Effect of Dangfei Liganning capsule (当飞利肝宁胶囊) on liver X receptor α/steroid regulatory element binding protein-1/fatty acid synthase signal pathway in rats with metabolic- associated fatty liver disease

doi:10.19852/j.cnki.jtcm.2022.06.007

Abstract

Abstract:

OBJECTIVE: To study the mechanism of Dangfei Liganning capsule (当飞利肝宁胶囊) in the treatment of rats with metabolic associated fatty liver disease (MAFLD).

METHODS: Totally 48 specific pathogen free Sprague-Dawley male rats were randomly divided into normal Group, model group, Dangfei Liganning high, moderate, and low-dose groups and Essentiale group which were fed with high fat diet for 8 weeks, and gavage and molding were carried out simultaneously. Dangfei Liganning high, middle and low-dose group were given 0.27, 0.135 and 0.0675 g·kg^-1·d^-1 respectively by gavage, Essentiale group was given 0.123 g·kg^-1·d^-1 by gavage, the same amount of distilled water was given by gavage in the normal group and the model group. The rats were weighed at the 0th week, 2nd week, 4th week, 6th week and 8th weekend respectively. The rats were sacrificed at the end of the 8th week. Serum levels of alanine aminotransferase (ALT), alanine aminotransferase (AST), triglyceride (TG), total cholesterol (CHO), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein (LDL-C), total protein (TP), albumin (Alb), globulin (GLB), total bilirubin (TBIL), direct bilirubin (DBIL), tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) were measured. The levels of liver tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and liver pathology [hematoxylin and eosin (HE) staining, oil red O staining] were detected. The expression levels of liver X receptor α (LXRα), steroid regulatory element binding protein-1 (SREBP-1) and fatty acid synthase (FAS) were detected by immunohistochemistry, Western blot and reverse transcription-polymerase chain reaction reverse transcription-polymerase chain reaction.

RESULTS: From the beginning to the 8th week, the growth rate of body weight in the Dangfei Liganning high-dose group was slower than all other groups. There was no significant difference in ALB level in all groups (P > 0.05). Compared with the model group, the levels of ALT, AST, LDL-C, TG, CHO, TP, GLB, TBIL, DBIL, IL-6, TNF-α were significantly decreased and HDL-C were significantly increased in Dangfei Liganning high-dose group (P < 0.01, < 0.05). HE and oil red O staining showed that the fatty lesions in rat liver were alleviated, while the expressions of LXRα, SREBP-1, FAS mRNA and protein were significantly decreased (P < 0.01).

CONCLUSIONS: Dangfei Liganning capsule can slow down the increase of body weight of MAFLD rats, reduce the levels of transaminase, Lipid and inflammatory factors in MAFLD rats, promote the synthesis of liver protein and bile metabolism, and improve the liver fatty lesion of MAFLD rats, among which the Dangfei Liganning high-dose group is more effective. The mechanism of action may be through blocking LXR-SREBP-1-FAS signal pathway.

Key words: metabolic associated fatty liver disease, liver X receptors, steroid regulatory element binding protein-1, fatty acid synthases, signal transduction, Dangfei Liganning capsule.

LI Xiaoling, SUN Fengxia, SHANG Zimeng, ZHANG Yingxue, LI Jie, ZHANG Qiuxiang. Effect of Dangfei Liganning capsule (当飞利肝宁胶囊) on liver X receptor α/steroid regulatory element binding protein-1/fatty acid synthase signal pathway in rats with metabolic- associated fatty liver disease[J]. Journal of Traditional Chinese Medicine, 2022, 42(6): 940-947.

Figures/Tables 13

Table 1 Changes in body weight of rats in each group (g, $\bar{x} \pm s$)

Group	n	Week 0	Week 2	Week 4	Week 6	Week 8
Normal	8	218±7	318±8	405±5	502±4	578±8
Model	8	218±6	324±5^a	421±3^a	524±7	602±7
Dangfei-L	8	216±6	322±4^a	418±6^a	524±7	601±8
Dangfei-M	8	220±6	323±4^a	419±4^a	522±6	601±8
Dangfei-H	8	219±3	313±4^b	404±4^b	476±4^c	537±4^c
Essentiale	8	217±7	317±11^b	414±6^b	516±7	578±4

