Network pharmacology and experimental study of modified Guizhi Fuling Pill in the treatment of benign prostatic hyperplasia
Benign prostatic hyperplasia (BPH) is referred to as benign prostatic hyperplasia (BPH). The clinical incidence group is mostly middle-aged and elderly men. According to statistics, the incidence rate of BPH increases with age, reaching 80% at the age of 80, and the data is still increasing year by year. The treatment methods for BPH include Western medicine and surgical treatment, but Western medicine therapy can cause damage to neurocognition and sexual function, while surgical treatment is accompanied by postoperative complications. Therefore, it is urgent to explore new treatment approaches for BPH.

Traditional Chinese medicine emphasizes a holistic approach and dialectical treatment, with rich experience and unique advantages in disease treatment. Guizhi Fuling Pill originated from Zhang Zhongjing, a medical saint in the Han Dynasty. In this study, the modified Guizhi Fuling Pill (MGFP) was composed of eight traditional Chinese medicines: Guizhi, Fuling, Taoren, Mudan Skin, Chishao, Wangbuliuxing, Wuyao, and Leech. It was the core formula extracted by the research team based on data mining for the treatment of BPH by Academician Wang Qi, a master of Chinese medicine. According to traditional Chinese medicine theory, the pathogenesis of BPH is “kidney qi deficiency and blood stasis obstruction”. MGFP is good at dispersing blood stasis, reducing symptoms and accumulation, and promoting water circulation, and has significant therapeutic effects in clinical use. However, the complex composition of traditional Chinese medicine formulas greatly hinders the study of its mechanism of action.
In recent years, network pharmacology has been widely used in the study of traditional Chinese medicine pharmacology. Its concept revolves around the effective drug treatment of diseases by targeting multiple molecules in the interconnected network, which coincides with the multi-component, multi-target, and multi pathway theory of traditional Chinese medicine. Based on this, this study uses network pharmacology and molecular docking techniques to explore the potential mechanism of action of MGFP on BPH, and establishes a BPH model rat to verify the target with the best binding energy, in order to provide data support for clinical treatment.

 

