Evaluation indicators of Traditional Chinese Medicine syndromes for gouty arthritis with damp heat accumulation and the effect of administering Tongfeng Qingxiao formula (痛风清消方)

doi:10.19852/j.cnki.jtcm.20240706.001

Abstract

Abstract:

OBJECTIVE: To evaluate the indicators of an animal model of gouty arthritis (GA) with dampness heat accumulation and the intervention effect of Tongfeng Qingxiao formula (痛风清消方, TFQXF).

METHODS: Seventy-two healthy adult Sprague?Dawley male rats were selected and randomly divided into a normal group, model group, low-dose group, medium-dose group, high-dose group, and diclofenac group using a random number table method, with 12 rats in each group. After group intervention, the general condition of the rats in each group was monitored and recorded, and the swelling index was measured. After separating the serum, the changes in glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), carbamide (UREA), creatinine (CREA), triglyceride (TG), total serum cholesterol (TC), low density lipoprotein cholesterol (LDL-C), and high density lipoprotein cholesterol (HDL-C) in the serum of the rats in each group were measured using an automatic biochemical analyzer. The levels of motilin (MTL), gastrin (GAS), endothelin (ET), calcitonin gene-related peptide (CGRP), heat shock protein 70 (HSP70), interleukin (IL)-1β, and nuclear factor kappa-B (NF-κB) in the serum of the rats in each group were evaluated using enzyme-linked immunosorbent assay (ELISA) kits. Kidney tissues were used to evaluate the protein and mRNA expression of aquaporin (AQP) 1 and AQP2. Colon tissue was used to evaluate the protein and mRNA expression of AQP3 and AQP4 by Western blotting (WB) assay and real-time quantitative polymerase chain reaction (RT?qPCR). The levels of ALT, AST, UREA, and CREA were used to evaluate the liver and kidney function of rats. The levels of MTL and GAS were used to evaluate the gastrointestinal function of rats. The levels of TG, TC, LDL-C, HDL-C, AQP1, AQP2, AQP3, and AQP4 were used to evaluate the "dampness" syndrome performance in rats. The levels of ET, CGRP, and HSP70 were used to evaluate the "heat" syndrome performance in rats. The levels of IL-1β and NF-κB were used to evaluate the degree of inflammation in rats. The pathological changes in synovial and colonic tissues were observed by hematoxylin and eosin staining.

RESULTS: Except for the normal group, after modeling treatment, the ankle joint of rats in both the model group and drug treatment groups gradually swelled, reaching a peak at 12 h, and then gradually began to subside. The results of biochemical analyzer detection indicated that the serum ALT, AST, UREA, CREA, TG, TC and LDL-C levels were significantly higher, but the HDL-C level was significantly lower in the rats of the model group than in the rats of the normal group (P < 0.05). The serum ALT, AST, UREA, CREA, MTL, TC and LDL-C levels were significantly lower, but the HDL-C level was significantly higher in the rats of all drug treatment groups than in the rats of the model group (P < 0.05). The results of ELISA detection indicated that the MTL, GAS, ET, HSP70, IL-1β, and NF-κB levels were significantly higher, but the CGRP level was significantly lower in the rats of the model group than in the rats of the normal group (P < 0.05). The levels of MTL, GAS, ET, HSP70, IL-1β, and NF-κB were significantly lower, but CGRP was significantly higher in the rats of the drug treatment groups than in the rats of the model group (P < 0.05). The results of WB and RT-qPCR indicated that compared to the normal group, the levels of AQP1 and AQP2 in the model group were significantly higher in the kidney tissue, whereas the levels of AQP3 and AQP4 were significantly lower in the colon tissue (P < 0.05). Compared to those in the model group, the levels of AQP1 and AQP2 in the drug treatment groups were significantly lower in the kidney tissue, whereas the levels of AQP3 and AQP4 were significantly higher in the colon tissue (P < 0.05). In the model group, erosion of the colonic mucosal surface and inflammatory exudate occurred. Some mucosal epithelium had fallen off, the number of glands in the lamina propria was lower, many inflammatory cells infiltrated the interstitial layer, the connective tissue in the submucosa became loose and edematous, and lymph follicles developed. We found a significant proliferation of synovial cells in the ankle joint, an increase in cell density and neovascularization, and visible infiltration of inflammatory cells. The cartilage surface was not smooth. However, each drug group could improve the pathological changes in intestinal and synovial tissues to varying degrees.

CONCLUSIONS: Blood lipid metabolism indexes and AQPs could be used as objective evaluation indexes for the "dampness" syndrome performance of damp-heat accumulation type GA. ET, HSP70 and CGRP could be used as objective evaluation indexes for the "heat" syndrome performance, and the immune inflammation index could be used as objective evaluation indexes for the inflammation degree. The overall efficacy of TFQXF in the treatment of damp-heat accumulation-type GA could be determined by adjusting the above objective evaluation indexes. It provided some ideas and directions for clinical risk assessments and drug development of GA.

