Rheumatoid arthritis is a common clinical disease. The author believes that the main pathogenesis in Chinese medicine during the active stage of RA is paralysis of joints by damp-heat and toxic evil, and the clinical application of Ermiao San is effective. In this paper, we observed the anti-inflammatory effect of different combinations of Ermiao San using AA animal model, and provided animal experimental basis for its clinical application and mechanism of action.
1. Materials and methods
1.1 Animals and grouping Ninety female 8-week-old W istar rats were purchased from the Experimental Animal Center of Shandong University of Traditional Chinese Medicine, weighing (150±20) g, SPF class, animal license number: SCXK(Lu) 20111118. Cangzhi 2 Huangbai 1, Cangzhi group, Huangbai group, and Huangbai Cangzhi single decoction combined group, all were given equal amounts of distilled water and fed with normal chow.
1.2 Drugs and reagents Ermusan was composed of Atractylodes cantonensis and Phellodendron cantonensis. They were uniformly equipped by the preparation room of our hospital, and were blended into a raw drug concentration of 0.64g/mL, 0.43g/mL, 0.43g/mL, 0.32g/mL, 0.32g/mL, 0.64g/mL. Leigengenin polysaccharide tablets: produced by Huangshi Feiyun Pharmaceutical Co., Ltd. with the dose specification of 10mg/tablet. The experimental reagents were complete Freund’s adjuvant, interleukin 1 (IL-1), interleukin 4 (IL-4), interleukin 6 (IL-6) and alpha tumor necrosis factor (TNF-α).
2. Modeling and drug administration
Except for the blank group, rats in each group were injected with 0.1 ml of complete Freund’s adjuvant subcutaneously in the right hind toe.
After successful modeling, no drug was administered to the blank group, and all other groups were administered on the first day after modeling at a uniform dose of 2.5 mL. Details were as follows: 2.5 ml of saline was administered daily to each rat in the model group; 4 mg/2.5 mL of Regulus polysaccharide tablets was administered daily to each rat in the control group; 1.6 g/2.5 mL of herbal solution was administered daily to each rat in the Atractylodes macrocephala 1 and Atractylodes macrocephala 2 groups; 1.6 g/2.5 mL of herbal solution was administered to each rat in the Atractylodes macrocephala 1 and Atractylodes macrocephala 2 groups. The doses were calculated according to the raw drug concentration.
Except for the blank group, all rats were gavaged daily until the day before the end of the experiment, for a total of 22 days.
3.Experimental testing items
(1)Measurement of joint swelling The thickness of the middle and posterior 1/3 of the right lower extremity of the foot was measured with vernier calipers on the day of modeling and every 3 days after modeling, and the average value was taken as the swelling value and recorded.
(2) Examination of serum specimens After the drug administration phase, the animals were anesthetized with 2% pentobarbital sodium by intraperitoneal injection, and the lower vena cava blood was drawn by dissection, centrifuged and the serum was taken, and IL-1, IL-4, IL-6 and TNF-α kits were taken and spiked for laboratory tests.
(3) Joint pathological examination Two rats were taken from each group, and routine pathological sections were sent to the right knee joint capsule for examination.
4. Experimental results
(1) Swelling of the foot and plantar of each group of rats
On the day of modeling, the right hindfoot and plantar injection site of rats in each group showed obvious swelling. After that, the swelling of the injection site of all the rats reached the peak within 4-7 days one after another. At about one week, most rats began to show swelling at the left knee joint. By two weeks, the left knee swelling tended to stabilize. After that, the swelling of the left knee joint in some rats was relieved to different degrees as the number of days of instillation increased, and the swelling of the left knee joint disappeared in very few rats before the specimen was cut. The detailed values of swelling and the measurements after drug administration are shown in Table 1.
