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INTRODUCTION
Inflammation refers to the complex defense response of vascular system tissue to external, harmful stimulation, which is an important pathological process in the body. Usually, the manifestations of inflammation are redness, swelling, heat, pain, and dysfunction [1], and the pain, swelling, and other symptoms often become chronic. In severe cases, it will affect normal daily activities and reduce quality of life. At present, the development of inflammatory pain drugs is mainly focused on non-opioid drugs, especially non-steroidal anti-inflammatory and analgesic drugs, but they are limited by their addictive properties, serious cardiovascular effects, and other side effects [2-4]. Some externally used drugs will cause skin allergies, and possess other undesirable traits [5]. Compared with chemical drugs, extracts of natural substances have the advantages of low development cost and few side effects.
Zhongyi paste is an external traditional Chinese medicine, and it has been improved by applying modern technology to the traditional Chinese medicine formula. All the ingredients used in Zhongyi paste are derived from natural plants. Extracts from more than ten different natural plants are used in certain proportions to obtain a paste-like medicine that has been clinically verified to be beneficial for arthritis [6]. However, no studies have been conducted to determine the mechanism of Zhongyi paste. The carrageenan-induced foot-swelling model in rodents is the most commonly used acute inflammatory model to evaluate the activity of anti-inflammatory drugs [7].
The inflammatory process of foot swelling induced by carrageenan can be divided into two stages: in the early stage of inflammation, histamine, 5-hydroxytryptamine, bradykinin, and a small amount of prostaglandins are released from local tissues; in the later stage, neutrophil infiltration occurs, and a large amount of prostaglandins is produced [8]. Neutrophils can release inflammatory factors and participate in the body’s defense response. Inflammatory factors themselves not only damage the body cells, but also induce other cells to produce additional inflammatory factors, resulting in a more severe inflammatory response [9].
Prostaglandin E2 (PGE2) plays an important role in the regulation of inflammatory factors. It is produced by arachidonic acid under the catalysis of cyclooxygenase-2 (COX-2) and other proteases [10]. The hyperphosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) stimulates the expression of COX-2, promotes the release of PGE2, and then stimulates and mediates the occurrence and development of inflammatory pain [11]. The ERK1/2–COX-2–PGE2 pathway is closely related to foot swelling and inflammation [12].
In the current study, an acute foot swelling and inflammation model using C57BL/6J mice, induced by carrageenan, was created to study the effect of Zhongyi paste on foot swelling and inflammation, and the regulatory role of the ERK1/2–COX-2–PGE2 pathway. Through this study, we can observe the mechanism of action of the Zhongyi paste, improve its technology and efficacy, and develop a more scientific application of the ointment for more optimal results.
MATERIALS AND METHODS
1. Production technology of Zhongyi paste
Ten traditional Chinese medicine plants (Table 1) as well as gypsum (1,600 g), red lead (40 g), and borneol (80 g) were ultrafine comminuted and mixed. When a mixture of vaseline and vegetable oil was heated to 120℃ and cooled to 60℃, the comminuted powder of traditional Chinese medicine was added and stirred until it was condensed.
2. Foot model of swelling and inflammation
Fifty six-week-old C57BL/6J mice (half male and half female) were randomly divided into five groups: the normal group, model group, indomethacin group, voltaren (diclofenac diethylamine emulsion) group, and Zhongyi paste group, with 10 mice in each group. One week after adaptive feeding, the right hind paw of the mice in the normal group were injected subcutaneously into the plantar tissues with 25 μL 0.9% saline. The subcutaneous tissues of the right hind paws of the other groups of mice were injected with 25 μL 1% (w/v) carrageenan solution to induce acute swelling of the foot. Immediately afterward, administration of 10 mg/kg (body weight) indomethacin by gavage was performed for the mice in the indomethacin group; the right paws of the mice in the voltaren and Zhongyi paste groups were evenly coated with 0.2 g voltaren or Zhongyi paste (Table 1), respectively, and after application of the drug, the right paws of all mice except the normal group were wrapped with gauze [13]. The right paws of the mice were then observed hourly for 5 hours, and then, the mice were killed by cervical amputation, and the whole blood and right paw of the mice were harvested for experimentation. This study was approved by the ethics committee of Chongqing Functional Food Collaborative Innovation Center, Chongqing University of Education, China (201911035B).
