Rheumatoid arthritis is an autoimmune disease characterized by polyarthritis, progressive joint damage, and swelling deformities (Pu et al., 2016; Sparks, 2019; Shi et al., 2020). The pathological features of RA are joint synovial lesions, including excessive synovial cell proliferation, inflammatory cell infiltration, pannus formation, joint bone and cartilage destruction (Aletaha et al., 2011; Xing et al., 2018). According to relevant investigations and studies, the incidence of this disease worldwide is as high as 1%–2%. There are more than 5 million RA patients in China, most of whom are in the 30–50 years old age group. It is the second largest cause of disability in the Chinese population (van der Woude and van der Helm-van Mil, 2018). It can quickly develop into multi-system inflammation and irreversible joint damage, leading to a decline in quality of life, disability and death without proper treatment (Malmstrom et al., 2017). Because the etiology and pathogenesis of RA are not clearly understood (Mcinnes and Schett, 2011). The targets and methods of clinical treatment are not clear, which brings certain difficulties to treatment. There are no highly targeted therapeutic drugs in clinical practice, the treatment of RA still from the aspects of analgesia, anti-inflammatory, preventing or reduce joint deformation and damage and increasing joint mobility (Yuan et al., 2015; Shams et al., 2021). These drugs reduce joint inflammation, inhibit the development of lesions and irreversible bone destruction, but long-term use of these drugs lead to immune system decline, bone marrow suppression, liver and kidney function damage, gastrointestinal function decline, cartilage degeneration, infection (Abbasi et al., 2019; Koller et al., 2019). Therefore, it is clinically necessary to find effective drugs with low toxicity to treat RA. In recent years, ethnomedicine and some natural medicine products have attracted more and more researchers’ attention because of their unique safe and effective pharmacological activities (Jang et al., 2014). Ethnomedicine can be a new resource for the treatment of rheumatoid arthritis with more and more evidences.

P. aculeata Mill is a plant of the genus Pereskia in the Cactaceae family, also known as leaf cactus and tiger thorn. It is mainly produced in Yunnan, Guangdong, Guangxi and other regions south of the Yangtze River. It has a long history of folk medicine, with low side effects, and anti-inflammatory, antioxidant, antibacterial and other pharmacological activities (Tan et al., 2005; Kazama et al., 2012; Pinto and Scio, 2014; Pinto et al., 2015). Its medicinal liquor can treat bruises and rib pain. According to reports in the literature, there are as many as 30 chemical components in the essential oil of P. aculeata Mill leaves, among which the higher content includes phytol, palmitic acid, linoleic acid (Souza et al., 2014). In addition, the plant essential oil has the highest content of oxidized sesquiterpenes (44.92%), acorus spirenone comes next. The current research on the pharmacological effects of P. aculeata Mill mainly focuses on analgesia, cardiovascular protection and other related aspects. There are few studies on its anti-RA effect (Peng et al., 2018). The previous research show that P. aculeata Mill ethanol extract inhibited acetic acid-induced increase in vascular permeability and writhing behavior of mice in acetic acid-induced writhing reaction experiment, reduce the swelling of the feet of CFA rats and the level of IL-6 and other inflammatory cytokines in the rat serum, inhibit LPS-induced inflammatory response in RAW264.7 macrophages (Kanno et al., 2006).

This study aimed to screen effective sites by establishing an LPS-induced RAW264.7 macrophage inflammation model, and through the FCA rat model to evaluate the therapeutic effect and possible mechanism of PEEP, provide a more adequate theoretical basis for the development and utilization of PEEP.

