studied.[2]
The synovial membrane is an anatomical structure in the body that covers most parts of the joint cavities. Mechanical disruption of the subcutaneous connective tissue by injecting air repeatedly can form a cavity with a synovial membrane-like lining structure.[8] Previously, it was suggested that carrageenan could induce sterile acute inflammation in the synovial membrane.[9],[10] Therefore, this study evaluated the anti-inflammatory effect of DHPs on sterile carrageenan-induced air pouch synovitis to provide an experimental basis for using DHPs for the auxiliary treatment and healthcare of patients suffering from sterile inflammation such as those with gout, pyrophosphate arthropathy, asbestosis, atherosclerosis, and aging.
Materials and MethodsAnimals
Specific pathogenic-free grade ICR mice with an average body weight of 18–22 g were obtained from the Laboratory Animal Center of Anhui Medical University (Hefei, China). All experiments were conducted in accordance with the National Guidelines for the Care and Use of Experimental Animals and approved by the Bioethics Committee of West Anhui University (the approval number was 202101001), China. The mice were kept in an air-conditioned room at 23°C under natural light and free intake of feed and water.
Preparation of Dendrobium huoshanense polysaccharides
The stems of D. huoshanense provided by the Anhui Dendrobium Huoshan Industrial Development Collaborative Innovation Center were identified as authentic by Prof. Chen Naifu, West Anhui University. DHPs were prepared as a method previously described.[11] Briefly, D. huoshanense stems were dried and crushed to powder; then, the crude polysaccharide was prepared by water precipitation method from the D. huoshanense powder. The crude polysaccharide was refined after being deproteinized by the Sevag method and freeze-dried. The polysaccharide contents of D. huoshanense stem and refined DHPs determined by anthrone sulfuric acid colorimetry were 29.4% and 90.6%, respectively. The protein content of refined DHPs measured by Coomassie brilliant blue method was 1.1%.
Reagents and kits
λ-Carrageenan (C24H36O39S6) with a molecular weight of 1140.9 was purchased from Sigma-Aldrich, USA. Total superoxide dismutase activity (T-SOD activity) test kit, malondialdehyde (MDA) test kit, and bicinchoninic acid (BCA) protein quantification test kit were obtained from Nanjing Jiancheng Bioengineering Co., Ltd., Nanjing, China. Mouse interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α) ELISA Kits were obtained from Wuxi Donglin Sci and Tech Development Co., Ltd., Wuxi, China. All other reagents used were of analytical grade.
Experimental design
Thirty ICR mice were randomly assigned into the control, carrageenan-induced air pouch synovitis model (model), and carrageenan-induced air pouch synovitis model + DHP (model + DHP) groups, with 10 mice per group (five males and five females). The carrageenan-induced air pouch synovitis model was prepared according to the previously described methods.[8],[12],[13] Briefly, 5 mL sterile air was injected into the subcutaneous tissue of the back of the midline of all mice on the 1st day of the experiment, and an injection of 3 mL air was repeated on days 3, 6, and 9 to keep the air pouch inflated. On day 9, there was no obvious inflammatory reaction at the injection site, such as redness, purulence, induration, or exudate, indicating that the air pouch was successful.
Starting on day 1, DHP solution at a dose of 200 mg/kg/BW was intragastrically administered daily for mice in the model + DHP group. Mice in the control and model groups were intragastrically administered the same amount of distilled water. Two hours after intragastric administration on day 10, 1 mL of 1% carrageenan solution in sterile 0.9% saline solution was injected into the air pouch of mice in the model and model + DHP groups, and the same volume of sterile saline was injected into the control group. Six hours later, the mice were sacrificed by cervical dislocation. Then, 4 mL of ice-cold sterile 0.9% saline solution was injected into the air pouch to fully wash its inner wall. The lavage fluid was collected into a heparin lithium anticoagulation tube. The color and turbidity of the lavage fluid, as well as the appearance of the backside of the air pouch, were observed. The lavage fluid volume was accurately measured to calculate the exudate volume, which was calculated as the lavage fluid volume – 4 mL. Next, 1 mL of the lavage fluid was taken to determine the total number of leukocytes. The rest of the lavage fluid was centrifuged at 4°C at 1006 × g for 15 min. The supernatant was collected and stored at −80°C to determine the protein content, levels of IL-1β, TNF-α, and MDA, and T-SOD activity.
Determination of the number of total leukocytes in the lavage fluid
To reduce the turbidity of the lavage fluid, it was diluted 5 times with normal saline and then used to determine the total number of leukocytes using HC Haimai blood test system (Meiyilin Electronic Instrument Co., Ltd., Jinan, Shandong Province, China). The results were expressed as the number of cells (×109) per liter.