3. Determination of pain threshold by the hot plate method
The hot-plate method uses thermal stimulation to produce pain in mice. The mice were placed on a 55℃ hot plate to stimulate a painful reaction in the foot, i.e., a licking reaction. The time of licking (latency) in mice was used as an indicator of the pain response to determine whether the drug had an antihyperalgesic effect. The latency of the mice in each group was determined before administration of the drugs, and again 5 hours after the injection of carrageenan to evaluate the antihyperalgesic effect of drugs on the carrageenan-induced foot swelling in the mice [14].
4. Measurement of foot swelling in mice
The right hind paw volume of the mice was measured at 5 hours after injection of carrageenan or saline. Seventy mL pure water was added into the measuring cup of the paw volume meter (KW-7C; Nanjing Calvin Biotechnology Co., Ltd, Nanjing, Jiangsu, China, Fig. 1). The mouse ankle joint was marked. The mouse’s hind knee joint was held by hand to make the hind foot straighten. Then it was slowly put into the measuring cup. When the horizontal plane overlapped with the measurement mark on the mouse foot, the measurement of was carried out [15].
5. Detection of serum tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) cytokine levels
After 5 hours of inducing foot swelling the blood was collected from the mice using the retro-orbital blood sampling method [16], then the mice were killed by neck amputation. The blood was centrifuged at 3,000 rpm for 10 minutes after 30 minutes at 4℃ and the supernatant serum was collected. The serum levels of TNF-α and IL-1β were detected using enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).
6. Detection of mRNA expression in injured foot tissues by quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
TRIzol reagent was used to extract total RNA from right hind plantar tissues. For the reaction, 1 μL of Oligo(dT), 18 μL of primer with a concentration of 500 ng/μL, and 1.0 μL of total RNA with a concentration of 1.0 μg/μL were added to 10.0 μL of nuclease-free water and then heated at 65℃ for 5 minutes. Next, 4.0 μL of 5× reaction buffer, 1.0 μL of ribocock RNase inhibitor with a concentration of 20 U, 2.0 μL of 10 mM dNTP Mix, and 1.0 μL of reveraid reverse transcriptase with a concentration of 200 U/μL were added to the above reaction system solution for transcription of cDNA at 42℃ for 60 minutes and 70℃ for 5 minutes. Then, 1.0 μL of this reaction body fluid was added to 1 μL of 10.0 μM upstream primer, 1 μL of 10.0 μM downstream primer (Table 2) and 7.0 μL of sterile double-distilled water for amplification. The reaction conditions were denaturation at 95℃ for 3 minutes, annealing at 60℃ for 30 secconds, and extension at 95℃ for 1 minute, for 40 cycles. The relative expression intensity of each gene to be tested for mice in the model group was calculated by the 2-∆∆Ct method through the measured cycle threshold value [17].
7. Detection of protein expression in injured foot tissues by western blot
The right hind plantar tissues of mice underwent tissue homogenization. Then, 1 mL of precooled lysate was added to each 100 mg of tissue for a 15-minutes incubation on ice, and subsequently centrifuged at 10,000 rpm at 4℃ for 15 minutes. After centrifugation, the supernatant was removed, and the protein was quantified by the bicinchoninic acid method. The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins in the polyacrylamide gel were transferred to a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk powder solution at room temperature for 1 hour and incubated overnight on a shaker with primary antibody at 4℃. Subsequently, the membrane was washed three times with phosphate-buffered saline with Tween 20 (PBST) for 5 minutes each time. Then, secondary antibody was added, the membrane was incubated on a shaker for 2 hours, and was washed three times with PBST for 5 minutes each time to enhance the chemiluminescent agent’s luminescence, development, and imaging [18].
8. Statistical analysis
Statistical software SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was used for analysis, and the measurement data are expressed as the mean ± standard deviation. The t-test was used for comparison between two samples, and the one-way analysis of variance (ANOVA) test was used for comparison between multiple samples. Duncan’s new multiple range test distribution was used to test the hypothesis, and the total “false” error probability is adjusted according to the mean number to be tested, and the total “false” error probability does not exceed the set significance level of P < 0.05.