The etiology and pathogenesis of RA have not been clearly elucidated. The number of macrophages is one of the main factors in RA because the severity of the disease and the degree of joint damage are significantly related to the number of macrophages (Kinne et al., 2007). When macrophages are stimulated by LPS, they will activate MAPK and other related signal pathways, and then secrete a large number of inflammatory factors, such as TNF-α, IL-6, IL-1β, and PGE₂ (Xu et al., 2017; Yonezawa et al., 2018). RAW264.7 mouse macrophages are a type of macrophages. Because of their strong adhesion and ability to swallow antigens, they are generally used to build inflammation models and conduct research on inflammatory responses (Adams and Hamilton, 1984; Yang et al., 2014). LPS was used for stimulation to establish a macrophage inflammation model to study the effect of PEEP on inflammation and its molecular mechanism. According to the literature, the preliminary experimental results of the research group, 1 μg/ml LPS was used to stimulate RAW264.7 macrophages to construct as vitro inflammation model. The CCK-8 method was used to detect the effects of different concentrations of P. aculeata Mill on the proliferation of RAW264.7 macrophages. The results showed that, the concentrations of PEEP (0, 15, 30, and 60 μg/ml), EEEP (0, 5, 15, and 30 μg/ml), and BEEP (0, 10, 20, and 40 μg/ml) no significant effect on the survival rate of RAW264.7 macrophages when compared with the blank group. Therefore, PEEP (60 μg/ml), EEEP (30 μg/ml), and BEEP (40 μg/ml) are selected as the highest concentrations for subsequent experiments.

In the early stage of inflammation, inflammatory cytokines such as TNF-α and IL-6 released by activated macrophages play an extremely important role in initiating local inflammation (Ueda et al., 2015). TNF-α is a potent pro-inflammatory cytokine released by macrophages, and its secretion reflect the severity of inflammation in the body (Caldwell et al., 1999; Su et al., 2015), and the excessive release of TNF-α can induce the production of other cytokines (such as IL-6), to further amplify the damage and destruction of TNF-α to inflamed tissues. Therefore, inhibiting the expression of TNF-α has a good control effect on the inflammatory response (Jayaraman et al., 2008). IL-6 plays an important role in chronic inflammatory diseases, and promotes the proliferation and differentiation of cells in inflammation (Tanaka and Kishimoto, 2014; Takeuchi et al., 2021). It is associated with high levels of IL-6 in a variety of inflammation-related diseases. Therefore, IL-6 is used as an indicator of anti-inflammatory efficacy (Mesquida et al., 2014). NO is a highly reactive oxidative free radical in organisms, which is generated by endogenous L-arginine under the catalysis of iNOS (Raij, 2006). It has been reported in the literature that NO is directly involved in the inflammatory response and joint damage process of RA (Mccartney-Francis et al., 2001). High expression levels of NO can cause hyperdilatation and increased exudation of blood, accompanied by the release of inflammatory cytokines (TNF-α, IL-6). TNF-α induces the synthesis of iNOS and further increases the level of NO (Cohen et al., 2017). Therefore, the level of NO can also be used as one of the important indicators for observing the inflammatory response. The NO colorimetric method to detect the effect of different concentrations of P. aculeata Mill extracts on the LPS-induced NO expression in RAW264.7 macrophages. The results show that the extracts from P. aculeata Mill significantly reduce NO. PEEP has the most significant effect which is dose-dependent. It is suggested that PEEP more effectively reduce the production of NO, and play a better therapeutic effect on intracellular inflammation and oxidative stress. Next, the Q-PCR experiment was conducted to investigate the effects of each extraction site of P. aculeata Mill on the expression of inflammatory factors in RAW264.7 macrophages induced by LPS. The results showed that PEEP significantly inhibit TNF-α and IL-6. EEEP and BEEP have no significant effect on the levels of TNF-α and IL-6 in cells. Based on the experimental results, PEEP significantly reduce the levels of NO, TNF-α, and IL-6 in activated RAW264.7 macrophage expression level and inhibit cell inflammatory response.