Determination of the protein content, malondialdehyde content, and total superoxide dismutase activity in the lavage fluid
A BCA kit (biuret reaction method) was used to quantify the protein content in the lavage fluid under the guidance of the manual. The MDA level was determined using a test kit (double antibody one-step sandwich ELISA assay, with intra- and inter-assay Coefficient of Variation. (CV) <15%), and T-SOD activity was examined using a test kit (hydroxylamine method) as instructions described.
Determination of the content of interleukin-1β and tumor necrosis factor-alpha in the lavage fluid
The contents of IL-1β and TNF-α in the lavage fluid were determined using mouse IL-1β and TNF-α ELISA kits. The intra and inter assay CV were under 10% and 12%, respectively, both for IL 1β and TNF α. The concentrations of the cytokines were expressed as pg/mL and ng/mL of the lavage fluid for IL-1β and TNF-α, respectively.
Statistical analysis
All data were expressed as mean ± standard deviation. Data processing system software (Hangzhou Ruifeng Information Technology Co., Ltd., China) was used for one-way analysis of variance, and the Duncan test was used for multiple comparisons.
Results and DiscussionAppearance of carrageenan-induced air pouch synovitis
Air pouch lavage fluid in the control group was colorless and transparent, but it became yellow and turbid with many suspended floccules in the model group, indicating that it was an inflammatory exudate [Table 1]. The air pouch lavage fluid in the model + DHP group was yellowish and mildly turbid with few suspended floccules, indicating that it was a mild inflammatory exudate. Congestion in the inner back of the air pouch was not observed in the control group but was obvious in the model group [Figure 1]. In the model + DHP group, the inner back of the air pouch was slightly congested [Figure 1]. These results indicate that carrageenan could induce air pouch synovitis successfully and pretreatment of DHPs relieved this inflammation.
Figure 1: Appearance of the inner side of air pouch 6 h after the injection of carrageenanInflammation is the body's defensive response to various inflammatory factors and the damage caused by them. The basic pathological changes of inflammation were degeneration, exudation, and proliferation of local tissues and cells in the inflammatory focus. At the same time, it is often accompanied by systemic reactions such as fever and leukocytosis. On the one hand, inflammation can eliminate various inflammatory factors and promote damage repair;[14],[15] on the other hand, it can also cause blood circulation disorders, release of inflammatory mediators, tissue degeneration, and necrosis in the process of anti-injury. Therefore, the inflammatory process must be analyzed and treated dialectically. For example, persistent chronic inflammation causes serious harm to tissues and organs.[16] Many pathological processes, such as obesity, atherosclerosis, and aging, are accompanied by chronic sterile inflammation that cannot self-heal.[16] Therefore, it is necessary to develop drugs or healthcare products with few side effects to control sterile chronic inflammation that cannot self-heal.
Carrageenan-induced air pouch synovitis is a subacute sterile inflammation.[8],[17] IL-1β, a proinflammatory cytokine mainly produced by macrophages, has important biological functions in sterile inflammation. For example, it can upregulate the expression of endothelial cell adhesion molecules, which plays important roles in attracting phagocytes, and induce the secretion of other proinflammatory mediators.[18],[19] TNF-α is also an important mediator of inflammation, and their concentrations reflect the severity of inflammation. In this study, DHPs reduced the levels of these inflammatory mediators, the protein content, the total number of leukocytes in the air pouch lavage fluid, and the exudate volume of model mice [Figure 2], indicating that DHPs effectively alleviated carrageenan-induced air pouch synovitis.
Figure 2: Effects of DHPs on MDA content, T-SOD activity, and inflammation-related indexes including exudate volume of the air pouch, number of total leukocytes, and the contents of protein, IL-1β, and TNF-α in the air pouch lavage fluid. Each column represents the mean ± SD obtained from 10 mice. One-way ANOVA followed by Duncan's test: *P < 0.05, **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus model group. DHP: Dendrobium huoshanense polysaccharide, MDA: Malondialdehyde, IL-1β: Interleukin-1β, TNF-α: Tumor necrosis factor-alpha, T-SOD: Total superoxide dismutase, SD: Standard deviation, ANOVA: Analysis of varianceAlthough the proinflammatory action of carrageenan is well known,[20],[21] the underlying mechanism has not been fully clarified. It was found that 1% carrageenan solution had high viscosity, especially that it could be jelly like after cooling. After being injected into the mouse air pouch, carrageenan will undoubtedly adhere to the tissue cells on the inner wall of the air pouch. It is not sure whether this adhesion will interfere with the activity of cell membrane, or even cause cell death, and then cause inflammatory reaction. In other words, whether high-viscosity carrageenan solution can cause aseptic inflammation directly is awaited further investigation.
