Computational Systems Pharmacology, Molecular Docking and Experiments Reveal the Protective Mechanism of Li-Da-Qian Mixture in the Treatment of Glomerulonephritis

Figures & data

Figure 1 Twenty core compounds of four herbs from Li-Da-Qian mixture.

Table 1 Potential Targets of Li-Da-Qian Mixture Against Glomerulonephritis

No.	Gene	No.	Gene	No.	Gene
1	RXRA	41	PPARG	81	TGFB1
2	PTGS1	42	KCNH2	82	IL2
3	PTGS2	43	F10	83	PLAT
4	ADRB2	44	MMP3	84	THBD
5	AKR1B1	45	EGFR	85	SERPINE1
6	PLAU	46	AKT1	86	COL1A1
7	LTA4H	47	VEGFA	87	IFNG
8	CHRM3	48	CCND1	88	PTEN
9	HTR2A	49	FOS	89	IL1A
10	NOS2	50	MMP2	90	MPO
11	DPP4	51	MMP9	91	NQO1
12	HSP90AA1	52	MAPK1	92	PARP1
13	PIK3CG	53	IL10	93	COL3A1
14	PRSS1	54	EGF	94	CXCL11
15	F2	55	RB1	95	CXCL2
16	NOS3	56	CD40LG	96	INSR
17	RHO	57	JUN	97	PPARA
18	AR	58	IL6	98	PPARD
19	APOB	59	CDKN2A	99	CRP
20	HMGCR	60	ELK1	100	CXCL10
21	MTTP	61	NFKBIA	101	SPP1
22	PREP	62	ODC1	102	E2F1
23	CYP19A1	63	XDH	103	IRF1
24	NR3C1	64	SOD1	104	PON1
25	CYP2C19	65	MMP1	105	CDK4
26	EPAS1	66	STAT1	106	PCNA
27	ALOX5	67	HSPA5	107	IL4
28	HIF1A	68	HMOX1	108	FOSL1
29	PRKCA	69	CYP3A4	109	FOSL2
30	C5AR1	70	CYP1A2	110	MME
31	MAPK14	71	MYC	111	SLC5A2
32	CDK2	72	F3	112	SLC5A1
33	RELA	73	GJA1	113	TNF
34	BCL2	74	ICAM1
35	CDKN1A	75	IL1B
36	BAX	76	CCL2
37	CASP3	77	SELE
38	TP53	78	VCAM1
39	CASP8	79	CXCL8
40	FASLG	80	PRKCB

Figure 2 The GO enrichment analysis of genes of Li-Da-Qian mixture against glomerulonephritis, including three domains: biological process (A), cellular component (B), and molecular function (C). The enrichment ration, size and color of the dots represent the degree of GO enrichment analysis.

Table 2 KEGG Pathway Analysis of Li-Da-Qian Mixture in Treating Glomerulonephritis

Gene Set	Description	Size	Expect	Ratio	P Value	FDR
hsa05200	Pathways in cancer	524	7.8553	6.1105	0	0
hsa05163	Human cytomegalovirus infection	225	3.3730	8.3012	0	0
hsa05167	Kaposi sarcoma-associated herpesvirus infection	186	2.7883	9.3246	0	0
hsa05161	Hepatitis B	144	2.1587	13.897	0	0
hsa05418	Fluid shear stress and atherosclerosis	138	2.0688	11.601	0	0
hsa04668	TNF signaling pathway	110	1.6490	13.341	0	0
hsa05142	Chagas disease (American trypanosomiasis)	102	1.5291	13.08	0	0
hsa04933	AGE-RAGE signaling pathway in diabetic complications	99	1.4841	20.888	0	0
hsa05215	Prostate cancer	97	1.4541	13.754	0	0
hsa04657	IL-17 signaling pathway	93	1.3942	16.497	0	0
hsa05140	Leishmaniasis	74	1.1093	16.226	0	0
hsa05219	Bladder cancer	41	0.61463	26.032	0	0
hsa05222	Small cell lung cancer	92	1.3792	13.051	8.88E-16	2.23E-14
hsa04066	HIF-1 signaling pathway	100	1.4991	12.007	3.89E-15	9.05E-14
hsa05212	Pancreatic cancer	75	1.1243	14.231	8.66E-15	1.88E-13

Figure 3 The KEGG analysis of genes of Li-Da-Qian mixture against glomerulonephritis. Different colors represent different pathways. The size of red dots represents amounts of enriched genes.