p38/MAPK is an important member of the MAPK family. It triggers a chain reaction of intracellular protein kinases under the stimulation of inflammatory factors and external factors, thereby affecting biological effects such as cell transcription, protein synthesis and cell surface receptor expression (Munoz and Ammit, 2010). The p38/MAPK signaling pathway is one of the most important family members that control inflammation, and plays an important role in the physiological and pathological processes such as inflammation, cell stress, apoptosis, cell cycle and growth (Niranjan et al., 2012). Under LPS stimulation, macrophages and the p38/MAPK signaling pathway be activated, and cause the secretion of a large number of inflammatory factors (TNF-α, IL-6) (Zhu et al., 2016; Dong et al., 2018). The activation of p38/MAPK lead to the overexpression of p-MK2 which downstream substrate of the pathway, and activated MK2 phosphorylate zinc finger protein 36 (Tristetraprolin, TTP), thereby over-promoting and regulating cytokines such as TNF-α and IL-6 hyperplasia leads to the continuous expression of inflammatory factors and causes a series of inflammatory reactions. Therefore, the p38-MK2-TTP axis mediate pro-inflammatory signals, which also explains why the knockout mouse MK2 gene cannot be successfully prepared into experimental arthritis (Hegen et al., 2006). We explored the effects of different concentrations of PEEP on the expression of p-p38, p-MK2 and TTP in RAW264.7 macrophags stimulated by LPS in order to further explore the molecular mechanism of PEEP inhibiting inflammation. Western blot was used to detect the effect of PEEP on the p38/MAPK signaling pathway of RAW264.7 macrophages stimulated by LPS. The results showed that the expression of p38 in activated RAW264.7 macrophages was not significantly different The levels of p-p38 and p-MK2 in the cells induced by LPS increased significantly when compared with the blank group. The expressions of p-p38 and p-MK2 in PEEP (30 and 60 μg/ml) were lower than those in the model group. The expression of TTP in each PEEP administration group increased when compared with the model group. The results suggest that PEEP exerts anti-inflammatory effects by regulating the p38/MAPK signaling pathway. PEEP can reduce the expression of NO, TNF-α, and IL-6 in RAW264.7 macrophages induced by LPS. The mechanism may be through regulating the p38/MAPK signaling pathway, thereby inhibiting LPS-induced inflammatory response in RAW264.7 macrophages.

CFA-induced arthritis model is the basic method of the immune arthritis rat model. It is an inflammatory response mediated by immune complexes and its pathogenesis is mainly based on molecular simulation theory. Intradermal injection of CFA delays the body’s absorption and causes continuous stimulation of the body to produce a secondary autoimmune response (Shakeel et al., 2021). The primary lesion of adjuvant arthritis model rats is an acute stress response, it will appear red and swollen joints and foot soles after sensitization. The secondary lesions appeared about 10 days ago, which was an autoimmune reaction, generally manifested as the swelling of joints and plantar joints on the inflamed side and non-inflamed side after modeling. The rat adjuvant arthritis model established by CFA is a simple method, with high success rate and stable model after establishment. Its pathological process and laboratory indicators are similar to those of RA and it is an ideal model for screening and researching drugs for the treatment of RA (Wang et al., 2019). Therefore, we established an CFA-induced arthritis rat model in order to further verify the anti-RA effective part of P. aculeata Mill. The results of this experiment show that, rats in the model group were injected with CFA intracutaneously on the right hind foot plantar. The inflamed right paw of the rat began to swell within 5–9 h, and the swelling degree of the rat’s foot gradually became swollen. It swelled again 14 days after modeling, secondary lesions generally appear at about 11 days after inflammation, and the swelling of the contralateral foot gradually increases to a peak. P. aculeata Mill petroleum ether had a significant inhibitory effect on the swelling of the left and right paws of arthritis after 10 days of administration when compared with the model group.