RESULTS
1. Effects of drugs on the pain threshold in normal mice
There was no significant difference in pain thresholds between groups without inducing foot swelling (Fig. 2). From this finding, we could assume that the individual differences between each group of mice would not have affected the results of other experiments in this study.
2. Effects of drugs on the pain threshold in mice with carrageenan-induced foot swelling
After inducing foot swelling, the pain threshold of the mice significantly decreased (P < 0.05, Fig. 3). However, the pain threshold of the mice was increased after drug treatment. The effect of Zhongyi paste on raising the pain threshold of the mice was significantly (P < 0.05) greater than that of the other two drugs.
3. Effect of drugs on swelling degree of the foot in mice with carrageenan-induced foot swelling
The paw volume of the mice was measured by the foot volume meter, and the swelling degree in each group was compared (Fig. 4). Compared with the normal group (paw volume: 12.5 ± 0.2 mm3), the swelling of the foot (paw volume: 18.9 ± 0.6 mm3) in the carrageenan model group significantly increased (P < 0.05). Compared with the model group, the three drugs reduced the degree of swelling in the foot, and the effect of Zhongyi paste (paw volume: 14.7 ± 0.3 mm3) was the most robust, and was significantly (P < 0.05) higher than that of indomethacin (paw volume: 17.3 ± 0.4 mm3) and voltaren (paw volume: 16.6 ± 0.5 mm3).
4. Effect of drugs on serum TNF-α, IL-1β, and PGE2 cytokine levels in mice with carrageenan-induced foot swelling
The experimental results show that the levels of TNF-α and IL-1β in the serum of normal mice were the lowest (Table 3), and the levels of foot swelling in the model group were the highest. All three drugs significantly (P < 0.05) reduced the serum levels of TNF-α and IL-1β in the mice as compared to those in the model group, and the effects of Zhongyi paste, voltaren, and indomethacin, in turn, increased.
5. Effect of drugs on paw tissue PGE2 levels in mice with carrageenan-induced foot swelling
Fig. 5 shows that the paw tissue PGE2 levels in mice with carrageenan-induced foot swelling (the model group) were highest (135.43 ± 10.95 ng/g), and the levels in normal mice were the lowest (41.01 ± 4.26 ng/g). Zhongyi paste (61.13 ± 5.23 ng/g), voltaren (77.71 ± 7.01 ng/g), and indomethacin (97.87 ± 9.66 ng/g) decreased the paw tissue PGE2 levels compared to the model group, and the effect of Zhongyi paste was stronger than that of voltaren or indomethacin.
6. Effect of drugs on ERK1/2, COX-2, and PGE2 mRNA expression of paw tissues in mice with carrageenan-induced foot swelling
Fig. 6 shows that carrageenan-induced foot swelling significantly (P < 0.05) increased expression of the ERK1/2, COX-2, and PGE2 mRNA in the paw tissues compared to the mice in the normal group. Zhongyi paste, voltaren, and indomethacin altered the expression of the mice with foot swelling so that it was similar to that of the normal mice, and the expression of mice in the Zhongyi paste group was most similar to that of mice in the normal group.
7. Effect of drugs on ERK1/2, and COX-2 protein expression of paw paw tissues in mice with carrageenan-induced foot swelling
Fig. 7 shows that the ERK1/2, and COX-2 protein expression of paw tissues in mice with carrageenan-induced foot swelling was highest, and was lowest in mice of the normal group. Zhongyi paste downregulated the expression compared to that of mice in the model group, and these downregulation abilities of Zhongyi paste were stronger that those of voltaren and indomethacin.
DISCUSSION
Carrageenan is an inflammatory agent that produces a stimulation reaction when subcutaneously injected into the paws of animals. It expands the capillaries of the feet of mice, increases the permeability of blood vessels, increases the inflammatory exudation, and causes a series of inflammatory reactions such as edema. Carrageenan can be used to establish a mouse foot swelling animal model to observe the pain and anti-inflammatory effect of the drug to be tested [19]. Therefore, this animal model was also used to observe the effect of Zhongyi paste. Indomethacin is an oral anti-inflammatory drug that can be used as a positive control drug for screening and activity evaluation [20]. Voltaren (diclofenac diethylamine emulsion) is a drug that is used externally to relieve mild to moderate pain in muscles, soft tissues, and joints, and also can be used as a positive control drug for research [21]. In this study, these two commonly used drugs were used as positive controls for comparing the efficacy of Zhongyi paste.
