Calculus bovis inhibits M2 tumor-associated macrophage polarization via Wnt/β-catenin pathway modulation to suppress liver cancer

BACKGROUND Calculus bovis(CB),used in traditional Chinese medicine,exhibits anti-tumor effects in various cancer models.It also constitutes an integral component of a compound formulation known as Pien Tze Huang,which is indicated for the treatment of liver cancer.However,its impact on the liver cancer tumor microenvironment,particularly on tumor-associated macrophages(TAMs),is not well understood.AIM To elucidate the anti-liver cancer effect of CB by inhibiting M2-TAM polarization via Wnt/β-catenin pathway modulation.METHODS This study identified the active components of CB using UPLC-Q-TOF-MS,evaluated its anti-neoplastic effects in a nude mouse model,and elucidated the underlying mechanisms via network pharmacology,transcriptomics,and molecular docking.In vitro assays were used to investigate the effects of CB-containing serum on HepG2 cells and M2-TAMs,and Wnt pathway modulation was validated by real-time reverse transcriptase-polymerase chain reaction and Western blot analysis.RESULTS This study identified 22 active components in CB,11 of which were detected in the bloodstream.Preclinical investigations have demonstrated the ability of CB to effectively inhibit liver tumor growth.An integrated approach employing network pharmacology,transcriptomics,and molecular docking implicated the Wnt signaling pathway as a target of the antineoplastic activity of CB by suppressing M2-TAM polarization.In vitro and in vivo experiments further confirmed that CB significantly hinders M2-TAM polarization and suppresses Wnt/β-catenin pathway activation.The inhibitory effect of CB on M2-TAMs was reversed when treated with the Wnt agonist SKL2001,confirming its pathway specificity.CONCLUSION This study demonstrated that CB mediates inhibition of M2-TAM polarization through the Wnt/β-catenin pathway,contributing to the suppression of liver cancer growth.

Promise and challenges of traditional Chinese medicine,specifically Calculus bovis,in liver cancer treatment

Liver cancer,one of the most common malignancies worldwide,ranks sixth in incidence and third in mortality.Liver cancer treatment options are diverse,inclu-ding surgical resection,liver transplantation,percutaneous ablation,transarterial chemoembolization,radiotherapy,chemotherapy,targeted therapy,immuno-therapy,and traditional Chinese medicine(TCM).A multidisciplinary team(MDT)is essential to customize treatment plans based on tumor staging,liver function,and performance status(PS),ensuring individualized patient care.Treatment decisions require a MDT to tailor strategies based on tumor staging,liver function,and PS,ensuring personalized care.The approval of new first-line and second-line drugs and the establishment of standard treatments based on immune checkpoint inhibitors have significantly expanded treatment options for advanced liver cancer,improving overall prognosis.However,many patients do not respond effectively to these treatments and ultimately succumb to the disease.Modern oncology treatments,while extending patient survival,often come with severe side effects,resistance,and damage to the body,negatively impacting quality of life.Huang et al's study published at World Journal of Gastroenterology rigorously validates the anticancer properties of Calculus bovis,enhancing our understanding of TCM and contributing to new liver cancer treatment strategies.For over 5000 years,TCM has been used in East Asian countries like China to treat various diseases,including liver conditions.Analysis of real-world clinical data suggests that for patients with advanced-stage tumors lacking effective treatments,integrated TCM therapies could provide significant breakthroughs.

Calculus bovis in hepatocellular carcinoma:Tumor molecular basis,Wnt/β-catenin pathway role,and protective mechanism