Key words: arthritis, gouty, uric acid, damp heat accumulation, Tongfeng Qingxiao formula

ZHANG Xiaoyun, LI Yongjin, LI Huanan, CHAI Yuan, CHENG Feng, LI Binglin, ZHOU Yi, LAI Yu. Evaluation indicators of Traditional Chinese Medicine syndromes for gouty arthritis with damp heat accumulation and the effect of administering Tongfeng Qingxiao formula (痛风清消方)[J]. Journal of Traditional Chinese Medicine, 2024, 44(6): 1204-1216.

Figures/Tables 9

Table 1 Ankle swelling index of rats at different time (mm, $\bar{x}±s$)

Group	n	4 h	12 h	24 h	48 h	72 h
Normal	12	0.27±0.10	0.11±0.05	0.11±0.07	0.13±0.08	0.17±0.10
Model	10	2.13±0.30^a	4.98±0.32^a	3.74±0.28^a	2.87±0.29^a	1.92±0.42^a
TFQXF-H	11	1.20±0.46^ab	3.65±0.38^abc	2.59±0.40^abc	1.35±0.36^ab	0.56±0.17^ab
TFQXF-M	11	1.35±0.29^abc	3.81±0.37^abc	2.66±0.43^abc	1.56±0.33^abc	0.61±0.16^ab
TFQXF-L	10	1.73±0.31^abc	4.17±0.17^abc	3.11±0.29^abc	2.00±0.45^abc	1.06±0.24^abc
Diclofenac	11	1.15±0.32^ab	3.39±0.36^ab	2.32±0.24^ab	1.22±0.21^ab	0.52±0.19^ab

Table 2 Liver and kidney function of rats in each group ($\bar{x}±s$)

Group	n	ALT (U/L)	AST (U/L)	UREA (mmol/L)	CREA (μmol/L)
Normal	12	34.25±4.56	54.17±2.66	3.70±0.35	52.25±4.73
Model	10	72.10±4.48^a	97.70±6.82^a	10.68±1.02^a	80.40±3.86^a
TFQXF-H	11	46.91±3.30^ab	76.36±3.72^ab	6.07±0.10^abc	63.18±3.54^abc
TFQXF-M	11	47.73±1.62^ab	77.91±3.02^ab	6.16±0.18^abc	63.64±2.25^abc
TFQXF-L	10	56.55±3.88^abc	81.91±3.18^abc	6.65±0.26^abc	67.64±2.38^abc
Diclofenac	11	49.18±2.89^ab	75.64±6.58^ab	5.37±0.24^ab	58.82±2.27^ab

Table 3 Blood lipid metabolism of rats in each group (mmol/L, $\bar{x}±s$)

Group	n	TG	TC	HDL-C	LDL-C
Normal	12	0.64±0.09	1.39±0.13	0.90±0.07	0.36±0.06
Model	10	0.95±0.13^a	1.92±0.19^a	0.51±0.06^a	0.71±0.03^a
TFQXF-H	11	0.72±0.06^ab	1.54±0.11^ab	0.78±0.06^ab	0.50±0.07^ab
TFQXF-M	11	0.74±0.08^ab	1.57±0.12^ab	0.76±0.06^ab	0.52±0.05^ab
TFQXF-L	10	0.81±0.05^abc	1.73±0.11^abc	0.61±0.07^abc	0.62±0.04^abc
Diclofenac	11	0.72±0.05^ab	1.52±0.12^ab	0.81±0.05^ab	0.46±0.10^ab

Table 4 Gastrointestinal function of rats in each group (pg/mL, $\bar{x}±s$)

Group	n	MTL	GAS
Normal	8	23.3±3.2	18.8±2.0
Model	8	43.9±7.7^a	32.1±5.2^a
TFQXF-H	8	28.1±3.2^b	22.7±3.2^ab
TFQXF-M	8	29.8±5.3^ab	23.5±3.6^ab
TFQXF-L	8	35.9±6.8^abc	27.9±3.8^abc
Diclofenac	8	27.7±6.1^b	22.0±2.9^b

Table 5 Objective indexes of heat syndrome in each group (pg/mL, $\bar{x}±s$)

Group	n	ET	CGRP	HSP70
Normal	8	7.24±1.14	20.03±3.19	29.84±4.60
Model	8	16.75±2.71^a	10.22±1.55^a	89.41±10.87^a
TFQXF-H	8	9.32±1.50^ab	16.89±2.63^ab	51.63±10.78^abc
TFQXF-M	8	9.56±1.55^ab	16.55±2.15^ab	52.10±7.81^abc
TFQXF-L	8	12.82±2.15^abc	14.01±1.94^abc	74.25±9.90^abc
Diclofenac	8	8.25±1.41^b	17.45±2.28^ab	39.94±5.10^ab

Table 6 Serum inflammatory indexes of rats in each group ($\bar{x}±s$)

Group	n	IL-1β (ng/L)	NF-κB (μmol/L)
Normal	8	25.03±4.06	31.01±5.09
Model	8	108.74±8.70^a	124.79±18.94^a
TFQXF-H	8	65.50±11.46^abc	54.53±7.92^abc
TFQXF-M	8	59.41±10.50^abc	39.30±8.05^bc
TFQXF-L	8	72.57±7.94^abc	92.32±9.55^abc
Diclofenac	8	26.94±4.10^b	79.26±12.67^ab

