Study on the mechanism of ischemic stroke treatment based on network pharmacology and Raman spectroscopy in the larval zebrafish model, Calculus Bovis as a case.

Calculus Bovis (C. bovis) is a precious traditional Chinese medicine of animal origin, and it is one of the traditional medicines for treating cerebral inflammatory diseases such as stroke. However, the pharmacological action of C. bovis on ischemic stroke (IS) and its mechanism are still unclear. The purpose of this study was to investigate the potential mechanism to treat IS. Chemical constituents of different varieties of C. bovis were analyzed and confirmed by HPLC-MS/MS technique. We constructed a component and corresponding target network and drug-disease target protein-protein interaction (PPI) network. GO and KEGG enrichment analysis were performed. The molecular docking of the main compound with the target protein. Subsequently, the potential mechanism of therapy for IS was verified in vivo by zebrafish model. We introduced Raman spectroscopy to detect changes in the biochemical composition of zebrafish. 13 active chemical constituents and 129 potential targets were selected. 122 KEGG signaling pathways were obtained. The binding energy of the main compounds is less than -4.5. The results of animal experiments showed that C. bovis could significantly improve Ponatinib-induced IS, decrease the aggregation degree of brain macrophages, reduce the number of macrophage migrations, and significantly increase the expression level of NR3C1. Raman information indicated that the biochemical composition in the brain of the Ponatinib-induced group shifted to the control group. The mechanism may be related to anti-inflammatory process and regulation of lipid metabolism. This study demonstrates that Raman spectroscopy has great potential as a drug evaluation tool in living larval zebrafish.

Calculus bovis inhibits M2 tumor-associated macrophage polarization via Wnt/ß-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/ß-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/ß-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/ß-catenin pathway, contributing to the suppression of liver cancer growth.

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.

Study on the acute and subchronic oral toxicity of Calculus Bovis Sativus in rats.

This study aims to assess the acute and subchronic toxicity of Calculus Bovis Sativus (CBS), which is an ideal substitute for natural Calculus Bovis. After conducting a test of acute toxicity with KM mice of both sexes, it was determined that oral CBS had a lethal dosage (LD50) of greater than 9.26 g/kg BW. For ninety days, Wistar rats were fed on CBS orally at dosages of 0, 167, 501, and 1503 mg/kg BW/day, respectively, as part of the subchronic investigation. A comparison of the controls with the 1503 mg/kg and 501 mg/kg dosage groups revealed significant differences in the hematological and serum biochemical parameters, such as RBC, HGB, MONO%, PLT, LYMPH% and GLU, TP, ALB, and Ca2+, were observed. However, values of the above parameters fell within our laboratory's normal range. In terms of body weight, food intake, urinalysis, clinical chemistry, and pathology, no other adverse effects were observed. After 90 days of exposure, the no observed adverse effect level (NOAEL) of CBS in rats was determined to be 1503 mg/kg BW/day.

Quality study of animal-derived traditional Chinese medicinal materials based on spectral technology: Calculus bovis as a case.

INTRODUCTION: Calculus bovis (C. bovis) is a typical traditional Chinese medicine (TCM) derived from animals, which has a remarkable curative effect and high price. OBJECTIVES: Rapid identification of C. bovis from different types was realized based on spectral technology, and a rapid quantitative analysis method for the main quality control indicator bilirubin was established. METHODS: We conducted a supervised and unsupervised pattern recognition study on 44 batches of different types of C. bovis by five spectral pretreatment methods. Three variable selection methods were used to extract the essential information, and the partial least squares regression (PLSR) quantitative model of bilirubin by near-infrared (NIR) spectroscopy was constructed. RESULTS: The partial least squares discriminant analysis (PLS-DA) model could achieve 100% accuracy in identifying different types of C. bovis. The R2 of the NIR quantitative model was 0.979, which is close to 1, and the root mean square error of calibration (RMSEC) was 2.3515, indicating the good prediction ability of the model. CONCLUSION: The study was carried out to further improve the basic data of quality control of C. bovis and help the high-quality development of TCM derived from animals.

Taurocholic Acid and Glycocholic Acid Inhibit Inflammation and Activate Farnesoid X Receptor Expression in LPS-Stimulated Zebrafish and Macrophages.