Table 1 Effect on swelling of right hind foot and plantar (mm)
Group
Days
Blank group
Model group
Control group
Atractylodis Macrocephalae1
Cyperus rotundus1
Atractylodis Macrocephalae1
Phellodendron 2
Atractylodis Macrocephalae2
Huangbai1
Atractylodes group
Phellodendron group
Single decoction combined group
0
3.93±0.27
4.09±0.40
3.85±0.46
4.07±0.58
4.29±0.31
4.43±0.32
4.32±0.16
4.08±0.49
4.36±0.59
1
3.96±0.30
7.38±0.40
7.45±0.46
7.26±0.39
7.29±0.52
7.62±0.47
7.60±0.55
7.57±0.27
7.35±0.15
4
4.02±0.46
7.99±0.65
8.22±0.54
8.02±0.57○
7.96±0.59○
8.15±0.67○
7.88±0.45○
7.74±0.52○
7.92±0.32○
7
3.94±0.33
7.90±0.39
8.31±0.24
7.95±0.55○
8.05±0.49○
8.21±0.67○
7.92±0.60○
7.83±0.48○
7.98±0.45○
10
3.92±0.40
7.87±0.40
8.26±0.30
7.68±0.45○
7.98±0.40○
8.12±0.56○
7..88±0.56○
7.75±0.48○
7.87±0.43○
13
3.95±0.41
7..78±0.42
8.12±0.33
7.49±0.42○
7.76±0.41○
7.98±0.45○
7.72±0.48○
7.62±0.34○
7.62±0.38○
16
3.93±0.35
7.69±0.38
7.98±0.42
7.26±0.35○
7.59±0.39○
7.82±0.40○
7.60±0.36○
7.58±0.43○
7.52±0.33○
19
3.95±0.40
7.60±0.41
7.82±0.35
7.02±0.45○
7.45±0.36○
7.69±0.30○
7.50±0.46○
7.49±0.37○
7.29±0.45○
22
3.94±0.35
7.56±0.42
7.70±0.42
6.80±0.36○
7.36±0.42○
7.59±0.46○
7.41±0.35○
7.38±0.42○
7.12±0.38○
Note: By t-test analysis, all medication groups were compared with the model group, P<0.05, and the Chinese herbal medicine group was compared with the control group, ○ P<0.05.
As can be seen from Table 1, the swelling of the foot and plantar of rats in all groups appeared to be gradually relieved around the time of the 2nd week after medication, suggesting that both Chinese and Western drugs had an inhibitory effect on the inflammation of the foot and plantar of rats, and the comparison of all medication groups with the model group was statistically significant (P<0.05). At the same time, there was statistical significance (p<0.05) in the comparison of different preparation groups of Ermiao San and single herb groups with the control group. There was also a statistically significant (p<0.05) between the group with equal amounts of Chinese herbs and all other groups. It can be seen that in terms of the degree of relief of joint swelling, the treatment effect was best in the Cangzhi1Huangbai1 group. < span="">
(2) Effects of drugs on inflammatory cytokines
The results of the effects of Chinese and Western drugs on serum inflammatory cytokines in rats are detailed in Table 2.
Table 2 Effects of drugs on serum inflammatory cytokines
Cytokine
Group
IL-1β
IL-4
IL-6
TNF-α
Blank group
54.01±13.19
307.04±12.48
59.64±8.39
1.19±0.25
Model group
112.55±10.54
41.36±4.15
68.31±6.72
1.95±0.05
Control group
85.90±13.89○
115.64±12.18
62.03±5.44
1.75±0.09
Atractylodis Macrocephalae 1 Huangbai 1
69.29±10.90○
170.40±10.76○
38.69±8.97□
1.23±0.12○
Atractylodes1Phellodendron2
79.39±1229○
139.09±10.61○
49.60±5.02□
1.42±0.25○
Atractylodes 2 Phellodendron 1
76.64±15.89○
140.26±9.18○
45.88±8.43□
1.47±0.05○
Atractylodes group
87.56±9.32
123.66±15.23
46.26±9.33□
1.60±0.38
Phellodendron group
90.78±13.56
130.55±13.56
51.69±5.43
1.58±0.24
Single decoction combined group
86.34±12.75
125.36±15.33
56.59±4.29
1.56±0.08
Note: By t-test analysis, P<0.01 for all medication groups compared with the model group; ○P<0.05 and □P<0.01 for the Chinese herbal medicine group compared with the control group.< span="">
As can be seen from Table 2, IL-1β, IL-6 and TNF-α, which can reflect the severity of inflammation, were the highest in the model group, and IL-4, a cytokine reflecting the inhibition of inflammation, was the lowest in the model group, all of which were statistically significant when compared with the blank group (P<0.01). The effects on serum cytokines were statistically significant (p<0.01) in the different dose groups of Ermusan compared with the model group and also compared with the control group (p<0.01), including il-1, il-4, tnf-α (p<0.05) and il-6 (p<0.01), which fully illustrated that the efficacy of different doses of Ermusan was better than that of the control group. And < span="">Cangzhu 1 Huangbai 1 group was statistically significant (p<0.05) compared with all other medication groups, indicating the best efficacy of 1:1 ratio of the original formula of Ermusan. < span="">
(3) Effect of drugs on joint pathological sections
Blank group: There were occasional small amounts of fibrous connective tissue and occasional chronic inflammatory cells in the excised joint cavity, and there was no congestion or edema in the joint capsule.
