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Abstract

Objective: To analyze the mechanism of Huanglian Jiedu Decoction (HLJDD) in the treatment of metabolism-associated fatty liver disease (MAFLD). Methods: The main components, targets, and pathways for treating MAFLD of HLJDD were screened through network pharmacology and molecular docking validation was done; HLJDD was used to intervene MAFLD model of rat, the levels of ALT, AST, TC, TG, GLU, HDL, and LDL were identified, HE staining was used to observe the pathological changes, lipid deposition in liver was detected by oil red O staining. MAFLD model of HepG2 (hepatocellular carcinoma cell line) was constructed by PA (palmitate-acid) incubating, and HLJDD was administered with drug-containing serum intervention, lipid droplets in HepG2 cells was observed by oil red O staining, TG and FFA of HepG2 were detected, the expressions of AMPK, mToR, and Beclin-1 were detected through Western blot. Results: Seventy components and 229 targets were obtained, and 85 targets were used to treat MAFLD, which focus on the signal passways of AMPK/mToR/PI3K-AKt/MAPK, NAFLD, autophagy-animal, insulin, etc. Molecular docking outcomes showed quercetin, kaempferol, and baicalein that were successfully docked with AMPK and mToR, and had good binding activity, compared with MAFLD group of rats, the levels of ALT, AST, TC, TG, GLU, HDL, and LDL were significantly decreased in Silybin group and each dos group of HLJDD, liver pathology and lipid deposition were significantly improved; the results in vitro experiments showed that drug-containing serum of HLJDD and Silybin could improve intracellular lipid accumulation and reduce the increase of TG and FFA levels in HepG2 cells, the therapeutic effect of HLJDD was significantly attenuated after application of AMPK inhibitor; the results of Western blot showed that HLJDD could up-regulate the protein expression of AMPK and Beclin-1,down-regulate the protein expression of mToR. Conclusion: Within process of MAFLD intervention, HLJDD could regulate AMPK-mToR signaling pathway to treat MAFLD.

Keywords

Huanglian Jiedu Decoction(HLJDD)metabolism-associated fatty liver disease (MAFLD)network pharmacology molecular docking autophagy

Introduction

In 2020, NAFLD was officially renamed metabolism-associated fatty liver disease (MAFLD) and its definitive diagnosis was clearly stated to be a pathology characterized through hepatic lipid accumulation with metabolic disorders.^1,2The pathogenesis of MAFLD is closely related to “multiple strikes,” involving multiple processes such as insulin resistance (IR), lipotoxicity, oxidative stress and endoplasmic reticulum stress.³ A recent study found that the global prevalence of MAFLD is 39%,⁴ a cross-sectional study of 139,170 subjects in China according to newly defined diagnostic criteria through Chen et al⁵ found the prevalence of MAFLD to be 26.1%. MAFLD strongly associated with advanced age, obesity and other factors.⁶ With the urbanization of human society, MAFLD also tends to be younger and could induce other chronic metabolic diseases,⁷ MAFLD seriously jeopardizes human health and brings a huge burden to society and families. However, there is no specific drug for MAFLD, and lifestyle intervention is the first line of treatment, taking into account prophylaxis and therapy for metabolic cardiovascular conditions and hepatic inflammatory injury.

Huanglian Jiedu Decoction (HLJDD), recorded in “Zhouhou Beiji Fang,” is a representative formular of clearing heat and detoxifying. The formula is composed of Huanglian (Coptis chinensis Franch), Huangqin (Scutellaria baicalensis Georgi), Huangbo (Phellodendron amurense Rupr), and Zhizi (Gardenia jasminoides Ellis). Recent pharmacological investigations found that HLJDD has a series of functions such as anti-inflammatory, antioxidant, anti-tumor and immune regulation.⁸ However, current studies have only demonstrated that HLJDD could improve disorders of glucose and lipid metabolism, but its mechanism has not been clarified. This research project focuses on the preliminary investigation of the target and molecular mechanism of HLJDD for treating MAFLD through network pharmacology combined with molecular docking technology, validated through animal pharmacodynamic studies and cellular experiments.

Methods

Targets of Action and Main Active Components for Huanglian Jiedu Decoction

The chemical compositions of the four Chinese herbs of HLJDD were searched in TCMSP2.3 Pharmacological Analysis Platform for Traditional Chinese Medicine Systems (https://old.tcmsp-e.com/tcmsp.php), TCMSP allowed for retrieving additional targets for active components obtained within previous step, and the screening was completed through mapping the targets to standard gene names within UniProt (https://www.uniprot.org) mapped targets to standard gene names.

Targets for Huanglian Jiedu Decoction for the Treatment of MAFLD

Using “metabolism-related fatty liver disease,” “NAFLD,” and “MAFLD” as keywords, we searched the GeneCards5.0 database for potential targets for MAFLD. After searching the GeneCards5.0 database for the potential targets of MAFLD, combining the targets within disease database, deleting the duplicates to the MAFLD targets, and the intersection of MAFLD target genes and HLJDD principal component targets was screened, key targets for HLJDD for treating this condition were finally obtained.

Network Construction of Target Interactions

Construction of a Target Network Concerning Principal Components in Huanglian Jiedu Decoction

Principal components for HLJDD and their action targeted networks were developed through Cytoscape 3.7.2 package. Throughout the network, the nodes are the target proteins of the drug during disease intervention, and the edges represent the interactions across drug components and these target proteins. Topological parameters of the interaction network were calculated using the “Analyze Network” function in Cytoscape, and the important active ingredients of HLJDD for treating MAFLD were identified according to the degree of connectivity of the nodes, and increased node value raised active ingredient importance.