A hyperactive immune response can be observed in patients with bacterial or viral infection, which may lead to the overproduction of proinflammatory cytokines, or "cytokine storm", and a poor clinical outcome. Extensive research efforts have been devoted to the discovery of effective immune modulators, yet the therapeutic options are still very limited. Here, we focused on the clinically indicated anti-inflammatory natural product Calculus bovis and its related patent drug Babaodan to investigate the major active molecules in the medicinal mixture. Combined with high-resolution mass spectrometry, transgenic zebrafish-based phenotypic screening, and mouse macrophage models, taurochiolic acid (TCA) and glycoholic acid (GCA) were identified as two naturally derived anti-inflammatory agents with high efficacy and safety. Both bile acids significantly inhibited the lipopolysaccharide-induced macrophage recruitment and the secretion of proinflammatory cytokines/chemokines in in vivo and in vitro models. Further studies identified strongly increased expression of the farnesoid X receptor at both the mRNA and protein levels upon the administration of TCA or GCA, which may be essential for mediating the anti-inflammatory effects of the two bile acids. In conclusion, we identified TCA and GCA as two major anti-inflammatory compounds in Calculus bovis and Babaodan, which could be important quality markers for the future development of Calculus bovis, as well as promising lead compounds in the treatment of overactive immune responses.

Characterization of Calculus bovis by principal component analysis assisted qHNMR profiling to distinguish nefarious frauds.

A new approach is developed for the reliable classification of Calculus bovis along with the identification of willfully contaminated C. bovis species and the quantification of unclaimed adulterants. Guided by a principal component analysis, NMR data mining achieved a near-holistic chemical characterization of three types of authenticated C. bovis, including natural C. bovis (NCB), in vitro cultured C. bovis (Ivt-CCB), and artificial C. bovis (ACB). In addition, species-specific markers used for quality evaluation and species classification were confirmed. That is, the content of taurine in NCB is near negligible, while choline and hyodeoxycholic acid are characteristic for identifying Ivt-CCB and ACB, respectively. Besides, the peak shapes and chemical shifts of H2-25 of glycocholic acid could assist in the recognition of the origins of C. bovis. Based on these discoveries, a set of commercial NCB samples, macroscopically identified as problematic species, was examined with deliberately added sugars and outliers discovered. Absolute quantification of the identified sugars was realized by qHNMR using a single, nonidentical internal calibrant (IC). This study represents the first systematic study of C. bovis metabolomics via an NMR-driven methodology, which advances the toolbox for quality control of TCM and provides a more definitive reference point for future chemical and biological studies of C. bovis as a valuable materia medica.

Calculus Bovis Sativus alleviates estrogen cholestasis-induced gut and liver injury in rats by regulating inflammation, oxidative stress, apoptosis, and bile acid profiles.

ETHNOPHARMACOLOGICAL RELEVANCE: Natural Calculus Bovis (NCB) is a traditional Chinese medicine used for anti-inflammation, treating fever, pain, sedation, and recovering hepatobiliary function. Calculus Bovis Sativus (CBS), produced from in vitro artificial cultivation by bioengineering techniques, acts as an ideal substitute for NCB when treating various diseases. AIM OF THE STUDY: Gut-liver injury is an important pathological feature of several cholestatic liver diseases, including estrogen-induced cholestasis (EIC). The strong link between cholestatic liver injury and intestinal damage emphasizes the need of considering gut-liver integrity during treatment. The purpose of this study is to look into the pharmacological activities of CBS on EIC-induced gut and liver damage. MATERIALS AND METHODS: EIC-induced cholestatic rats were given oral gavage daily for five days with or without CBS (150 mg/kg). The liver/body weight, serum biochemistry, and tissue histopathology were then evaluated. Quantitative real-time PCR, Western blot analyses, and immunofluorescence were used to determine the gene expression associated with pathological alterations of the liver and intestine in EIC-induced cholestatic rats. Bile acid profiles within enterohepatic circulation were detected by liquid chromatography-mass spectrometry. RESULTS: CBS significantly reduced relative liver weight, restored serum biochemistry levels, and improved the hepatic and intestinal pathological damage in EIC model rats. CBS reduced EIC-induced hepatic inflammation by inactivation of the NF-κB signaling and inhibition of TNFα, IL-1ß, and IL-6 expression. CBS alleviated EIC-induced hepatic and intestinal oxidative stress by regulating Nrf2-GCLM/GCLC and Nrf2-HO-1 pathways, respectively. CBS treatment upregulated Bcl-2 and downregulated Bax and cleaved caspase3 to improve EIC-induced hepatic and intestinal cell apoptosis. Additionally, CBS reversed the disorders of bile acid profiles in the enterohepatic circulation by reducing bile acid accumulation in the liver and plasma and increasing bile excretion and intestinal reabsorption of bile acids. CONCLUSION: CBS alleviates EIC-induced hepatic and intestinal injury through regulating inflammation, oxidative stress, apoptosis, and bile acid profiles. These results suggest that CBS or drugs targeting the gut-liver axis may be effective therapeutic agents for cholestasis.

Comparative Review of Effects of Pien Tze Huang and AnGong NiuHuang Pill and their Potential on Treatment of Central Nervous System Diseases.

