Network pharmacology and animal experiments revealed the protective effects of Guilong prescription (归龙方) on chronic prostatitis and its possible mechanisms

doi:10.19852/j.cnki.jtcm.20240423.002

Abstract

Abstract:

OBJECTIVE: To investigate the protective effects of Guilong prescription (归龙方, GL) on chronic prostatitis (CP) and unravel the underlying mechanisms of its pharmacological effects.

METHODS: The composition of GL was determined via linear ion trap/electrostatic field orbital trap tandem high-resolution mass spectrometry, and the identified compounds were performed network pharmacological analysis to predict possible pathways of the effects of GL on CP. A CP rat model was established by carrageenan, and rats were randomly assigned into a Control group, Sham group, CP group, GL low dose (3.5 g/kg) group, GL medium dose (7 g/kg) group, and GL high dose (14 g/kg) group. Hematoxylin-eosin staining of the prostate, and prostate blood -perfusion measured by laser speckle contrast analysis were used to evaluate the efficacy of GL. Expression of intercellular cell adhesion molecule-1 (ICAM-1) and induce nitric oxide synthase (iNOS) were determined by immunohistochemistry, and the content of interferon-γ (IFN-γ), interleukin-1β (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), chemokine ligand 1 (CXCL1) and tumor necrosis factor-α (TNF-α) were determined by electro-chemiluminescence assays. The expression of p38 mitogen-activated protein kinase (p38 MAPK), phosphatidylinositol 3-kinase (PI3K), ribosome-associated complex-alpha serine/threonine-protein kinase (Akt), nuclear factor-κ-gene binding p65 (NF-κB p65), inhibitor of NF-κB-α (IκBα), glycogen synthase kinase-3β (GSK-3β), and their phosphorylated forms were tested by Western blot.

RESULTS: In GL, a total of 48 compounds were identified, including 14 flavonoids, 14 alkaloids, 11 carboxylic acids, 4 lactones, 2 glycosides, 2 terpenoids and 1 aldehyde. Network pharmacological analysis suggested that the mechanism of GL may be related to PI3K-Akt signaling pathway and cytokine expression. After treatment with GL, inflammatory pathological changes in the prostate of rats were significantly improved, and blood perfusion of the prostate was significantly decreased. GL reduced the expression of IFN-γ, CXCL1, TNF-α, IL-1β, iNOS, ICAM-1, p38 MAPK, p-p38 MAPK, PI3K, p-PI3K, NF-κB, p-NF-κB, IκBα, p-IκBα, GSK-3β, p-GSK-3β, p-Akt in CP rats, and increased the expression of IL-4 and IL-10 in CP rats.

CONCLUSION: The chemical compositions of GL were first identified. GL can improve pathological changes in the prostate and recover the prostate blood perfusion of CP rats. The possible mechanisms of GL on CP involve increasing the expression of anti-inflammatory cytokines IL-4 and IL-10, inhibiting pro-inflammatory cytokines TNF-α, IL-1β, and IFN-γ, and down regulating the expression of CXCL1, iNOS, and ICAM-1 via inhibiting PI3K-Akt and NF-κB signaling pathway.

Key words: Prostatitis, cytokines, anti-inflammatory agents, carrageenan, Guilong prescription, network pharmacology

ZHU Peixuan, SU Zeqi, FAN Qiongyin, ZHANG Cai, WANG Ting. Network pharmacology and animal experiments revealed the protective effects of Guilong prescription (归龙方) on chronic prostatitis and its possible mechanisms[J]. Journal of Traditional Chinese Medicine, 2025, 45(1): 89-99.

Figures/Tables 8

Figure 1 Possible mechanisms of GL in the treatment of CP based on network pharmacology A: common targets of compositions and disease; B: KEGG analysis of GL in the treatment of CP. GL: Guilong prescription; CP: chronic prostatitis; KEGG: Kyoto encyclopedia of genes and genomes.