Table 2 Changes of liver function and blood lipids of rats in each group ($\bar{x} \pm s$)

Group	n	TG (mmol/L)	CHO (mmol/L)	HDL-C (mmol/L)	LDL-C (mmol/L)	ALT (U/L)	AST (U/L)
Normal	8	0.33±0.12	2.10±0.42	1.06±0.16	0.33±0.10	3.40±0.81	10.61±1.17
Model	8	0.89±0.23^a	5.68±0.71^a	0.61±0.14^a	0.72±0.11^a	9.98±2.26^a	23.62±4.82^a
Dangfei-L	8	0.95±0.26	5.61±0.71	0.58±0.12	0.69±0.11	9.71±2.18	22.43±4.53
Dangfei-M	8	1.01±0.29	5.47±0.65	0.57±0.10	0.70±0.10	9.24±1.81	22.34±4.19
Dangfei-H	8	0.55±0.19^b	3.93±1.07^b	0.81±0.13^b	0.41±0.10^b	4.81±0.21^bc	13.21±0.89^bc
Essentiale	8	0.56±0.13^b	3.41±0.81^b	0.78±0.11^b	0.42±0.09^b	6.42±1.88^b	16.41±3.29^b

Table 3 Changes in the levels of inflammatory factors in rats of each group (pg/mL, $\bar{x} \pm s$)

Group	n	IL-6	TNF-α
Normal	8	0.94±0.80	2.40±0.96
Model	8	7.88±3.07^a	6.63±0.91^a
Dangfei-L	8	7.55±3.17	6.45±0.97
Dangfei-M	8	7.66±1.19	5.98±1.04
Dangfei-H	8	1.15±0.20^b	3.99±0.81^b
Essentiale	8	1.64±0.55^b	4.44±1.38^b

Table 4 Changes of serum protein level in rats of each group (g/L, $\bar{x} \pm s$)

Group	n	TP	ALB	GLB
Normal	8	61.4±2.7	33.0±2.0	28.4±1.2
Model	8	74.5±1.8^a	33.2±0.7	41.3±1.7^a
Dangfei-L	8	73.3±1.6	33.2±0.9	39.9±1.9
Dangfei-M	8	73.7±0.9	33.4±0.8	40.3±1.3
Dangfei-H	8	61.8±1.7^bc	33.4±0.8	28.6±1.8^bc
Essentiale	8	64.4±2.9^b	33.3±0.9	31.1±3.4^b

Table 5 Changes of serum bilirubin level in rats of each group (umol/L, $\bar{x} \pm s$)

Group	n	TBIL	DBIL
Normal	8	2.08±0.12	0.49±0.06
Model	8	6.05±0.34^a	1.51±0.07^a
Dangfei-L	8	6.01±0.31	1.45±0.06
Dangfei-M	8	5.82±0.11	1.51±0.06
Dangfei-H	8	4.00±0.21^bc	0.99±0.08^b
Essentiale	8	4.43±0.20^b	1.03±0.03^b

Table 6 Changes of inflammatory factors in liver tissue of rats in each group (pg/mL, $\bar{x} \pm s$)

Group	n	IL-6	TNF-α
Normal	8	16.3±2.0	40.6±3.0
Model	8	30.6±3.6	85.4±3.4
Dangfei-L	8	29.3±3.0	84.7±5.5
Dangfei-M	8	28.2±3.2	80.8±2.9
Dangfei-H	8	18.8±2.4^a	52.4±2.7^a
Essentiale	8	19.6±1.5	52.4±2.1

Figure 1 Pathological changes in rat livers according to treatment (hematoxylin and eosin staining, × 200) A: liver pathology of normal rat after HE staining (× 200); B: liver pathology of model rat after HE staining (× 200); C: liver pathology of Dangfei Liganning low-dose group rat after HE staining (× 200); D: liver pathology of Dangfei Liganning middle-dose group rat after after HE staining (× 200); E: liver pathology of Dangfei Liganning high-dose group rat after HE staining (× 200); F: liver pathology of Essentiale group rat after HE staining (× 200); The arrow points to fat vacuoles. The normal group and the model group were given the same amount of distilled water by gavage; Dangfei Liganning low, medium and high-dose group was given Dangfei Liganning 0.0675, 0.135 and 0.27 g•kg-1•d-1 by gavage; Essentiale group was given Essentiale 0.123 g•kg-1•d-1 by gavage.