Protein Interaction Networks and Important Targets

The targets of HLJDD for treating MAFLD were imported into STRING11.0 platform, and the species type was set as “Homo sapiens,” the confidence level was set as “0.9,” and all other parameters are filtered through default settings. Finally, the target protein interaction information files obtained after screening were inserted within Cytoscape to construct PPI networks.

KEGG Pathway Annotation and Enrichment Analyses

The drug–disease intersection targets were imported within DAVID 6.8 (https://david.ncifcrf.gov), with species selected as “Homo sapiens,” which was then subjected to KEGG pathway annotation and enrichment analysis, and the results were visualized.

Molecular Docking

3D frameworks for AMPK and mToR proteins were downloaded from the PDB database, and the proteins were dehydrated and hydrogenated through AutoDock Tools 1.5.6 package. Active ingredients of AMPK and mToR were obtained from TCMSP database depending upon composition of HLJDD (International Compound Identifier, the InChIKey of each compound is unique), and were searched through PubChem. InChIKey to obtain the SDF format of the 3D structure of the active ingredient of the drug, and the format conversion of mol2 was performed using OpenBabel2.4.1. The processed proteins and active ingredients were molecularly docked through AutoDock Vina, and the binding capacity below 0, indicating that the ligand and receptor could spontaneously bind, and these results were analyzed and visualized using Pymol 2.4.0.

Animal Pharmacodynamic Study

Laboratory Animals and Drugs

Sixty male SD rats of SPF grade are all weighing (180 ± 20) g. These rats were procured through Changsha Tianqin Biotechnology Co, License No. SCXK (Xiang) 2019-0013; HLJDD was purchased from Jiangyin Tianjiang Pharmaceutical Co. Ltd of Sinopharm Group, Su 20160102; high-fat diet was purchased from Suzhou Shuangshi Experimental Animal Stall Food Technology Co., Ltd, License No. Su Feeding License (2017) 05005(high-fat diet was made up of 20% lard, 4% sugar, 2% powdered milk, 1% cholesterol, and 73% normal diet). The study protocol was approved by the Ethics Committee of Hainan Medical University, approval number: HYLL-2021-009.

Preparation of Pharmaceutical Solution

The human clinical dose (Drug1) was converted into the equivalent dose for rats (Drug2) according to the body surface area, the formula: $Drug 2 (g / kg) = Drug 1 g \div 70 kg \times 6.3$ .^9,10 The high, medium, and low doses of HLJDD were 5.4 g/kg, 2.7 g/kg, and 1.35 g/kg, respectively. HLJDD was fully dissolved in distilled water at 40 °C and heated to 37 °C before administration. The equivalent dose of Silybin group was 37.8 mg/kg.

Establishment of Rat Model

Sixty male SD rats were fed adaptively for one week and divided into normal group (N group, n = 10) and model group (MAFLD group, n = 50) according to random number table method. The rats in group N were fed with ordinary diet, the rats in the MAFLD group were fed high-fat diet and all rats were given free water. To detect whether the model of MAFLD was built successfully, we detected the pathological change of a rat’s liver after 4 weeks of modeling by HE and oil red O staining. HE staining results showed that there is an abnormal arrangement of hepatocytes, small vacuoles in some cells, and a large number of vacuole-like structures around liver cells in the model group compared with the control group. In addition, Oil red O staining results showed a large amount of oil red O precipitation with a large number of lipid droplets in the liver of the model group compared with the control group. The above results indicate that the animal model of MAFLD has been established successfully (see Appendix Figure 1 for details). Rats in MAFLD group were randomly divided into model group (M, n = 10), HLJDD low-dose group (HL-L, n = 10), HLJDD medium-dose group (HL-M, n = 10), HLJDD high-dose group (HL-H, n = 10), and silybin group (SF, n = 10). The feeding of all groups remained unchanged. HL-L group, HL-M group, HL-H group, and SF group were administered the corresponding drug solution (10 mL/kg/d) by gavage for 6 weeks, N and M groups were administered the same volume of normal saline.

Specimen Collection and Processing

After modeling was completed, rats were deeply anesthetized with 1% sodium pentobarbital given at 4 ml/kg, and abdominal aortic blood and liver tissue were taken. The rat serum was allowed to stand for 30 minutes and then centrifuged (3000 rpm; 10 minutes) and the supernatant was poured into EP tubes. The treated serum and part of the liver were stored in an ultra-low temperature refrigerator at −80 °C for subsequent experiments. Part of the liver was soaked in 4% paraformaldehyde and stored at room temperature.

Liver HE Staining

After the fresh tissue was treated with 4% paraformaldehyde overnight, it was cleaned with PBS buffer, paraffin embedded, sliced, and then stained through HE.

Liver Oil Red O Staining

Fresh tissue was washed in 4% paraformaldehyde overnight with PBS buffer, and liver tissue was directly glued to the sample table coated with OCT glue and placed within frozen microtome for direct freezing. The frozen sample table with tissue was clamped on the microtome clamp, and the section was performed with a thickness of about 5 to 10 µm.