RA mainly manifests as persistent joint and synovial inflammation, accompanied by bone and bone erosion, joint adhesion, pannus formation, and inflammatory cell infiltration (Turesson et al., 2003; Lee et al., 2012). HE staining and X-ray observation of rat foot and ankle joints showed that the synovial tissue of the model group was proliferated, inflammatory cells infiltrated, and pannus formed. X-rays showed that the soft tissues of the model group were swollen, the joint space was narrow, and the articular surface was blurred. PEEP significantly improve synovial hyperplasia, inflammatory cell infiltration and pannus formation in rat foot and ankle joint disease, reduce the swelling of soft tissue around the foot joint, and improve the bone surface erosion in rats and the degree of joint space stenosis after administration when compared with the model group.

The spleen is the body’s immune organ and plays a very important role in the body’s immune function (Xu et al., 2021). The results of this experiment showed that the spleen index of the model group increased. PEEP reduce the spleen index of CFA rats when compared with the model group. It shows that CFA rats have immune disorders and damaged immune organs. A certain dose of PEEP has a protective effect on the immune function of CFA rats.

In the process of inflammation in CFA rats, the release of inflammatory cytokines (TNF-α, IL-6, IL-8, etc.) played an important role in the occurrence and development of inflammation, the disintegration of articular cartilage matrix and bone destruction (Scott et al., 2010; Wang et al., 2017; Uttra et al., 2018). TNF-α is one of the earliest and most important inflammatory mediators in the process of inflammation. It can activate neutrophils and lymphocytes, increase the permeability of vascular endothelial cells, regulate the metabolic activity of other tissues and promote the synthesis and release of other cytokines (Moelants et al., 2013). PGE₂ is an important inflammatory mediator as a second messenger. The inflammatory response is closely related to the part PGE₂ expression level. Studies have shown that high concentrations of PGE₂ will dilate capillaries, increase vascular permeability, tissue congestion and edema, aggravate inflammation, and even produce collagenase, erode cartilage, and destroy bone (Akaogi et al., 2006; Chen et al., 2012). Further studies have found that PGE₂ induce the production of IL-6, and these cytokines usually interact with each other to form a network and jointly participate in the pathogenesis of RA (Matsuno et al., 2002; Axmann et al., 2009; Takeuchi et al., 2020). Therefore, it can help determine the improvement or deterioration of the inflammatory state in rats when studying the levels of TNF-α, IL-6, and PGE₂. The results showed that the expression levels of inflammatory factors TNF-α, IL-6, and PGE₂ in the serum of the model group increased significantly. PEEP reduce the levels of inflammatory factors TNF-α, IL-6, and PGE₂ in the serum of rats when compared with the model group.

We explored the regulation of PEEP on the p38/MAPK signaling pathway by detecting the protein expressions of p38, p-p38, p-MK2, and TTP in the ankle joints. It preliminarily proves that PEEP regulate the p38/MAPK pathway by inhibiting the expression levels of p-p38 and p-MK2 and reduce the phosphorylation of TTP, and ultimately slow down the inflammatory response and the progression of RA disease.

Each group of PEEP can reduce the index of the spleen of the immune organ, significantly improve the synovial hyperplasia, inflammatory cell infiltration and pannus formation in the foot and ankle joint disease of rats, reduce the swelling of the soft tissue around the foot and ankle joint, and improve the bone of the foot and ankle joint in rats surface erosion and joint space stenosis alleviate various inflammations in rats with adjuvant arthritis. The mechanism of action is that PEEP downregulates the levels of TNF-α, IL-6, and PGE₂ in rat serum by regulating the p38/MAPK pathway, thereby alleviating the RA inflammation and damage.

The pharmacodynamic evaluation in vitro and in vivo strongly demonstrated that the P. aculeata Mill petroleum ether site is the active site for anti-rheumatoid arthritis. P. aculeata Mill petroleum ether site regulate the p38/MAPK pathway by inhibiting the expression levels of p-p38 and p-MK2 and reduce the phosphorylation of TTP, and further inhibit the secretion of NO, TNF-α, IL-6, and PGE₂ inflammatory cytokines, eventually slow down the inflammatory response and the progression of RA disease (Figure 10).