In this editorial,we comment on the recent article by Huang et al.The editorial focuses specifically on the molecular mechanisms of hepatocellular carcinoma(HCC),mechanism of Wnt/β-catenin pathway in HCC,and protective mechanism of Calculus bovis(CB)in HCC.Liver cancer is the fourth most common cause of cancer-related deaths globally.The most prevalent kind of primary liver cancer,HCC,is typically brought on by long-term viral infections(hepatitis B and C),non-alcoholic steatohepatitis,excessive alcohol consumption,and other conditions that can cause the liver to become chronically inflamed and cirrhotic.CB is a wellknown traditional remedy in China and Japan and has been used extensively to treat a variety of diseases,such as high fever,convulsions,and stroke.Disturbances in lipid metabolism,cholesterol metabolism,bile acid metabolism,alcohol metabolism,and xenobiotic detoxification lead to fatty liver disease and liver cirrhosis.Succinate,which is a tricarboxylic acid cycle intermediate,is vital to energy production and mitochondrial metabolism.It is also thought to be a signaling molecule in metabolism and in the development and spread of liver malignancies.The Wnt/β-catenin pathway is made up of a group of proteins that are essential for both adult tissue homeostasis and embryonic development.Cancer is frequently caused by the dysregulation of the Wnt/β-catenin signaling pathway.In HCC liver carcinogenesis,Wnt/β-catenin signaling is activated by the expression of downstream target genes.Communication between the liver and the gut exists via the portal vein,biliary tract,and systemic circulation.This"gutliver axis"controls intestinal physiology.One of the main factors contributing to the development,progression,and treatment resistance of HCC is the abnormal activation of the Wnt/β-Catenin signaling pathway.Therefore,understanding this pathway is essential to treating HCC.Eleven ingredients of CB,particularly oleanolic acid,ergosterol,and ursolic acid,have anti-primary liver cancer properties.Additionally,CB is important in the treatment of primary liver cancer through pathways linked to immune system function and apoptosis.CB also inhibits the proliferation of cancer stem cells and tumor cells and controls the tumor microenvironment.In the future,clinicians may be able to recommend one of many potential new drugs from CB ingredients to treat HCC expression,development,and progress.

Anti-tumor efficacy of Calculus bovis: Suppressing liver cancer by targeting tumor-associated macrophages

Despite significant advances in our understanding of the molecular pathogenesis of liver cancer and the availability of novel pharmacotherapies,liver cancer remains the fourth leading cause of cancer-related mortality worldwide.Tumor relapse,resistance to current anti-cancer drugs,metastasis,and organ toxicity are the major challenges that prevent considerable improvements in patient survival and quality of life.Calculus bovis(CB),an ancient Chinese medicinal drug,has been used to treat various pathologies,including stroke,convulsion,epilepsy,pain,and cancer.In this editorial,we discuss the research findings recently published by Huang et al on the therapeutic effects of CB in inhibiting the development of liver cancer.Utilizing the comprehensive transcriptomic analyses,in vitro experiments,and in vivo studies,the authors demonstrated that CB treatment inhibits the tumor-promoting M2 phenotype of tumor-associated macrophages via downregulating Wnt pathway.While multiple studies have been performed to explore the molecular mechanisms regulated by CB,this study uniquely shows its role in modulating the M2 phenotype of macrophages present within the tumor microenvironment.This study opens new avenues of future investigations aimed at investigating this drug’s efficacy in various mouse models including the effects of combination therapy,and against drug-resistant tumors.

From traditional Chinese medicine formulations to effective anticancer agents:Insights from Calculus bovis

This editorial examines the therapeutic potential of traditional Chinese medicine(TCM)for aggressive cancers,particularly liver cancer.It highlights the study by Huang et al,which shows how Calculus bovis,a component of the TCM Pien Tze Huang,suppresses liver cancer by inhibiting M2 macrophage polarization via the Wnt/β-catenin pathway.This research emphasizes the importance of transitioning from effective TCM formulations to isolating active components and understanding their mechanisms.While the study provides valuable insights,it primarily focuses on the Wnt/β-catenin pathway and does not delve deeply into the mechanisms of individual components.Future research should aim to comprehensively study these components,explore their interactions,and validate findings through clinical trials.This approach will integrate traditional wisdom with modern scientific validation,advancing the development of innovative cancer treatments based on TCM formulations.