Verification Through Cell Experiment

Palmitic acid (PA)-induced MAFLD model was established using human hepatocellular carcinoma cell line (HepG2), and intervention of drug-containing serum of HLJDD and drug-containing serum of silybin was given. The specific group are as follows: Normal group (N), Model group (M), HLJDD treatment group (HL), silybin treatment group (SF), and AMPK inhibitor (drug-containing serum of HLJDD + Compound C) group (HL + CC), for details, see Table 1. Lipid droplet changes of HepG2 cells were detected through oil red O staining; the levels of TG and FFA in HepG2 cells were detected; the expressions of AMPK, mToR, and Beclin-1 were detected through Western blot.

Table 1.

(1) Common Culture medium Preparation System. (2) Cell Grouping and Intervention.

	MEM Base medium (Contains 0.5% penicillin + streptomycin)	0.3 mmol/L PA	Serum
Blank serum medium	+	-	10% Blank serum
PA medium	+	+	–
HLJDD medium	+	-	10% drug-containing serum of Huanglian Jiedu Decoction
Silybin medium	+	-	10% drug-containing serum of Silybin
(2)
group	modeling	Treatment
N	Blank serum medium	Blank serum medium
M	PA medium	Blank serum medium
HL	PA medium	drug-containing serum of Huanglian Jiedu Decoction
SF HL + CC	PA medium PA medium	drug-containing serum of Silybin HLJDD medium + Compound C (0.2 μmol reaction for 30 min)

Statistical Analysis

Data were analyzed using GraphPad Prism 8.0, and the results were expressed as mean ± standard deviation (±sd). One-way analysis of variance compared inter-group data, P < .05 indicates statistical difference, P < .01 indicates a statistically significant difference, P < .001 indicates a statistically significant difference.

Results

After screening, 102 substances were obtained through analyzing the active ingredients of each group of HLJDD, including 14 substances of Huanglian, 37 substances of Huangbo, 36 substances of Huangqin, and 15 substances of Zhizi, and after removing duplicate entries, we obtained 70 of the active ingredients in HLJDD (Table 2), 229 targets (Table 3); and 1090 targets of MAFLD; Using Venny2.1.0 to map the common targets for HLJDD and MAFLD (Figure 1), 85 common targets of HLJDD-MAFLD were obtained (Table 4).

Figure 1.

Huanglian Jiedu decoction-MAFLD target Wayne's plot.

Table 2.

Main Constituents of Huanglian Jiedu Decoction.