The acute inflammatory model of foot swelling induced by carrageenan results in a series of symptoms similar to human acute inflammation, such as edema, and it can also show an experimental decrease in the pain threshold [22]. In the current study, the paws of the model mice exhibited significant swelling, and the pain threshold significantly decreased. Zhongyi paste significantly inhibited the morphological changes of paws caused by swelling and reduced the pain in the feet of the mice, with effects that were stronger as compared to the other two drugs.
TNF-α stimulates endothelial cells, leading to inflammation, coagulation, and tissue damage, including tissue edema [23]. IL-1β attracts neutrophils, causes the release of inflammatory mediators, and stimulates a variety of different stromal cells to release proteolytic enzymes with various effects. IL-1β also causes synovial lesions (collagen destruction and bone resorption) in rheumatoid arthritis, and can also affect chondrocytes, fibroblasts, and bone metabolism [24]. In the process of acute inflammation, we can apply external agents that will activate monocyte macrophages, induce and release TNF-α, IL-1β, and other inflammatory factors, and then induce other cells to produce additional inflammatory factors [25,26]. In the current study, Zhongyi paste also promoted the decrease in TNF-α and IL-1β levels in mice, so as to reduce inflammation and foot swelling.
Pain is one of the main manifestations of inflammation, and inflammation is often accompanied by pain. Both interact with each other and contribute to the formation of inflammatory pain. Tissue damage induces an inflammatory response, and phospholipase A2 is then activated to release arachidonic acid from membrane phospholipids [27]. A series of prostaglandin derivatives and thromboxan A2 are produced from free arachidonic acid by COX [28]. PGE2 is a strong pro-inflammatory factor that can cause local vasodilation, increase microvascular permeability, increase the concentration of granulocytes, escalate the pain induced by bradykinin or histamine, and also cause fever. It can also induce the synthesis of COX-2, promote the production and release of inflammatory factors such as IL-6, TNF-α, and IL-1β, further enhance the intensity and duration of inflammation, enlarge the inflammatory response, and promote the amplification of the pain level [29-31]. In this study, it was found that the Zhongyi paste significantly inhibited the upregulation of COX-2 and PGE2 expression in mouse swollen paw tissues induced by carrageenan, and exerted anti-inflammatory and antihyperalgesic actions.
ERKs are important signal transduction proteins in the mitogen-activated protein kinase family that transmit mitogen signals. ERK1/2, as a mitogen-activated protein kinase, is expressed in response to extracellular stimuli. Activated ERK1/2-mediated inflammation or injury causes peripheral sensitization and hyperalgesia [32]. Overphosphorylation of ERK1/2 during inflammation stimulates the expression of COX-2 mRNA and protein to promote the release of PGE2, which in turn stimulates and mediates the occurrence and development of inflammatory pain [33,34]. In this study, we found that the ERK1/2 phosphorylation level substantially increased in injured tissues, while the intervention with Zhongyi paste inhibited the hyperphosphorylation of ERK1/2, blocked the signal transmission of inflammatory pain, reduced COX-2 synthesis, and effectively inhibited the expression of the inflammatory factor PGE2. The results showed that the anti-inflammatory and antihyperalgesic mechanism of Zhongyi paste might be closely related to the regulation of the ERK1/2–COX-2–PGE2 signaling pathway.
In conclusion, the results showed that Zhongyi paste clearly decreased foot swelling in mice due to its anti-inflammatory and antihyperalgesic action. The mechanism of action of Zhongyi paste was derived from its ability to regulate the ERK1/2–COX-2–PGE2 pathway. There was a more marked effect of Zhongyi paste when compared to that of the commonly used oral drug indomethacin and the externally used drug voltaren. Although the mechanism was elucidated, Zhongyi paste can be further optimized, so that the efficacy is improved, which will require additional studies for further discovery.
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