Figure 4 Drug targets network of Li-Da-Qian mixture. The potential target network of Li-Da-Qian is constructed using 20 compounds and 244 potential targets. Lines stand for the interactions between compounds and target nodes. A stands for MOL001735, which belongs to Daji, Cheqiancao and Lizhicao. B1 stands for MOL000359, which belongs to Daji, Xiaoji and Cheqiancao. B2 stands for MOL000449, which belongs to Daji, Xiaoji and Cheqiancao.

Figure 5 (A) PPI network of Li-Da-Qian mixture-glomerulonephritis genes. The network (A) contains 113 nodes and 1967 edges. Core genes have a higher degree and are positively correlated with node size and color depth, including, IL-6, VEGFA, EGF, MAPK1, MMP9, TP53, TNF, and (B–D) MCODE analysis of PPI network in (A). Cluster 5 (B) includes 52 nodes and 1068 edges, Cluster 5 (C) includes 3 nodes and 3 edges and Cluster5 (D) includes 6 nodes and 7 edges.

Figure 6 Continue.

Figure 6 1. Virtual 3D docking of bioactive ingredients from Li-Da-Qian mixture for glomerulonephritis targets. The molecular docking of baicalein, luteolin and quercetin with MMP9 (A, E, H), MAPK1 (/, D, B) and VEGFA (G, F, C) is shown. 2. Virtual 2D docking of bioactive ingredients from Li-Da-Qian mixture for glomerulonephritis targets. The molecular docking of baicalein, luteolin and quercetin with MMP9 (A, E, H), MAPK1 (/, D, B) and VEGFA (G, F, C) is shown.

Table 3 Docking Results of Three Bioactive Ingredients from Li-Da-Qian Mixture for Glomerulonephritis Targets

Ingredients	Structure	Binding Energy/(kcal/mol)
		AKT1	IL6	VEGFA	TNF	TP53	EGF	MAPK1	MMP9
Quercetin		−6.4	−6.4	−8.2	−6.8	−6.5	−5.9	−8.3	−7.6
Baicalein		−6.8	/	−8.1	/	−6.7	−5.9	/	−8.9
luteolin		−6.2	−6.5	−8.2	−6.8	−6.6	−5.9	−8.3	−8.3

Figure 7 Baicalein treatment protected from LPS-induced acute kidney injury. (A) Serum biochemical levels in mice (n=5–6) treated with different doses of baicalein after 24h LPS administration. (B) The inflammatory factors in the renal cortex of LPS-induced acute kidney injury model with or without different doses of baicalein (n=5–6). (C) Representative images of hematoxylin and eosin staining (magnification: ×200, scalebar: 50μm) of kidneys after 24h LPS administration. **p < 0.01, ***p < 0.001 versus the control group, #p < 0.05 versus the LPS group.

Figure 8 Effects of baicalein and luteolin treatment for LPS-induced glomerulonephritis by Western blotting and qPCR. Cells were grown on 6-well or 12-well plates until 60–70% confluence, pretreated with baicalein or luteolin for 2h, and sequentially stimulated with LPS (20μg/mL) for another 24 h. Western blotting results for baicalein therapeutic effect (A), densitometric analysis (B) and qPCR results for baicalein therapeutic effect (C and D). Western blotting results for luteolin therapeutic effect (E), densitometric analysis (F) and qPCR results for luteolin therapeutic effect (G and H). Protein results are shown as mean ± SEM (n = 3) and mRNA results are displayed as mean ± SEM (n=6). *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 versus the LPS group.

Figure 9 Effects of baicalein and luteolin treatment for TGFβ1-induced renal fibrosis by Western blotting and qPCR. Cells were grown on 6-well or 12-well plates until 60–70% confluence, pretreated with baicalein or luteolin for 2h, and sequentially stimulated with TGFβ1 (10ng/mL) for another 24 h. Western blotting results for baicalein therapeutic effect (A), densitometric analysis (B) and qPCR results for baicalein therapeutic effect (C). Western blotting results for luteolin therapeutic effect (D), densitometric analysis (E) and qPCR results for luteolin therapeutic effect (F). Protein and mRNA results are displayed as mean ± SEM (n=3). *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 versus the TGFβ1 group.

Figure 10 Possible mechanisms of Li-Da-Qian mixture core compounds on glomerulonephritis.