Chinese medicine	Mol ID	Main active ingredients	OB (%)	DL
Huanglian (14 substances)	MOL001454	Berberine	36.86	0.78
	MOL013352	Obacunone	43.29	0.77
	MOL002894	Berberrubine	35.74	0.73
	MOL002897	Epiberberine	43.09	0.78
	MOL002903	(R)-Canadine	55.37	0.77
	MOL002904	Berlambine	36.68	0.82
	MOL002907	Corchoroside A_qt	104.95	0.78
	MOL000622	Magnograndiolide	63.71	0.19
	MOL000762	Palmidin A	35.36	0.65
	MOL000785	Palmatine	64.60	0.65
	MOL000098	Quercetin	46.43	0.28
	MOL001458	Coptisine	30.67	0.86
	MOL002668	Worenine	45.83	0.87
	MOL008647	Moupinamide	86.71	0.26
Huangbo (37 substances)	MOL001454	Berberine	36.86	0.78
	MOL001458	Coptisine	30.67	0.86
	MOL002636	Kihadalactone A	34.21	0.82
	MOL013352	Obacunone	43.29	0.77
	MOL002641	Phellavin_qt	35.86	0.44
	MOL002643	delta7-Stigmastenol	37.42	0.75
	MOL002644	Phellopterin	40.19	0.28
	MOL002651	Dehydrotanshinone II A	43.76	0.40
	MOL002652	delta7-Dehydrosophoramine	54.45	0.25
	MOL002656	Dihydroniloticin	36.43	0.81
	MOL002659	Kihadanin A	31.60	0.70
	MOL002660	Niloticin	41.41	0.82
	MOL002662	Rutaecarpine	40.30	0.60
	MOL002663	Skimmianin	40.14	0.20
	MOL002666	Chelerythrine	34.18	0.78
	MOL000449	Stigmasterol	43.83	0.76
	MOL002668	Worenine	45.83	0.87
	MOL002670	Cavidine	35.64	0.81
	MOL002671	Candletoxin A	31.81	0.69
	MOL002672	Hericenone H	39.00	0.63
	MOL002673	Hispidone	36.18	0.83
	MOL000358	beta-Sitosterol	36.91	0.75
	MOL000622	Magnograndiolide	63.71	0.19
	MOL000762	Palmidin A	35.36	0.65
	MOL000785	Palmatine	64.60	0.65
	MOL000787	Fumarine	59.26	0.83
	MOL000790	Isocorypalmine	35.77	0.59
	MOL000098	Quercetin	46.43	0.28
	MOL001131	Phellamurin_qt	56.60	0.39
	MOL001455	(S)-Canadine	53.83	0.77
	MOL001771	Poriferast-5-en-3beta-ol	36.91	0.75
	MOL002894	Berberrubine	35.74	0.73
	MOL005438	Campesterol	37.58	0.71
	MOL006392	Dihydroniloticin	36.43	0.82
	MOL006401	Melianone	40.53	0.78
	MOL006413	Phellochin	35.41	0.82
	MOL006422	Thalifendine	44.41	0.73
Huangqin（36 substances）	MOL001689	Acacetin	34.97	0.24
	MOL000173	Wogonin	30.68	0.23
	MOL000228	(2R)-7-Hydroxy-5-methoxy-2-phenylchroman-4-one	55.23	0.20
	MOL002714	Baicalein	33.52	0.21
	MOL002908	5,8,2′-Trihydroxy-7-methoxyflavone	37.01	0.27
	MOL002909	5,7,2,5-Tetrahydroxy-8,6-dimethoxyflavone	33.82	0.45
	MOL002910	Carthamidin	41.15	0.24
	MOL002911	2,6,2′,4′-Tetrahydroxy-6′-methoxychaleone	69.04	0.22
	MOL002913	Dihydrobaicalin_qt	40.04	0.21
	MOL002914	Eriodyctiol (flavanone)	41.35	0.24
	MOL002915	Salvigenin	49.07	0.33
	MOL002917	5,2′,6′-Trihydroxy-7,8-dimethoxyflavone	45.05	0.33
	MOL002925	5,7,2′,6′-Tetrahydroxyflavone	37.01	0.24
	MOL002926	Dihydrooroxylin A	38.72	0.23
	MOL002927	Skullcapflavone II	69.51	0.44
	MOL002928	Oroxylina	41.37	0.23
	MOL002932	Panicolin	76.26	0.29
	MOL002933	5,7,4′-Trihydroxy-8-methoxyflavone	36.56	0.27
	MOL002934	NEOBAICALEIN	104.34	0.44
	MOL002937	DIHYDROOROXYLIN	66.06	0.23
	MOL000358	beta-Sitosterol	36.91	0.75
	MOL000359	Sitosterol	36.91	0.75
	MOL000525	Norwogonin	39.40	0.21
	MOL000552	5,2′-Dihydroxy-6,7,8-trimethoxyflavone	31.71	0.35
	MOL000073	ent-Epicatechin	48.96	0.24
	MOL000449	Stigmasterol	43.83	0.76
	MOL001458	Coptisine	30.67	0.86
	MOL001490	Bis[(2S)-2-ethylhexyl] benzene-1,2-dicarboxylate	43.59	0.35
	MOL001506	Supraene	33.55	0.42
	MOL002879	Diop	43.59	0.39
	MOL002897	Epiberberine	43.09	0.78
	MOL008206	Moslosooflavone	44.09	0.25
	MOL010415	11,13-Eicosadienoic acid, methyl ester	39.28	0.23
	MOL012245	5,7,4′-Trihydroxy-6-methoxyflavanone	36.63	0.27
	MOL012246	5,7,4′-Trihydroxy-8-methoxyflavanone	74.24	0.26
	MOL012266	Rivularin	37.94	0.37
Zhizi (15 substances)	MOL001406	Crocetin	35.30	0.26
	MOL001663	(4aS,6aR,6aS,6bR,8aR,10R,12aR,14bS)-10-Hydroxy-2,2,6a,6b,9,9,12a-heptamethyl- 1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid	32.03	0.76
	MOL001941	Ammidin	34.55	0.22
	MOL004561	Sudan III	84.07	0.59
	MOL000098	Quercetin	46.43	0.28
	MOL000358	beta-Sitosterol	36.91	0.75
	MOL000422	Kaempferol	41.88	0.24
	MOL000449	Stigmasterol	43.83	0.76
	MOL001494	Mandenol	42.00	0.19
	MOL001506	Supraene	33.55	0.42
	MOL001942	Isoimperatorin	45.46	0.23
	MOL002883	Ethyl oleate (NF)	32.40	0.19
	MOL003095	5-Hydroxy-7-methoxy-2-(3,4,5-trimethoxyphenyl)chromone	51.96	0.41
	MOL007245	3-Methylkempferol	60.16	0.26
	MOL009038	GBGB	45.58	0.83

Table 3.

Targets of Huanglian Jiedu Decoction (229).

Total genes shared by Huanglian Jiedu Decoction
NOS2	PTGS1	KCNH2	ESR1	AR	SCN5A	F7	PTGS2	RXRA	ADRB2
HSP90AA1	PRSS1	NCOA2	PDE10A	CALM2	CHRM3	CHRM1	CHRM5	AMPK	HTR3A
ADRA2C	CHRM4	OPRD1	HTR2A	HTR2C	ADRA1B	BECLIN-1	SLC6A3	ADRA1D	SLC6A4
OPRM1	DRD1	DRD5	SLC6A2	CHRM2	NR3C2	GABRA1	GRIA2	ESR2	CDK2
PPARG	DPP4	AKR1B1	TOP2B	SERPIND1	MMP3	ACHE	MAOB	RELA	EGFR
AKT1	VEGFA	CCND1	BCL2	BCL2L1	FOS	CDKN1A	EIF6	BAX	CASP9
PLAU	LC3B	MMP2	MMP9	MAPK1	IL10	EGF	RB1	CD40LG	JUN
IL6	AHSA1	CASP3	TP53	ELK1	NFKBIA	POR	ODC1	CASP8	TOP1
SOD1	PRKCA	MMP1	HIF1A	STAT1	RUNX1T1	CDK1	HSPA5	ERBB2	HMOX1
CYP3A4	CYP1A2	CAV1	MYC	F3	GJA1	CYP1A1	ICAM1	IL1B	CCL2
SELE	VCAM1	PTGER3	CXCL8	PRKCB	BIRC5	DUOX2	NOS3	HSPB1	TGFB1
SULT1E1	MGAM	IL2	NR1I2	CYP1B1	CCNB1	PLAT	THBD	SERPINE1	COL1A1
IFNG	ALOX5	IL1A	MPO	TOP2A	NCF1	ABCG2	HAS2	GSTP1	NFE2L2
NQO1	PARP1	AHR	PSMD3	SLC2A4	COL3A1	CXCL11	CXCL2	DCAF5	NR1I3
CHEK2	INSR	CLDN4	PPARA	PPARD	HSF1	CRP	MTOR	CXCL10	CHUK
SPP1	RUNX2	RASSF1	E2F1	E2F2	ACPP	CTSD	IGFBP3	IGF2	IRF1
ERBB3	PON1	DIO1	PCOLCE	NPEPPS	HK2	NKX3-1	RASA1	GSTM1	GSTM2
CHEK1	NR3C3	NCOA1	IL4	SLC43A1	BEX2	MAGEA3	C1orf35	OCIAD1	MC3R
APLNR	C19orf10	PGR	ADH1C	ADRA2A	LTA4H	MAOA	CTRB1	ADRB1	RXRB
F2	Nr3c1	CACNA1S	PDE3A	KDR	CA2	ADRA2B	DRD3	NR3C1	FASN
FASLG	CYP19A1	MAPK14	GSK3B	BBC3	TEP1	PRKCD	FN1	MCL1	PKIA
FOSL1	FOSL2	CYCS	NFATC1	TDRD7	EGLN1	NOX5	FABP5	APOD	PYGM
CDK7	CYP2C9	LACTBL1	MAPK10	CCNA2	IKBKB	MAPK8	AKR1C3	SLPI

