Analgesic and anti-inflammatory effects and molecular mechanisms of Kadsura heteroclita stems, an anti-arthritic Chinese Tujia ethnomedicinal herb

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Abstract

Ethnopharmacological relevance

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by failure of spontaneous resolution of inflammation. The stem of Kadsura heteroclite (KHS) is a well-known anti-arthritic Tujia ethnomedicinal plant, which named Xuetong in folk, has long been used for the prevention and treatment of rheumatic and arthritic diseases.

Aim of the study

The analgesic and anti-inflammatory effects and the potential mechanisms behind such effects of KHS would be investigated by using different animal models.

Materials and methods

The abdominal writhing episodes of mice induced by intraperitoneal injection of acetic acid and the tail-flick response induced by radiant heat stimulation were used to evaluate the analgesic effect of KHS. The number of abdominal writhing episodes of mice and the latency of tail-flick in rats were measured and recorded. In acute inflammatory models, the ear edema of mice was induced by applying xylene on the ear surface, while the paw edema of male and female rats was induced by subcutaneous injection of carrageenan into the right hind paws of animals. The carrageenan-induced paw swelling in rats were selected as an anti-acute inflammatory mechanism of KHS. Serum levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) were measured by ELISA, and protein expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were detected by Western blot.

Results

The maximal tolerated single dose of KHS was determined to be 26 g/kg in both sexes of mice. Pharmacological studies showed that KHS at the dose of 200 mg/kg significantly prolonged the reaction time of rats to radiant heat stimulation and suppressed abdominal writhing episodes of mice induced by intraperitoneal injection of acetic acid. KHS at the dose of 200, 400, and 800 mg/kg, showed dose-dependent inhibition of xylene-induced ear swelling in mice. KHS at the dose of 100, 200, 400, and 800 mg/kg demonstrated dose- and time-dependent suppression of paw edema induced by subcutaneous injection of carrageenan in both all rats. Mechanistic studies revealed that the anti-inflammatory effect of KHS was associated with inhibition of the production of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α and effectively decreased the expression of COX and iNOS proteins in the carrageenan-injected rat serum, paw tissues and inflammatory exudates. The positive reference drug, rotundine at a dosage of 100 mg/kg and indomethacin at a dosage of 10 mg/kg were used in both mice and rat models.

Conclusion

These results suggested that KHS has significant effects on analgesia and anti-inflammation with decreasing the pro-inflammation cytokines of IL-1β, IL-6, and TNF-α and inhibiting the proteins expression of COX-2 and iNOS.

Introduction

Rheumatoid arthritis (RA) is a kind of chronic, systemic autoimmune inflammatory disease characterized by synovial lesions, cartilage and bone erosion, eventually joint destruction and deformity (McInnes and Schett, 2011, 2017). Epidemiological studies show that the global incidence of RA is about 0.5–1.2%, and the prevalence of RA is about 0.28% in China. However, disability rate of RA is as high as 30–50% during recent 5 years, which is one of the main factors leading to disability (Li, 2015; Myasoedova et al., 2010). At present, the clinical treatment of RA by using disease modifying anti-rheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoid, to ameliorate the symptoms. Unfortunately, during prolonged use of DMARDs such as methotrexate and leflunomide to treat RA, the problem of obvious side effects such as, gastric mucosal, liver and kidney damage, are difficult to be tolerated. Moreover, new biological agents are not only expensive, but are damaging to health and can lead to cancers (Smolen and Aletaha, 2015). Chinese medicine has a long history for the treatment of autoimmune diseases, which has obvious advantages and the low toxic side effects. There are nearly 20000 Chinese medicines with historically proven efficacies in human recorded in Chinese Materia Medica from which many successful and considerable therapeutic drugs have been discovered. Modern pharmacological studies discovered that Chinese medicines having triterpenes, lignans and flavonoids possess activating blood circulation to dissipate blood stasis, anti-inflammatory and analgesic functions, such as Panax notoginseng (Burk.) F. H. Chen, Kadsura coccinea Lem A. C. Smien, Polygonum orientale Linn and paeonia lactiflora pall, etc (Liu et al., 2018). Pharmacology research the extracts and isolated compounds of these plants showed the analgesic and anti-inflammatory activities in folk, Tujia people used them to treat RA and release the various pain. Thus, discovering and isolation of compounds from Chinese traditional medicinal sources having anti-inflammatory effects are safer and effective and can be said as valuable “shorcut” in modern drug development. Thus, the treatment of RA with TCM has gradually become a trend of treatment (Lu et al., 2015).