In the process of sterile inflammation, at least three nonexclusive pathways are involved in signaling pathways.[22],[23] Therefore, different sterile stimuli signal through different signal receptors. For example, the signal receptors of hyaluronic acid are Toll-like receptor (TLR) 2, TLR4, and CD44, and that of heparin sulfate is TLR4. The chemical composition of carrageenan is similar to that of hyaluronic acid or heparin sulfate and thus might also combine with TLR4 and/or TLR2 to cause sterile inflammation. Interestingly, hyaluronan was reported to relieve carrageenan-induced synovial inflammation in rats[24] although the mechanism involved has not been clarified. TLR4 is also an important receptor for LPS. DHP directly binds to TLR4 to activate the TLR4 signaling pathway in macrophages.[3] According to a study using biomembrane interference technology, LPS rapidly bound to TLR4 and reached saturation in 10 s in a concentration-dependent manner.[25] The yields of TNF-α, IL-6, and IL-10 in RAW264.7 macrophages stimulated by 5 μg/mL LPS were equivalent to cells stimulated by 50–200 μg/mL DHP-4a.[26] Therefore, LPS has a stronger ability to activate TLR4 than DHP. However, when DHP first occupies the binding site of TLR4, DHP may interfere with the binding between LPS and TLR4, thereby decreasing the strong inflammatory response induced by LPS.[27] This might also be the same for carrageenan. In this study, the dose of carrageenan injected into the air pouch was 1 mL at the 1% concentration, while the oral dose of DHPs was only 200 mg/kg•BW. On the face of it, since the local concentration of carrageenan in the air pouch was much higher than that of DHPs, it seemed difficult for DHPs to compete with carrageenan to bind TLR4 directly in the air pouch. However, since DHPs have been administrated for 10 days in advance, it may have occupied the binding receptor of TLR4 such that carrageenan cannot bind to TLR4 to induce strong inflammation. Furthermore, DHPs might downregulate the inflammatory effect of carrageenan by regulating its downstream inflammatory signaling pathway. For example, in a carbon tetrachloride-induced sterile hepatitis model, DHP-a1 downregulated the phosphorylation of nuclear factor kappa B (NF-κB) inhibitor , which inhibited activation of the NF-κB pathway.[2] Taken together, DHP might relieve carrageenan-induced inflammation by influencing the binding of TLR4 with carrageenan, as well as interfering with its downstream inflammatory signaling pathway. However, further direct proof is needed to verify this argument.
In addition, like polysaccharides derived from marine algae,[28]in vitro and in vivo experiments confirmed that DHP has a good antioxidant capacity.[11],[29] In this study, the MDA content was significantly increased (P < 0.01), and T-SOD activity was decreased (P < 0.01) in the air pouch lavage fluid of the model group compared with the control and model + DHPs groups [Figure 2], indicating that there was obvious oxidative stress, which might have been induced by inflammation. During inflammation, neutrophils produce reactive oxygen through an oxidative burst to destroy pathogens. However, under excessive cellular stress, high levels of reactive oxygen species lead to oxidative stress, resulting in cell death and necrosis, which in turn can induce sterile inflammation.[16] Therefore, this suggests that the inhibitory effect of DHPs on carrageenan-induced air pouch synovitis may be in part related to its excellent antioxidant effects.
ConclusionDHPs inhibited carrageenan-induced sterile air pouch synovitis, and its mechanism may be related to a reduction of oxidative stress and inhibition of the synthesis of inflammatory factors. However, how DHPs interfere with carrageenan-induced inflammatory signal transduction remains to be clarified.
Financial support and sponsorship
This work was supported by the Key Research and Development Projects of Anhui Province in 2022 under grant 202204c06020034, West Anhui University Outstanding Top Talent Training Project under grant gxgnfx 2019026, Anhui Undergraduate Quality Engineering Project under grant 2020szsfkc0908 and 2021jyxm1663, West Anhui University 2020 College Students' Innovation and Entrepreneurship Training Program (national level) under grant 202010376053, and the Key Collaborative Research Program of the Alliance of International Science Organizations (Grant No. ANSO-CR-KP-2021-10).
Conflicts of interest
There are no conflicts of interest.
References
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