Table 4.

Targets of Huanglian Jiedu Decoction for the Treatment of MAFLD (85).

Targets of action of Huanglian Jiedu Decoction in the treatment of MAFLD
ADRB2	AHR	AHSA1	AMPK	AKT1	AR	BBC3	BCL2
BECLIN-1	BIRC5	CASP3	CAV1	CCL2	CD40LG	CDKN1A	CHUK
COL1A1	COL3A1	CRP	CTSD	CXCL10	CXCL8	CYP1A1	CYP1A2
CYP2C9	CYP3A4	DPP4	E2F1	E2F2	EGFR	EIF6	ESR1
F2	F3	FASLG	FASN	FN1	GSK3B	GSTM1	GSTP1
HIF1A	HMOX1	HSP90AA1	HSPA5	HSPB1	HTR2A	ICAM1	IGFBP3
IKBKB	IL10	IL1A	IL1B	IL6	INSR	JUN	LC3B
MAPK14	MAPK8	MMP2	MMP9	MTOR	NFE2L2	NOS2	NOS3
NR1I2	NR1I3	PARP1	PLAU	PON1	PPARA	PPARD	PPARG
PRKCD	PTGS2	RELA	SERPINE1	SLPI	SOD1	SPP1	STAT1
TGFB1	THBD	TP53	VCAM1	VEGFA

CytoScape network analysis showed that quercetin (MOL000098) had a Degree of Connectedness (Degree) of 145, BetweennessCentrality of 0.5784, and ClosenessCentrality of 0.5547, which predicted that quercetin was the main component, followed through kaempferol (MOL000422) with a connectivity of 57, a BetweennessCentrality of 0.0892, and a ClosenessCentrality of 0.3953, and baicalein (MOL000173) with a connectivity of 44, a BetweennessCentrality of 0.0689, and a ClosenessCentrality of 0.3798, which suggests that quercetin, kaempferol, and baicalein may be principal active ingredients HLJDD in treating MAFLD (Figure 2).

Figure 2.

Component-MAFLD target-pathway network diagram of Huanglian Jiedu decoction.

Triangles indicate herbs, ovals indicate active ingredients, rectangles indicate potential therapeutic targets:

HL: Coptidis Rhizoma (huanglian) HQ: Scutellaria Radix (huangqin)

HB: Phellodendri Chinensis Cortex (huangbo) ZZ: Gardenia (zhizi) A: (HL + HB), B: (HL + HQ), D1: (HQ + ZZ), C1: (HL + HB + HQ), C2: (HL + HB + ZZ), C3: (HB + HQ + ZZ)

In order to better predict mode of function for HLJDD, 85 target proteins jointly contained in HLJDD and MAFLD were inputed into STRING platform, and PPI network of HLJDD was mapped as shown in Figure 3(a), which contained 85 nodes and 1535 edges, and the targets with the top 25 degree values were shown in Figure 3(b).

Figure 3.

(a). Huanglian Jiedu Decoction-MAFLD target network. (b) Top 25 targets for PPI network median value ranking.

KEGG pathway enrichment analysis within DAVID6.8 database, a total of 133 pathways were screened at P < .05, and after deleting some pathways not related to this disease and sorting them according to the P value from smallest to largest, the top 20 were truncated to do the enrichment analysis of the bubble diagram. The results are shown in Figure 4, these targets mainly focused on NAFLD, MAPK/PI3K-AKt/AMPK signaling pathways, animal autophagy, insulin signaling pathway, etc. Based on P-value magnitude, enriched therapeutic target quantities, together with correlation with MAFLD, the AMPK signaling pathway was considered to be the key pathway for HLJDD to treat MAFLD.

Figure 4.

KEGG pathway enrichment analysis of Huanglian Jiedu decoction for MAFLD treatment.