Effects of in vitro cultivated Calculus Bovis compound on pulmonary lesions in rabbits with schistosomiasis

AIM:To explore the interventional effects and mechanism of in vitro cultivated Calculus Bovis compound preparation(ICCBco) on pulmonary lesions in portal hypertensive rabbits with schistosomiasis. METHODS:The experimental group included 20 portal hypertensive rabbits with schistosomiasis treated by ICCBco.The control group included 20 portal hypertensive rabbits with schistosomiasis treated by praziquantel. The morphological changes of the pulmonary tissues were observed under light and electron microscopy.The expression of fibronectin(FN) and laminin(LN) in the lung tissues was analyzed by immunohistochemistry. RESULTS:Under light microscope,the alveolar exudation in the lung tissue was more frequently observed in the control group,while the alveolar space was fairly dry in the lung tissue of ICCBco group.Under electron microscope,more alveolar exudation in the lung tissue,and moremacrophages,alveolar angiotelectasis and the blurred three-tier structure of alveolar-capillary barrier could be seen in the control group.In ICCBco group,fibers within the alveolar interspace slightly increased in some lung regions,and the structure of typeⅠepithelium,basement membrane and endodermis was complete,and no obvious exudation from the alveolar space,and novascular congestion could be observed.There was a positive or strong positive expression of FN and LN in the lung tissue of the control group,while there was a negative or weak positive expression of FN and LN in ICCBco group. CONCLUSION:ICCBco can effectively prevent pulmonary complications in portal hypertensive rabbits with schistosomiasis by means of improving lung microcirculation and lowering the content of extracellular matrix.

The Protective Effects of In Vitro Cultivated Calculus Bovis on the Cerebral and Myocardial Cells in Hypoxic Mice

The protective effects of in vitro cultivated calculus bovis （ICCB） on the cerebral and myocardial cells in hypoxic mice and the mechanism were examined. In one group, mice were intragastrically （i.g.） given ICCB for 15 days and then they were subjected to acute cerebral ischemia by decapitation, and then the panting time was recorded. In the other group, 12 min after exposure to hypoxia, mice was administered the ICCB i.g. for 5 days, and then the blood serum and tissues of brain, heart, liver were harvested and examined for SOD, GSH-px and T-AOC activity and content of MDA. The tissues of brain and heart were observed electron-microscopically for ultrastructural changes. The corpus striatum and hippocampus of brain were collected and examined for content of dopamine （DA） and norepinephrine （NE）. The ultrastrural examination showed that the pathological change in brain and heart in the ICCB group was very slight, while abnormal changes in the control group were obviously more serious. ICCB significantly prolonged the panting time of the hypoxic mice （P〈0.001）, increased the activity of SOD, GSH-px, T-AOC in serum and tissues of brain, liver, heart and elevated the content of DA and NE. ICCB also pronouncedly reduced content of MDA in serum and tissues of brain, heart and liver. Significant differences in these parameters were noted between ICCB group and controls. It is concluded that ICCB can exert protective effect on the cells of brain and myocardium by enhancing the tolerance of the tissues to hypoxia and the body＇s ability to remove free radicals and regulating the neurotransmitters.

Niuhuang(Bovis Calculus)-Shexiang(Moschus)combination induces apoptosis and inhibits proliferation in hepatocellular carcinoma via PI3K/AKT/mTOR pathway