Figure 2 GL alleviated the pathological characteristics of prostate in CP rats induced by carrageenan A: histopathological changes in different groups, HE staining of prostate at × 40 magnification, scale bar = 100 μm. Red circle: interstitial edema, blue circle: interstitial hemorrhage, green circle: interstitial foam-like macrophages and infiltration of interstitial inflammatory cells (mainly lymphocyte). B: general observation of prostate, scale bar = 1 cm. C: Laser speckle pattern of blood perfusion in the prostate. A1, B1, C1: Control group, treated only with animal drinking water. A2, B2, C2: Sham group, the prostate was injected with sterile saline and treated with animal drinking water. A3, B3, C3: CP group, the prostate was injected with carrageenan and treated with animal drinking water. A4, B4, C4: GL low dose (GL-L) group, the prostate was injected with carrageenan and treated with low dose (3.5 g/kg) of GL. A5, B5, C5: GL medium dose (GL-M) group, the prostate was injected with carrageenan and treated with medium dose (7 g/kg) of GL. A6, B6, C6: GL high dose (GL-H) group, the prostate was injected with carrageenan and treated with high dose (14 g/kg) of GL. GL: Guilong prescription; CP: chronic prostatitis; HE: hematoxylin-eosin.

Table 1 Statistical results of prostate injury score and prostate index in each group ( x - ?± s)

Group	n	Prostate injury score	Prostate index
Control	10	37.00±10.85	1.60±0.23
Sham	10	36.50±13.75	1.61±0.23
CP	10	135.50±26.71^a	1.98±0.25^a
GL-L	10	61.50±24.39^b	1.63±0.23^b
GL-M	10	45.50±23.39^b	1.66±0.24^c
GL-H	10	44.50±14.42^b	1.58±0.16^b

Table 2 Statistical results of inflammatory cytokine levels in each group (pg/mg, x - ?± s)

Group	n	IL-4	IL-10	TNF-α	IL-1β	IFN-γ	CXCL1
Control	8	1.55±0.59	1.21±0.55	0.93±0.49	1.33±0.54	0.34±0.30	4.89±2.90
Sham	8	0.72±0.17	0.72±0.22	0.70±0.30	1.05±0.25	0.73±0.61	3.64±2.28
CP	8	0.30±0.14^a	0.52±0.12^a	3.71±1.48^a	2.29±0.61^a	2.26±0.83^a	13.45±10.84^d
GL-L	8	0.75±0.27^b	0.80±0.26^c	1.73±0.80^b	1.14±0.32^b	0.93±0.25^b	4.11±2.46^b
GL-M	8	0.65±0.34^c	0.82±0.23^b	1.23±0.84^b	0.71±0.38^b	0.85±0.79^c	2.85±2.04^b
GL-H	8	0.47±0.22	0.51±0.21	1.90±0.98^c	1.31±0.71^c	1.21±0.86^c	6.39±4.19

Figure 3 GL down regulated the expression of proteins associated with inflammation in CP rats induced by carrageenan A: immunohistochemical staining of ICAM-1 in the prostate at × 40 magnification, scale bar = 50 μm; B: immunohistochemical staining of iNOS in the prostate at × 40 magnification, scale bar = 50 μm. A1, B1: Control group, treated only with animal drinking water. A2, B2: Sham group, the prostate was injected with sterile saline and treated with animal drinking water. A3, B3: CP group, the prostate was injected with carrageenan and treated with animal drinking water. A4, B4: GL low dose (GL-L) group, the prostate was injected with carrageenan and treated with low dose (3.5 g/kg) of GL. A5, B5: GL medium dose (GL-M) group, the prostate was injected with carrageenan and treated with medium dose (7 g/kg) of GL. A6, B6: GL high dose (GL-H) group, the prostate was injected with carrageenan and treated with high dose (14 g/kg) of GL. GL: Guilong prescription; CP: chronic prostatitis; ICAM-1: intercellular cell adhesion molecule-1; iNOS: induce nitric oxide synthase.