The molecular docking dataset outcomes illustrated free binding energy for quercetin to AMPK was −6.4 kcal/mol and that of quercetin to mToR was −7.7 kcal/mol; the free binding energy of baicalein to AMPK was −6.9 kcal/mol and that of baicalein to mToR was −7.7 kcal/mol; the free binding energies of kaempferol and AMPK were −6.5 kcal/mol, and that of kaempferol and mToR were −7.9 kcal/mol. It is generally accepted that the stability of the bound conformation is negatively correlated with binding energy, whereby reduced binding energy increased likelihood that such protein will bind to the compound. Molecular docking simulation shows that quercetin, kaempferol, and hansenin successfully established docking with AMPK and mToR, confirming the reliability of the predicted results (Figure 5).

Figure 5.

Molecular docking diagram. (a) Quercetin with AMPK, green for quercetin. (b) Quercetin with mToR, green for quercetin. (c) Baicalein with AMPK, green for baicalein. (d). Baicalein with mToR, blue for baicalein. (e) Kaempferol with AMPK, green for kaempferol. (e) Kaempferol with AMPK, green for kaempferol. (f) Kaempferol with mToR, orange for kaempferol.

Effect of HLJDD on Lipid Level and Liver Function in MAFLD Rats

Compared with group N, the contents of ALT, AST, TC, TG, GLU, HDL, and LDL in serum of group M rats were significantly increased, after drug intervention, the levels of ALT, AST, TC, TG, GLU, HDL, and LDL were significantly decreased in Silybin group and each dose group of HLJDD (Figure 6).

Figure 6.

Effect of HLJDD on lipid level and liver function in MAFLD rats(χ±s, n = 8). Note: compared with group N: ^#P < .05, ^##P < .01, ^###P < .001, ^####P < .0001; compared with group M: **P < .01, ***P < .001, ****P < .0001; ns: no statistical significance.

HLJDD Alleviated Hepatic Lipopathology and Steatosis in MAFLD Rats

Compared with the normal group, the liver of rats in the model group turned yellow significantly with granular-like degeneration, and the liver of rats in Silybin group and each dose group of HLJDD was improved in different degree, as shown in Figure 7. HE staining of liver showed that hepatocytes within normal group were of normal size and morphology, with large and round nuclei located within center, and the inter-cell boundaries were clear and tightly arranged; while a large number of white round vacuoles of different sizes appeared within cytoplasm of hepatocytes within model group, and the boundaries were clear, the nuclei of the cells were pushed to the other side through the white vacuoles, and the cells became smaller and the boundaries of the inter-cells were blurred, and a small number of inflammatory cells infiltrated, presenting a mixed fatty cytopathy; significant improvement within in Silybin group and each dose group of HLJDD in comparison with model group. See Figure 8.

Figure 7.

Liver pictures of rats in each group.

Figure 8.

HE staining of liver in each group (400×).

Dataset outcomes for oil red O staining highlighted nuclei of hepatocytes within normal group were blue, and no red lipid droplets were seen within cells; a large number of red lipid droplets of different sizes were seen within hepatocytes of rats within model group. Compared with model group, the content of lipid droplets in hepatocytes of SF group, HL-L group, HL-M group, and HL-H group was significantly decreased. See Figure 9.

Figure 9.

Oil red O staining of rat liver in each group (400×).

Cell Experiment

As shown in Figure 10, the intracellular levels of TG and FFA in group M were significantly higher than those in group N (P < .05). HLJDD-containing serum and silybin-containing serum could reduce PA-induced increase of TG and FFA levels (P < .05), and HLJDD therapeutic effect was significantly attenuated after the application of AMPK inhibitor, suggesting that HLJDD and Silybin could both improve the intracellular lipid level in HepG2 cells and the therapeutic effect of HLJDD is closely related to AMPK.

Figure 10.

Levels of TG and FFA in HepG2 cells (n = 3). Note: Compared with group N: ^#P < .05, ^##P < .01; compared with group M: *P < .05; ns: no statistical significance.

Western blot results showed that HLJDD could up-regulate the protein expression of AMPK and Beclin-1, down-regulate the protein expression of mToR, as shown in Figure 11.

Figure 11.

Expression of AMPK and mToR-related proteins in HepG2 cells of each group.

Through observing the oil-red O staining results in Figure 12, it was found that there was no red lipid droplet development within group N, while a considerable population of red lipid droplets formed within group M cells. After the intervention of HLJDD and Silybin drug-containing serum, the color of intercellular lipid droplets became lighter and the number of lipid droplets was considerably reduced compared with that of group M. However, HLJDD therapeutic effect was considerably weakened after the application of inhibitors, indicating that HLJDD and Silybin could improve lipid accumulation in HepG2 cells and the therapeutic effect of HLJDD is closely related to AMPK.

Figure 12.

Lipid droplet changes in HepG2 cells (400×).

Discussion

Chinese Medicine Theory of “Lipid-Heat-Toxicity”

Traditional Chinese medicine points out that the addiction to fat, sweet and thick flavors, resulting within accumulation of cream and fat within liver, is a characteristic of the disease development of MAFLD. Therefore, MAFLD lipid metabolism disorders, oxidative stress and inflammatory imbalance, and other pathological processes could be attributed to the fat and sweet and thick flavors lead to the obstruction of lipid turbidity, and over time, it turns into heat and toxicity, damaging the liver, resulting in “lipid-heat-toxicity” interlinked. HLJDD is a reflective formula in clearing heat and removing toxins, with the efficacy of diarrhea and removing toxins, and it is mainly used for treating the evidence of tangible heat and toxins and San Jiao heat. Based upon traditional Chinese medicine theories, HLJDD conforms to treating MAFLD.