Objective To investigate the effects of Niuhuang(Bovis Calculus,BC)and Shexiang(Moschus)(BC-Moschus)on human hepatocellular carcinoma(HCC)cells SMMC-7721 and a nude mouse model of subcutaneous xenografts,and to explore its anti-HCC mechanism.Methods The BC-Moschus combination was applied to two liver cancer models in vivo and in vitro.SMMC-7721 was divided into the BC-Moschus group and the control group,and different doses(rude drug dosage 0.625,1.25,2.5,and 5 mg/m L)of BC-Moschus extract were used for the intervention.The proliferation ability of HCC cells was detected using the Cell Counting Kit-8(CCK-8)assay,and the migration ability was detected by a wound healing assay.A subcutaneous xenograft model was prepared using nude mice with human HCC.Specific pathogen-free-grade BALB/c nude mice(5-week-old)were randomly divided into the following groups(n=6 per group):control(0.9%physiological saline 0.2 m L/d),BC-Moschus[BC 45.5 mg/(kg·d)+Moschus 13 mg/(kg·d)],and cisplatin(DDP,intraperitoneal injection5 mg/kg per week)groups.All groups were administered for 14 d.The volume and mass of the subcutaneous xenografts in nude mice were observed.The expression levels of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway,apoptosis-associated factor p70 S6 Kinase(S6K),Bax,Bcl-2,caspase-3,and caspase-9 in nude mice subcutaneous xenografts were measured by real-time quantitative PCR(RT-qPCR)and Western blot.Terminal Deoxynucleotidy Transferase-Mediated d UTP NickEnd Labeling(TUNEL)was used for quantitative analysis of apoptotic cells.Results The CCK-8 assay demonstrated that the BC-Moschus combination inhibited HCC cell proliferation in a superior manner to the use of BC and Moschus alone,and the inhibition effect was dose-and time-dependent(P<0.01).The wound healing assay showed that the BC-Moschus combination inhibited HCC cell migration(P<0.01).In the subcutaneous xenograft model of nude mice with human HCC,we found that the tumor volume and weight of the BC-Moschus group were lower than those of the control group(P<0.01).The levels of the PI3K/AKT/m TOR signaling pathway and S6K protein in the BC-Moschus and DDP groups were significantly decreased(P<0.01).The expression level of the anti-apoptotic gene Bcl-2 was downregulated(P<0.05),and the expression of the pro-apoptotic gene Baxand apoptosis-related factors caspase-3 and caspase-9 were significantly upregulated(P<0.01).The TUNEL assays further confirmed that the combination of the BC-Moschuas could promote HCC(P<0.01).Conclusion The BC-Moschus combination inhibited the proliferation and migration ability of HCC cells SMMC-7721 and effectively inhibited the growth of subcutaneous xenografts in nude mice.The mechanism may be closely related to the downregulation of the PI3K/AKT/mTOR pathway,regulation of apoptosis-related protein caspase-3,caspase-9,Bcl-2,and Bax expression,and promotion of apoptosis.

Unveiling the anticancer effect of traditional Chinese herbal medicine

In this issue of World Journal of Gastroenterology,Huang et al reported that Calculus bovis(CB),a traditional Chinese herbal medicine,impedes the growth of liver cancers in vivo.Through further in vitro studies,they showed that CB suppressed the M2 polarization of tumor-associated macrophages by suppressing the Wnt signaling pathway,which consequently inhibited the growth of liver cancer.Although the effects of traditional Chinese herbal medicine are often not scientifically proven,Huang et al successfully identified the molecular mechanism involved in the anticancer effect of CB,and it is anticipated that the molecular mechanisms involved in the effects of other traditional Chinese herbal medicines will be scientifically elucidated,as demonstrated in this article.

Inhibition of M2 tumor-associated macrophages polarization by modulating the Wnt/β-catenin pathway as a possible liver cancer therapy method

The problem of liver cancer is becoming increasingly important due to the epi-demic of metabolic diseases and persistent high alcohol consumption.This deter-mines great attention to the development and improvement of methods for early diagnosis and treatment of liver cancer.Huang et al presented a study in the World Journal of Gastroenterology,in which they showed that the use of the traditional Chinese medicine Calculus bovis(CB)can suppress tumor growth in mice by inhibiting M2 tumor-associated macrophages(TAM)through modulating the activity of the Wnt/β-catenin pathway.The interaction of CB components with the Wnt/β-catenin pathway,M2 TAM polarization,and tumor dynamics were studied using network pharmacology,transcriptomics,and molecular docking.It is now generally accepted that the polarization of TAM and the differentiation of the functions of M1 and M2 phagocytes are of great importance for the progression of neoplasms.It is assumed that M2 TAM promote proliferation and migration of tumor cells.Attempts to medicinally influence the Wnt/β-catenin pathway in order to modulate phagocyte polarization now belong to one of the most promising areas of immunotherapy of oncological diseases.Undoubtedly,the work of the Chinese authors deserves attention and further development.