Figure 2 Pathological changes in rat livers according to treatment (Oil red O staining, × 200) A: liver pathology of normal rat after Oil red O staining (× 200); B: liver pathology of model rat after Oil red O staining (× 200); C: liver pathology of Dangfei Liganning low-dose group rat after Oil red O staining (× 200); D: liver pathology of Dangfei Liganning middle-dose group rat after Oil red O staining (× 200); E: liver pathology of Dangfei Liganning high-dose group rat after Oil red O staining (× 200); F: liver pathology of Essentiale group rat after Oil red O staining (× 200). The normal group and the model group were given the same amount of distilled water by gavage; Dangfei Liganning low, medium and high-dose group was given Dangfei Liganning 0.0675, 0.135 and 0.27 g·kg-1·d-1 by gavage; Essentiale group was given Essentiale 0.123 g·kg-1·d-1 by gavage.

Table 7 Changes of LXR α, SREBP-1 and FAS protein in liver tissues of rats in each group detected by immunohistochemistry ($\bar{x} \pm s$)

Group	n	LXRα	SREBP-1	FAS
Normal	8	345872±52737	259409±71143	201684±48205
Model	8	544998±55506^a	538701±62336^a	475218±78051^a
Dangfei-H	8	423402±55463^b	383871±103496^b	397673±61834^b
Essentiale	8	448588±104641^b	394167±158047^b	417853±84373^b

Figure 3 Expression levels of LXR α, SREBP-1 and FAS protein in each group (×400) A: normal group LXR α; B: model group LXR α; C: Dangfei Liganning high-dose group LXR α; D: Essentiale group LXR α; E: normal group SREBP-1; F: model group SREBP-1; G: Dangfei Liganning high-dose group SREBP-1; H: Essentiale group SREBP-1; I: normal group FAS; J: model group FAS; K: Dangfei Liganning high-dose group FAS; L: Essentiale group FAS. The normal group and the model group were given the same amount of distilled water by gavage; Dangfei Liganning low, medium and high-dose group was given Dangfei Liganning 0.0675, 0.135 and 0.27 g·kg-1·d-1 by gavage; Essentiale group was given Essentiale 0.123 g·kg-1·d-1 by gavage. LXR α: liver X receptor α; SREBP-1: steroid regulatory element binding protein-1; FAS: fatty acid synthase.

Figure 4 Expression levels of LXRα, SREBP-1, FAS protein in each group 1: normal group; 2: model group; 3: Dangfei Liganning high-dose group; 4: essentiale group. The normal group and the model group were given the same amount of distilled water by gavage; Dangfei Liganning high-dose group was given Dangfei Liganning 0.27 g·kg-1·d-1 by gavage; Essentiale group was given Essentiale 0.123 g·kg-1·d-1 by gavage. LXR α: liver X receptor α; SREBP-1: steroid regulatory element binding protein-1; FAS: fatty acid synthase; GAPDH: glyceraldehyde-3-phosphate dehy-drogenase.

Table 8 Changes of LXR α, SREBP-1 and FAS protein in liver tissues of rats in each group detected by Western Blot (pg/mL, $\bar{x} \pm s$)

Group	n	LXRα	SREBP-1	FAS
Normal	8	0.79±0.28	0.42±0.11	0.43±0.12
Model	8	1.51±0.19^a	1.14±0.45^a	1.10±0.40^a
Dangfei-H	8	0.99±0.21^b	0.82±0.18^b	0.76±0.22^b
Essentiale	8	1.09±0.35^b	0.83±0.20^b	0.79±0.27^b

Table 9 Changes of LXR α, SREBP-1 and FAS mRNA in liver tissues of rats in each group ($\bar{x} \pm s$)

Group	n	LXRα	SREBP-1	FAS
Normal	8	1.14±0.41	1.52±0.51	1.42±0.49
Model	8	4.29±0.52^a	6.13±1.17^a	4.35±1.97^a
Dangfei-H	8	2.27±0.21^b	3.32±1.52^b	2.30±0.88^b
Essentiale	8	2.25±0.34^b	2.64±1.97^b	2.61±1.81^b

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