Cyclooxygenases (COX), a necessary enzyme for prostaglandin synthesis, is also a rate limiting enzyme in PGs synthesis (Aoki and Narumiya, 2012). COX has two isoforms, namely COX-1 and COX-2. COX-1 is physiological enzyme constitutively expressed in virtually all tissues that are involved in the gastrointestinal, renal, and cardiovascular normal physiological activities. Unlike COX-1, COX-2 is usually expressed at very low levels in most tissues under physiological conditions, but it is up-regulated in inflammatory conditions (Ziegler et al., 2017). Some drugs for treating RA can exert anti-inflammatory effects by inhibiting the formation of prostaglandins synthesis by cyclooxygenase. NSAIDs work principally by inhibition of cyclooxygenase isoenzymes (COX-1 and COX-2), which mediate the formation of prostaglandins (PGs) and prostacyclins (PCs) from arachidonic acid (Moore et al., 2018). The combination of methotrexate and leflunomide shows inhibitory effects on the production of inflammatory mediators COX-1 and COX-2 from synovial macrophages co-cultured with T cell line. These observations support the positive results obtained in RA clinical studies by combination therapy (Abbasi and Mousavi, 2018). NSAIDs non-selectively inhibit COX-1 and COX-2 activities which results in adverse effects and induce of gastric ulcer and kidney failure. In addition, nitric oxide (NO) is also involved in inflammatory process. NO generation, mediated by excessive and prolonged inducible NOS (iNOS), acts as a cytotoxic agent in inflammatory disorders. It is well-known that pro-inflammation cytokines play a key role in inflammatory process. The classic pro-inflammatory cytokines include interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) that induce both acute and more chronic inflammation (Hussein et al., 2012; Khan et al., 2017).

Kadsura heteroclita (Roxb), alias “Xue tong”, “Xue qi” in Tujia ethnomedicine, belongs to family of Chinese schisandraceae. Kadsura heteroclite can expel wind-evil, remove wetness-evil, regulate Qi to alleviate pain, and activate blood circulation to dissipate blood stasis, in the terms of Chinese medical theories (Shen, Liu and Su, 2015). The stem of Kadsura heteroclite (KHS) has been long time used for the prevention and treatment of rheumatic and arthritic diseases, especially in the southern China (PH, 2007; Xiong, 2016). In this study, we research on KHS to exhibit the analgesic and anti-inflammatory effects on acetic acid induced writhing in mice, radiant heat induced tail flick in rats, xylene induced ear edema in mice, and carrageenan induced in rats, respectively.

Section snippets

Reagents and materials

The dosages of KHS employed in all experiments ranged from 50 to 800 mg/kg. The highest dosage 800 mg/kg is approximately 23 times of the equivalent dose of the human dosage in clinic and thus is an acceptable dosage for animal studies. All dilutions were obtained from the stock solution using a dilution vehicle that consisted of 0.3% CMC-Na. Indomethacin was purchased from Shanxi Taiyuan Pharmaceutical Co., Ltd (Shanxi, China, Lot#A120401). Carrageenan was purchased from Sigma Chemical Aldrich

Observation of symptoms and body weight change of mice with acute toxicity of maximum tolerated dose (MTD)

After 4 h of intragastric administration of KHS, no deaths had been observed in animals. The MTD of mice to KHS was 26  g/kg, equivalent to 540 times clinical dosage of people. Weighing the body weight of each group of mice every three days after administration, the weight of the mice in the drug group initially increased slowly, but after seventh day, the weight growth rate of drug group was faster than vehicle group (Fig. 1).

Influences of KHS on writhing test induced by acetic acid of mice

Fig. 3 showed the numbers of the abdominal writhing episodes evoked

Discussion

KHS is commonly used traditional medicine to treat arthritis in the minority areas of Tujia Nationality in the northwest of Hunan Province in China. It expels wind-evil, and remove wetness-evil, activate blood circulation to dissipate blood stasis, regulate Qi to alleviate pain, and was commonly used in the treatment of rheumatic pain, RA, bone pain, and lumbar muscle strain. After close observation of the use of traditional medicines, it was found that plants belong to schisandrace has

Conclusion

In conclusion, the anti-nociceptive and anti-inflammatory effects of KHS, an anti-arthritic Tujia ethnomedicinal herb, have been effectively evaluated using four experimental animal models in rodents. At the same time, the safety of the drug was evaluated with the MTD. The molecular mechanisms by which KHS its anti-inflammatory effects are believed to involve the inhibition of the production cascade of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α and suppression of expression of the

Conflicts of interest

We wish to confirm that there are no known conflicts of interest with this publication and that there is no significant financial support for this work that may affect its results.

Author contribution

Dr. Wei Wang, Xiong Cai, and Huang-he Yu designed experiments; Huang-he Yu, Xin Li, Ye Lin, and Ting Zhang carried out experiments; Prof. Rong Zeng and Yu-hong Wang analyzed experimental results. Yi-xing Qiu, Bin Li, Cong Chen, and Jin Liao analyzed sequencing data and developed analysis tools. Huang-he Yu wrote the manuscript. Dr. Shumaila Tasneem, Prof. Wei Wang, Xiong Cai providing language help, writing assistance or proof reading the article.

Funding

This work was funded by grants from the National Natural Science Foundation of China [Grant numbers 81874369, 81703819, 81704065, 81673579], Hunan Provincial Natural Science Foundation [Grant numbers 2017JJ3239, 2018JJ2293], Hunan Education Department’s Science & Research Project [Grant numbers 17K069, 17A157], Hunan Provincial Science & Research Project of Chinese Medicine [Grant numbers 201790, 201909], Hunan Provincial Innovation Project for Outstanding Postgraduate Students [Grant numbers

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