According to reports, the pathogenesis of BPH is mainly related to sex hormones, growth factors, inflammatory response, abnormal cell apoptosis, etc. Its main pathological features are the proliferation of prostate epithelial cells and stromal cells. MGFP, when used in combination with cold and warm, has the effects of promoting blood circulation, eliminating symptoms, softening, strengthening, and promoting diuresis. It can significantly improve BPH caused by kidney qi deficiency and blood stasis obstruction. Modern pharmacological research has shown that the ingredients of Guizhi Fuling Pill mainly include paeonol, paeoniflorin, cinnamaldehyde, porin, and amygdalin. It can reduce blood viscosity by improving hemorheological parameters and is generally used to treat diseases such as BPH caused by blood stasis (turbidity) accumulation. In addition, it was found that Wang Buliuxing can prolong the clotting time of blood stasis model rats and reduce their whole blood viscosity; The hirudin contained in leeches has anti coagulation and anti thrombotic effects; The volatile oil, the main component of the black medicine, can also release the lung qi and lower the bladder to alleviate the retention of urinary stones, thus facilitating urination. From this, it can be seen that the chemical components in MGFP can achieve the goal of treating BPH through certain pathways, reflecting the mechanism of action of traditional Chinese medicine compound with multiple components, targets, and pathways.
This study obtained 212 intersecting targets through Venn diagram, and then reverse matched the core pathways in KOBAS analysis to identify the core targets for MGFP treatment of BPH. Combined with degree ranking and literature, core targets closely related to the pathogenesis of BPH were obtained, including AKT1 BAX、BCL2、JUN、TGFB1、TNF、IL6、VEGFA、EGFR。 The above targets are mainly related to cell apoptosis, vascular growth, and inflammation. According to reports, AKT is a serine/threonine protein kinase. In the AKT subtype, AKT1 can inhibit apoptosis and promote cell growth and survival, participating in the development of BPH. VEGFA is a highly specific vascular endothelial cell mitogen closely related to the formation and maintenance of prostate neovascularization. Activated AKT phosphorylates eNOS to produce NO, promoting VEGFA induced endothelial cell migration, vasodilation, increased blood flow, and angiogenesis. IL-6 is a multifunctional cytokine with a wide range of functions. In prostate tissue, IL-6 promotes the proliferation of prostate basal epithelial cells through the autocrine pathway, causing an imbalance between prostate cell proliferation and apoptosis in the body, thereby inducing the occurrence of BPH.
This study found through GO functional annotation analysis that the biological processes involved in MGFP are mainly enriched in vascular growth related processes, oxidative stress related processes, apoptosis related processes, coagulation related processes, and immune related processes. Its molecular functions in regulating BPH are mainly enriched in transcription factor binding, nuclear receptor activity, protein kinase activity, cytokine receptor binding, adrenergic receptor activity, RNA polymerase II transcription factor binding, etc.
This study found through KEGG pathway enrichment analysis that MGFP has regulatory effects on AGE-RAGE signaling pathway, PI3K-AKT signaling pathway, cell apoptosis, IL-17 signaling pathway, Th17 cell differentiation, TNF signaling pathway, VEGF signaling pathway, MAPK signaling pathway, Ras signaling pathway, Rap1 signaling pathway, etc. Research has shown that AGERAGE can stimulate the production of pro-inflammatory cytokines, leading to excessive cell proliferation and further development of BPH. The activation of the PI3K-AKT signaling pathway leads to phosphorylation of pro apoptotic proteins, which in turn blocks their binding to anti apoptotic molecules BCL-2 and BCL-XL, resulting in reduced cell apoptosis in BPH. The IL-17 signaling pathway, Th17 cell differentiation, and TNF signaling pathway can all mediate immune inflammatory responses. It has been reported that infection stimulation causes BPH stromal cells to produce pro-inflammatory factors IL-6, IL-8, and TNF, thereby activating infiltrating CD4+T cells and promoting their differentiation in Th1 and Th17 effector cells by secreting IL12 and IL-23; Activated Th1 and Th17 lymphocytes produce pro-inflammatory cytokines, such as IL-17, which promote immune inflammatory responses and lead to prostate cell hyperplasia. VEGF is an angiogenic factor mediated by the PI3K-AKT signaling pathway, which can promote angiogenesis and cell proliferation in interstitial tissues. It also plays a key role in the development of BPH. Combined with the results of the core target and Figure 6, we speculate that there is a close relationship between the PI3K-AKT signaling pathway and the VEGF signaling pathway. The MAPK and Ras signaling pathways are involved in regulating the basic biological activities of cells, including proliferation, apoptosis, differentiation, and aging. The MAPK cascade is Ras dependent, and abnormal activation can cause an imbalance between cell proliferation and apoptosis in the body. In addition, it was found that activation of the Rap1 signaling pathway promotes cell proliferation and migration, leading to an increase in prostate volume. It is speculated that MGFP may affect inflammatory response, cell apoptosis, and proliferation by regulating multiple signaling pathways, thus achieving the goal of treating BPH.
This study found through molecular docking that the core components of MGFP, including quercetin, β – sitosterol, kaempferol, baicalein, and tannic acid, have binding energies of less than 0 with AKT1, BAX, BCL2, JUN, TGFB1, TNF, IL6, VEGFA, and EGFR, indicating that they can spontaneously bind to each other. However, the binding energies of quercetin, β – sitosterol, kaempferol, baicalein, and tannic acid with AKT1, BAX, JUN, TNF, and VEGFA are all less than -20929 J/mol, indicating strong binding ability. The docking result between BAX and β – sitosterol is the best. Research has shown that β – sitosterol is a plant sterol that interferes with various cellular signaling pathways, affecting processes such as cell cycle, apoptosis, proliferation, and angiogenesis. It is commonly used clinically in diseases such as heart disease, tuberculosis, and cervical cancer. During the process of cell apoptosis, the pro apoptotic BAX translocates to the mitochondria and integrates with the outer membrane of the mitochondria, inducing outer membrane permeabilization and releasing cytochrome c, leading to normal cell death. Sharmila et al. established a rat kidney cancer model induced by diethylnitrosamine and found that the expression of BAX protein was significantly increased in the β – sitosterol treatment group. In addition, a multicenter, placebo-controlled, double-blind study showed that β – sitosterol also has a significant improvement effect on BPH, but its mechanism of action is still unclear.
This study investigated the expression of BAX in prostate tissue by establishing a BPH rat model. The results showed that compared with the sham surgery group, the model group rats had glandular epithelial cell proliferation and significantly reduced glandular lumen, while the other treatment groups showed varying degrees of reduction in prostate wall thickness compared with the model group, suggesting that MGFP has a certain therapeutic effect on BPH model rats. The semi quantitative results of immunohistochemistry showed no significant difference in the expression of BAX in prostate tissue between the blank group and the sham surgery group; Compared with the sham surgery group, the expression of BAX in the prostate tissue of the model group rats was significantly reduced (P<0.01), indicating that BPH may cause abnormal cell apoptosis by inhibiting BAX expression in prostate tissue. Compared with the model group, the low, medium, and high dose groups of MGFP, finasteride group, and Longbishu capsule group all significantly increased the expression of BAX (P<0.01), which may be due to the regulation of mitochondrial dependent apoptosis pathway by MGFP, which increases the expression of BAX gene related to promoting cell apoptosis, inhibits cell proliferation, and has a dose-dependent effect.
In summary, MGFP may regulate targets such as AKT1, BAX, JUN, TNF, VEGFA, as well as PI3K-AKT and VEGF signaling pathways through core components such as quercetin, β – sitosterol, kaempferol, baicalein, and tannic acid, thereby regulating related biological processes such as prostate vascular growth, apoptosis, and oxidative stress, and exerting a therapeutic effect on BPH. The preliminary prediction of the optimal binding mode between the core component and key targets by molecular docking was β – sitosterol BAX, and the effect of MGFP on BAX was explored through animal experiments, providing a pharmacological basis for the widespread application of MGFP. Given the limitations of insufficient inclusion of chemical components in the database and the changes in the components of traditional Chinese medicine after processing, further in-depth research on the chemical components of MGFP can be conducted through fingerprint technology, which will help improve the reliability of network pharmacology prediction results.