Autophagy Signaling Pathway AMPK-mToR and MAFLD

Autophagy is a metabolic process dependent on the lysosomal pathway, having pivotal parts within removing damaged or aged substances, structural remodeling, cell growth and development, maintenance of protein metabolic homeostasis, and stability of the intracellular environment.¹¹ The lipophagy theory, which has emerged in recent years, refers to a procedure whereby cells selectively identify and degrade lipid droplets of cholesteryl esters and triglycerides through the activation of autophagy-linked molecules, developing cholesterol, free fatty acids and glycerol, in order to circumvent the over-aggregation of lipids within the cells.¹² In fact, lipophagy is a specific form of autophagy. Overall, autophagy remains controlled through differing autophagy-related genes (ATGs), and approximately 40 autophagy-linked genes were identified. Such genes remain highly conserved within yeast and mammals, and are essential molecules implicated within different stages of autophagy. Including Akt, MAPK, P53, AMPK, mTOR, Beclin-1, LC3B, and others play a regulatory role in cellular autophagy.

Adenylate-activated protein kinase (AMPK), known as the “master switch of cellular energy metabolism,” is a major regulator for lipid and glucose metabolism, being one of the central regulators of cellular and body metabolism, which could effectively regulate cellular autophagy and inhibit the mToR, having pivotal parts in controlling cellular energy homeostasis. Mammalian target of rapamycin (mToR) complex is an evolutionarily conserved serine/threonine protein kinase complex, which is closely linked to the PI3K/AKT-related kinase family, and together with a variety of other kinase families, it constitutes a broad signaling pathway in cells.¹³ mToR plays a negative regulatory role in autophagy through regulating the biosynthesis of autophagy-related proteins and lysosomes.¹⁴ As a classical pathway related to cellular autophagy, AMPK/mToR is closely related to MAFLD, and MAFLD and various metabolic diseases caused through long-term overnutrition are associated with mToR over-activation. It has been reported that regulating the AMPK/mToR pathway could effectively reduce fatty liver degeneration.¹⁵ AMPK/mToR pathway may be an important target for autophagy-based prevention and treatment of MAFLD.¹⁶ The mToR signaling pathway serves as a signaling pathway that controls cell growth, metabolism, and autophagy. More and more experiments have demonstrated that the mToR signaling pathway is involved in adipogenesis.^17,18 The mToR complex contains two forms, mToRC1 and mToRC2. mToRC1 is mainly involved for controlling cellular development, survival, stress together with energy metabolism.¹⁹ Extremely sensitive to the specific inhibitor rapamycin, mToR complexes contain kinases that regulate lipid metabolism within their C-terminal region.²⁰ It has been shown that mToRC1 is able to activate lipolysis and autophagy, thereby affecting adipogenesis.²¹ Also, mToR downstream proteins are able to regulate the activity of multiple adipose transcription factors,²² the role of the mToR complex in regulating adipogenesis and lipid metabolism was established, whereas the activation of AMPK phosphorylation level could affect adenosine triphosphatase synthesis and metabolism to maintain the homeostatic balance of cellular energy,²³ targets and inhibits the activation of its downstream gene mToR molecule to activate cellular autophagy response,²⁴ lipid homeostatic balance was maintained.

Huanglian Jiedu Decoction and its Components

HLJDD and its components have some modulatory effects on the targets of action such as AMPK and mToR. Chen et al²⁵ found that berberine (the active ingredient of HLJDD) potentially lowers apoptosis in high glucose-stimulated rat retinal Müller cells through enhancing autophagy and AMPK/mToR signaling; Wang et al²⁶ found that berberine could induce autophagy within glioblastoma through affecting AMPK/mToR/ULK1 pathway; Sun et al²⁷ found that quercetin (the active ingredient of HLJDD) regulated glucose and lipid metabolism through GPRC6A/AMPK/mToR signaling pathway and attenuated osteoporosis within testisectomized mice; Cao et al²⁸ confirmed that quercetin shields oral mucosal keratinocytes against lipopolysaccharide-driven inflammatory toxicity through inhibiting AKT/AMPK/mToR pathway; Yuan et al²⁹ found that kaempferol (the active ingredient of HLJDD) orchestrated AMPK/mToR signaling pathway contributes prophylaxis influence against cerebral ischemia/reperfusion injury within rats through driving autophagy; Varshney et al³⁰ demonstrated that kaempferol modulates the cytoprotective influence by autophagy on palmitic acid-induced pancreatic β-cell necrosis via the AMPK/mToR signaling pathway. Chen et al³¹ demonstrated that baicalein (the active ingredient of HLJDD) induces rat mitochondrial autophagy via miR-30b and SIRT1/AMPK/mToR pathways to prevent Parkinson's disease.