A Rapid and Effective HPLC Method for Identification of Bovis Calculus in Chinese Patent Medicines

Objective Due to the limited resource and the large demand,many kinds of Bovis Calculus(BC) including artificial Bovis Calculus(ABC),in vivo cultured Bovis Calculus(in vivo CBC),and in vitro cultured Bovis Calculus(in vitro CBC)were used in Chinese patent medicines(CPMs).Previous studies have shown that the chemical constituents of ABC and their properties were different from other BC.The two types of CBC with much higher price than ABC were approximately equivalent with natural Bovis Calculus in quality and clinical effect.The aim of the study is to establish a rapid and effective method for the identification of BC in CPMs.Methods An HPLC method with the higher specificity for analyzing bilirubin was established to distinguish ABC from other three kinds of BC by comparing the change of bilirubin content with the addition of EDTA-2Na as the extraction solvent and stabilizer.Results The bilirubin content in CPMs containing ABC was basically unchanged,while that in CPMs containing other kinds of BC showed significant difference.The proposed method was employed to analyze a variety of CPMs containing Bovis Calculus(CPMBCs) and proven to be universal.Conclusion An effective analytical method is established for the quality control of CPMBCs and further ensures the safety and efficacy of these drugs in clinical practice.

Calculus Bovis Sativus up-regulates hepatic protein 2(Mrp2) and Mrp4 in 17α-ethynylestradiol-induced cholestasis via a regulatory effect on ER signaling

OBJECTIVE:To investigate the pathway through which Calculus Bovis Sativus (CBS) up-regulates hepatic multidrug resistance-associated protein 2 (Mrp2) and Mrp4 in 17α-ethynylestradiol (EE)-induced cholestasis.METHODS:Five groups of rats were designed:control group,EE+ICI182780 group,EE group,EE+CBS 50 mg/kg group and EE + CBS 150 mg/kg group.CBS (50 and 150 mg.kg-1· d-1) was orally given to rats by gavage for five consecutive days in coadministration with EE.The levels of cholestasis biomarkers,alanine aminotransferase (ALT),aspartate aminotransferase (AST),alkaline phosphatase (ALP) and total bilirubin (TBIL) were determined by biochemical methods.The bile flow was measured.The histopathology of the liver tissue was evaluated.The expression of Mrp2,Mrp3,Mrp4,estrogen receptor α(ERα) and ERβ was determined by Western blotting.RESULTS:CBS markedly improved EE-induced cholestasis.EE exposure significantly reduced hepatic Mrp2 and Mrp4 expression compared with the control group.EE also dramatically up-regulated the expression of Mrp3.Compared to the EE group,CBS notably up-regulated hepatic Mrp2 and Mrp4 but failed to influence the Mrp3 level significantly.ICI182780,an ER antagonist,showed similar beneficial effects as CBS.Decreased expression of Mrp2 and Mrp4 caused by EE was also restored by IC1182780.Additionally,EE significantly induced hepatic ERα expression,which was reversed by ICI182780 or CBS (150 mg/kg) treatment,suggesting that CB5 exerted a moderate regulatory effect on ER signaling.CONCLUSION:CBS up-regulated hepatic Mrp2 and Mrp4 expression in EE-induced cholestasis,which might be associated with its regulation of ER signaling.

新仿牛黄的抗惊厥作用

牛黄为脊椎动物哺乳纲牛科牛BostaursdomesticusGmelin的胆结石。由于天然牛黄极为短缺,价格昂贵,因而许多学者都在进行人工牛黄的研究。五十年代,唐德煊首先进行了人工合成牛黄的研究并获得成功。后经朱颜进行的药理实验表明,人工牛黄对古柯碱和咖啡碱引起的小鼠惊厥有对抗作用,此点与天然牛黄的药理作用是一致的。1971年,卫生部组织了人工牛黄药理实验协作小组进行了实验,观察到人工牛黄对戊四氮引起的小鼠惊厥也有较好的对抗作用。同年年底。