Figure 1 Expression levels of AQPs in kidney and colon tissues A: Western blot analyses of AQP1-4 in kidney and colon tissues; B: statistical graphs of AQP1 Protein expression levels; C: statistical graphs of AQP2 Protein expression levels; D: statistical graphs of AQP3 Protein expression levels; E: statistical graphs of AQP4 protein expression levels; F: statistical graphs of AQP1 mRNA expression levels; G: statistical graphs of AQP2 mRNA expression levels; H: statistical graphs of AQP3 mRNA expression levels; I: statistical graphs of AQP4 mRNA expression levels. Normal group: healthy cases; Model group: gouty arthritis animal model of Damp-Heat Accumulation type without treatment; TFQXF-H group: gouty arthritis animal model of Damp-Heat Accumulation type rats treated with Tongfeng Qingxiao formula at a daily dose of 4.5 g/mL (1 mL/100 g) for 1 week; TFQXF-M group: gouty arthritis animal model of Damp-Heat Accumulation type rats treated with TFQXF at a daily dose of 2.25 g/mL (1 mL/100 g) for 1 week; TFQXF-L group: gouty arthritis animal model of Damp-Heat Accumulation type rats treated with TFQXF at a daily dose of 1.125 g/mL (1 mL/100 g) for 1 week; Diclofenac group: gouty arthritis animal model of Damp-Heat Accumulation type rats treated with diclofenac sodium at a daily dose of 1 mL/100 g for 1 week. AQP: aquaporin; TFQXF-H: high-dose group of Tongfeng Qingxiao formula; TFQXF-M: medium-dose group of Tongfeng Qingxiao formula; TFQXF-L: low-dose group of Tongfeng Qingxiao formula. One-way analysis of variance was performed to determine differences between groups, and a least significant difference test was conducted to perform multiple comparisons between groups. The data were expressed as the mean ± standard deviation (n = 8). Compared with the normal group, aP < 0.05; compared with the model group, bP < 0.05; compared with diclofenac group, cP < 0.05.

Figure 2 Results of intestinal pathological examination A: Normal group; B: Model group; C: TFQXF-H group; D: TFQXF-M group; E: TFQXF-L group; F: Diclofenac group. Normal group: healthy cases; Model group: gouty arthritis animal model of Damp-Heat Accumulation type without treatment; TFQXF-H group: gouty arthritis animal model of Damp-Heat Accumulation type rats treated with Tongfeng Qingxiao formula at a daily dose of 4.5 g/mL (1 mL/100 g) for 1 week; TFQXF-M group: gouty arthritis animal model of Damp-Heat Accumulation type rats treated with TFQXF at a daily dose of 2.25 g/mL (1 mL/100 g) for 1 week; TFQXF-L group: gouty arthritis animal model of Damp-Heat Accumulation type rats treated with TFQXF at a daily dose of 1.125 g/mL (1 mL/100 g) for 1 week; Diclofenac group: gouty arthritis animal model of Damp-Heat Accumulation type rats treated with diclofenac sodium at a daily dose of 1 mL/100 g for 1 week. Colon sections from all groups were stained with HE to assess changes in morphology. Method multiplier, × 400. TFQXF-H: high-dose group of Tongfeng Qingxiao formula; TFQXF-M: medium-dose group of Tongfeng Qingxiao formula; TFQXF-L: low-dose group of Tongfeng Qingxiao formula; HE: hematoxylin and eosin.

Figure 3 Results of synovial tissues examination A: Normal group; B: Model group; C: TFQXF-H group; D: TFQXF-M group; E: TFQXF-L group; F: Diclofenac group. Normal group: healthy cases; Model group: gouty arthritis animal model of Damp-Heat Accumulation type without treatment; TFQXF-H group: gouty arthritis animal model of Damp-Heat Accumulation type rats treated with Tongfeng Qingxiao formula at a daily dose of 4.5 g/mL (1 mL/100 g) for 1 week; TFQXF-M group: gouty arthritis animal model of Damp-Heat Accumulation type rats treated with TFQXF at a daily dose of 2.25 g/mL (1 mL/100 g) for 1 week; TFQXF-L group: gouty arthritis animal model of Damp-Heat Accumulation type rats treated with TFQXF at a daily dose of 1.125 g/mL (1 mL/100 g) for 1 week; Diclofenac group: gouty arthritis animal model of Damp-Heat Accumulation type rats treated with diclofenac sodium at a daily dose of 1 mL/100 g for 1 week. Synovium sections from all groups were stained with HE to assess changes in morphology. Method multiplier, × 40. Blue arrow: synovial tissues. TFQXF-H: high-dose group of Tongfeng Qingxiao formula; TFQXF-M: medium-dose group of Tongfeng Qingxiao formula; TFQXF-L: low-dose group of Tongfeng Qingxiao formula; HE: hematoxylin and eosin.

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