Zhuoxi et al³² demonstrated that HLJDD has a certain intervention effect on atherosclerotic aortic plaque formation in ApoE-/- mice, and its mechanism may be related to the increase within levels of AMPK and PPARα involved in autophagy and apoptosis; Man³³ found that HLJDD could regulate the hepatic AMPK-ACC signaling pathway, promote the oxidation of fatty acids, regulate lipid metabolism, meanwhile, it could increase the expression and activity of AMPK in aortic tissues and protect endothelial function; Xiaohu³⁴ demonstrated that berberine (main-active ingredient in HLJDD) could ameliorate disorders of glucose and lipid metabolism in T2DM rats, with mechanistics potentially linked to regulation of the expression for LKB1-AMPK-TORC2 signaling pathway proteins within skeletal muscles and adipose tissues. Li³⁵ demonstrated experimentally that HLJDD could improve atherosclerosis through blocking PI3K-Akt-mToR pathway and increasing autophagy; Yu et al³⁶ found that HLJDD containing serum inhibited AKT/mToR/p70S6K signaling pathway together with overexpression of inflammatory factors IFN-γ and IL-18 in foam cells and promoted the autophagy and inhibition of inflammatory response in foam cells, this may be one of the mechanisms to prevent and control atherosclerosis; Liwang et al³⁷ confirmed that HLJDD may reduce CLP-induced liver injury in septic mice through inducing autophagy and inhibiting apoptosis; Li et al³⁸ found that HLJDD could effectively improve the damage of aortic tissues in SHR, while mechanism/s being possibly linked to thwarting AKT/mToR and Beclin-1 autophagy.

The above indicated that: AMPK-mToR signaling pathway is a classical pathway related to cellular autophagy and closely related to MAFLD; HLJDD is a representative formula for clearing heat and removing toxins, with anti-inflammatory and lipid-lowering effects, which is suitable for the theory of the pathogenesis of MAFLD “lipid-heat-toxicity”; HLJDD and its components are able to modulate AMPK-mToR signaling pathway. The results of molecular docking, animal experiments and cellular experiments also proved our hypothesis. Based on the above points, we believe that HLJDD could regulate the autophagy pathway AMPK-mToR signaling pathway, so as to achieve the effect of treating MAFLD.

Conclusion

This research project focuses on screening principal components of HLJDD and their targets from the perspective of network pharmacology to study mechanistics for HLJDD for treating MAFLD, providing a reliable theoretical basis for this study through combining the theory of “lipid-heat-toxicity” in traditional Chinese medicine, as well as carried out molecular docking and relevant experimental validation.

However, HLJDD has the characteristics of multi-components and multi-targets. The limitation of this study is that only the mechanism of HLJDD on MAFLD was verified, but the corresponding relationship between the components of HLJDD and its targets was not verified by in vivo and in vitro MAFLD models. In the future, we will use MAFLD animal and cell models to verify whether the components of HLJDD, such as berberine and quercetin, inhibit hepatic lipid deposition in MAFLD through lipophagy signaling. Although the results of this study are consistent with the advantages of multi-target, multi-pathway treatment of diseases in traditional Chinese medicine, there are many Chinese medicines and a variety of active ingredients in the prescription, so it is difficult to fully elucidate its therapeutic principle. However, this is precisely the advantage of traditional Chinese medicine, traditional Chinese medicine is never limited to the treatment of a single disease, but has a holistic view of syndrome differentiation and treatment, we should strengthen their research and application.

In conclusion, HLJDD could treat MAFLD through regulating the autophagy signaling pathway AMPK-mToR.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X241235604 - Supplemental material for Mechanism for Huanglian Jiedu Decoction-Based Therapy for MAFLD Analyzed Through Network Pharmacology and Experimental Verification

Supplemental material, sj-docx-1-npx-10.1177_1934578X241235604 for Mechanism for Huanglian Jiedu Decoction-Based Therapy for MAFLD Analyzed Through Network Pharmacology and Experimental Verification by Jixian Zheng, Anni Zheng, Sufei Song, Mengyu Lin and Tao Liu, Qiuling Xu in Natural Product Communications

Footnotes

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

This study was approved by the Ethics Committee of Hainan Medical University, approval number: HYLL-2021-009.

Funding

This study was supported through the Youth Qi Huang scholar project of state administration of traditional Chinese medicine, National Natural Science Foundation of China, Natural Science Foundation of Hainan Province (grant numbers 81760795, 821RC579).

Statement of Human and Animal Rights

All of the experimental procedures involving animals were conducted in accordance with the Animal Care Guidelines of Hainan Medical University, China and approved by the Ethics Committee of Hainan Medical University, approval number: HYLL-2021-009.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Supplemental Material

Supplemental material for this article is available online.

ORCID iD

Jixian Zheng

Appendix

Appendix Table A1.

The incoming blood components of HLJDD-containing serum.

	Name	Formula	RT [minutes]	mzCloud best match
1	2-Hydroxymyristic acid	C14 H28 O3	18.337	85.5
2	(+/-)9,10-Dihydroxy-12Z-octadecenoic acid	C18 H34 O4	18.333	85.1
3	4-Methylumbelliferone	C10 H8 O3	10.507	80.9
4	Propachlor ESA	C11 H15 N O4 S	14.725	78.3
5	2,4,6-Trihydroxy-2-(4-hydroxybenzyl)-1-benzofuran-3(2H)-one	C15 H12 O6	16.853	64.8
6	Geniposide	C17 H24 O10	10.509	64.3
7	Andrographolide	C20 H30 O5	19.358	64.1
8	1-Benzyl-8-(4-chloro-3-methylphenoxy)-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione	C21 H19 Cl N4 O3	10.515	58.7
9	Piceatannol	C14 H12 O4	13.821	45.9
10	trans-Clovamide	C18 H17 N O7	14.059	45.1
11	(+)-Syringaresinol	C22 H26 O8	10.684	39.4
12	Phenylacetylglycine	C10 H11 N O3	10.074	32.4
13	N-Isovalerylglycine	C7 H13 N O3	8.406	31.3
14	5-(4-Methylpiperazino)-4-nitrothiophene-2-sulfonamide	C9 H14 N4 O4 S2	12.922	30.5

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