Table 3 Statistical results of ICAM-1 and iNOS expression in each group (pg/mg, x - ?± s)

Group	n	IOD/Area of ICAM-1	IOD/Area of iNOS
Control	5	0.20±0.02	0.18±0.02
Sham	5	0.21±0.02	0.21±0.04
CP	5	0.37±0.04^a	0.28±0.03^c
GL-L	5	0.21±0.03^b	0.21±0.04^d
GL-M	5	0.27±0.03^b	0.22±0.03^d
GL-H	5	0.22±0.03^b	0.20±0.03^b

Figure 4 GL inhibited PI3K-Akt and NF-κB pathway related proteins in the prostate of CP rats induced by carrageenan A: the protein expression levels of PI3K, p-PI3K, Akt, p-Akt, GSK-3β, p-GSK-3β, and β-actin were tested by Western blot. B: The protein expression levels of P38, p-P38, p65, p-p65, IκBα, p-IκBα, and β-actin were tested by Western blot. Control: treated only with animal drinking water. Sham: the prostate was injected with sterile saline and treated with animal drinking water. CP: the prostate was injected with carrageenan and treated with animal drinking water. GL-L: the prostate was injected with carrageenan and treated with low dose (3.5 g/kg) of GL. GL-M: the prostate was injected with carrageenan and treated with medium dose (7 g/kg) of GL. GL-H: the prostate was injected with carrageenan and treated with high dose (14 g/kg) of GL. GL: Guilong prescription; CP: chronic prostatitis; PI3K: phosphatidylinositol 3-kinase; Akt: ribosome-associated complex-alpha serine/threonine-protein kinase; GSK-3β: glycogen synthase kinase-3β; P38: p38 mitogen-activated protein kinase; p65: nuclear factor-κ-gene binding p65; IκBα: inhibitor of NF-κB-α.

Table 4 Statistical results of the relative expression levels of PI3K, p-PI3K, Akt, p-Akt, GSK-3β, p-GSK-3β, P38, p-P38, p65, p-p65, IκBα, p-IκBα ( x - ?± s)

Group	n	PI3K	p-PI3K	Akt	p-Akt	GSK-3β	p-GSK-3β	P38	p-P38	p65	p-p65	IκBα	p-IκBα
Control	6	0.54±0.22	0.28±0.04	0.51±0.08	0.24±0.07	1.02±0.23	0.31±0.04	0.59±0.21	1.36±0.09	0.97±0.18	0.22±0.04	0.56±0.11	0.16±0.04
Sham	6	0.55±0.09	0.29±0.07	0.55±0.10	0.30±0.06	1.18±0.22	0.35±0.09	0.64±0.21	1.61±0.09	1.08±0.23	0.26±0.07	0.58±0.21	0.25±0.08
CP	6	0.93±0.36^a	0.41±0.05^b	0.57±0.10	0.39±0.09^b	1.43±0.31^a	0.40±0.04^b	0.92±0.20^a	1.78±0.12^b	1.39±0.28^a	0.41±0.10^b	0.98±0.38^a	0.55±0.06^b
GL-L	6	0.64±0.33	0.28±0.05^c	0.54±0.11	0.37±0.10	1.02±0.13^d	0.27±0.07^c	0.62±0.22^d	1.45±0.05^c	0.97±0.23^d	0.20±0.08^c	0.74±0.32	0.29±0.05^c
GL-M	6	0.67±0.28	0.35±0.05	0.50±0.10	0.21±0.07^c	1.03±0.22^d	0.28±0.05^c	0.69±0.13^d	1.43±0.10^c	0.91±0.17^c	0.28±0.09^d	0.59±0.29	0.35±0.08^c
GL-H	6	0.58±0.41	0.33±0.06^d	0.47±0.09	0.24±0.07^c	1.00±0.16^d	0.38±0.15	0.71±0.38	1.63±0.08^d	0.99±0.15^d	0.40±0.22	0.62±0.24	0.46±0.08

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