In the model group, fibrous tissue proliferated significantly in the excised joint cavity, and a large number of chronic inflammatory cells infiltrated with obvious granulation tissue and typical lymphoid follicle formation, and congestion and edema in the joint capsule were obvious. (See Figure 1 for illustration)
For each drug group: the pathology in the excised joint cavity was significantly altered compared to the model group. Fibrous tissue hyperplasia was less than in the model group, chronic inflammatory cells were significantly reduced in number, and congestion and edema were significantly reduced compared with the model group. The improvement of Ermiao San in different combination groups was obvious compared with the control group, the herbal single decoction group and the monomer mixture group. It indicates that the anti-inflammatory efficacy of Ermiao San in all groups with different combinations was better, especially the best efficacy was achieved with the original formula in the ratio of 1:1. (See Figure 2, 3 and 4 shown)
The results are shown in: pathological sections under light microscopy (as shown in Figures 1, 2, 3 and 4).
Figure 1 (model group ×100) Figure 2 (control group ×100)
Fig. 3 (Atractylodes1Huangbao1 group×100) Fig. 4 (Atractylodes group×100)
5. Discussion
RA belongs to the Chinese medicine “heat paralysis”, “Dong bath wu jie hug 1 Jing” animal gag Jing” oh honestly drips the questionable Jing Jing kitchen court sneakers (6) edge according to the enthusiastic scandium calcium Jia rate questionable detention ammonia zai fork confused with the water goblet wall mound any gourmets ± ram [1].
Modern medicine has found that IL-1 and TNF-α are cytokines that play an important role in the pathogenesis of RA and can lead to progressive joint destruction in addition to synovial inflammation [2]. It has been shown that the reduction of IL-1 and TNFα is closely related to the improvement of clinical activity (clinical and laboratory indices) [3].The anti-inflammatory effect of IL-4 is mainly reflected in its regulation of the activity of many different cell types in RA, thus inhibiting the activity of TNF-α, IL-1, IL-6, IL-8, etc., inhibiting the synthesis of NO, the production of PGE2, thus controlling the inflammation , protecting the integrity of articular cartilage and subarticular bone and other joint tissues. There is a growing view that inhibition of the activities of the inflammatory factors TNF-α, IL-1, IL-6, IL-8, etc., and upregulation of the activities of the anti-inflammatory factors IL-4 and IL-10 at the cellular and molecular levels may provide new clues to strategies for the treatment of RA.
The secret of TCM lies in the dosage, and the effects of different ratios of the preparation may vary greatly. In this study, we found that the effects of different preparations of Ermusan were different in size, and the effect of the ratio of 1:1 between Atractylodes macrocephala and Phellodendron chinensis was the most obvious in terms of swelling effect on the inflammatory toes of rats and the effect on serum inflammatory cytokines, which shows that the equal dosage of Atractylodes macrocephala and Phellodendron chinensis in Ermusan is significant.