The ancient composite formulae Angong Niuhuang pill and Pien Tze Huang, which were used a few hundred years ago to treat febrile disease and inflammation, respectively, are found to exert effects benefiting other neurological diseases and conditions. This short review introduces the main constituents of the two formulae, looking into both the cumulative synergetic and possible individual effects of each herb or animal apcoien. In essence, the main effects of Angong Niuhuang pill include anti-inflammation, antioxidation, anti-cell death, anticonvulsion, antiedema, antipyretic, antithrombotic, antimicrobial (bacteria, viruses, fungi), neuroprotective effects, and cardiovascular protection. The main effects of Pien Tze Huang include anti-inflammation, antioxidation, anti-cell death, antithrombotic, antimicrobial, neuroprotective effects, and cardiovascular protection. Comparing both composites, similarities in the effects and part of the components are found, showing some pharmacological evidence. This review casts light on research on the effects of neuroprotective and cardiovascular protective mechanisms as well as treatment mechanisms for cerebral accidents from the integrative medicine perspective.

Exploring the pharmacological mechanism of calculus bovis in cerebral ischaemic stroke using a network pharmacology approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Calculus bovis is commonly used in traditional Chinese medicine for the treatment of cerebrovascular diseases given its roles in clearing away heat, detoxification and pain relief. Calculus bovis is used the treatment of cerebral ischaemia, liver and gallbladder diseases and various inflammatory conditions. However, the mechanism of action of calculus bovis in the treatment of ischaemic stroke is not well understood. AIM OF THE STUDY: In this study, the anti-inflammatory, antioxidative and antiapoptotic effects of calculus bovis on neurovascular units were studied, and the mechanism of action of calculus bovis on neurovascular units was also discussed. MATERIALS AND METHODS: Neurons, astrocytes, and endothelial cells were used to construct models of brain neurovascular units in vitro. The oxygen-glucose deprivation/reoxygenation and glucose (OGD/R) model was used to assess the effects of in vitro cultured calculus bovis on inflammatory factors, oxidative stress, and apoptosis. ZO-1, Occludin, Claudin-5, HIF-1, VEGF, PI3K, Akt, Bax, Bcl-2, and Caspase-3 expression was detected. RESULTS: In vitro cultured calculus bovis protects the blood-brain barrier; repairs tight junction proteins; increases ZO-1, Occludin and Claudin-5 protein expression; maintains TEER(transepithelial electrical resistance) values; repairs damaged endothelial cells; increases γ-GT activity; reduces LDH and inflammatory injury; and reduces TNF-α, LI-6, and IL-1ß levels. In vitro cultured calculus bovis reduces oxidative stress damage and NO and improves SOD activity. In vitro cultured calculus bovis protects neurons through antiapoptotic activities, including reductions in the apoptotic proteins Bax and Caspase-3, increases in Bcl-2 protein expression, and protection of brain neurovascular units through the HIF/VEGF and PI3K/Akt signalling pathways. CONCLUSION: In summary, the protective effect of calculus bovis on neurovascular units is achieved through antioxidative, anti-inflammatory and antiapoptotic effects. The mechanism of action of in vitro cultured calculus bovis in ischaemic stroke involves multiple targets and signalling pathways. The PI3K/Akt, HIF-1α and VEGF pathways effectively protect neurovascular units in the brain.

Comprehensive evaluation integrating omics strategy and machine learning algorithms for consistency of calculus bovis from different sources.

In the clinical application of Traditional Chinese Medicine (TCM) substitutes, the consistency evaluation of TCM substitutes from different sources is recognized as the main bottleneck. As the most widely used analytical method in TCM consistency evaluation, fingerprint similarity evaluation suffers from insufficient method sensitivity and poor conformity with the actual characteristics of TCM, which is difficult to adapt to the analytical needs of complex substance systems of TCM. This work aims to develop an effective and more accurate method for consistency evaluation using omics strategy and machine learning algorithms. The natural calculus bovis (NCB) were graded into three groups according to the similarity to in vitro cultured bovis (IVCB), and chemical markers between samples of each grade were screened out. Support vector machine (SVM) models with different kernels were then constructed by using the chemical markers as feature variables. The results showed that the classification accuracy of the SVM classifier of NCB and the consistency evaluation SVM model classifier was 95.74% and 100.0%, respectively. The approach demonstrated in the study presented a good analytical performance with higher sensitivity, accuracy for consistency evaluation of TCM.

Exploration of the Potential Mechanism of Calculus Bovis in Treatment of Primary Liver Cancer by Network Pharmacology.

AIM AND OBJECTIVE: Calculus Bovis (CB) has been employed to treat diseases for a long time. It has been identified to play significant anti-inflammatory and anti-tumor roles. However, the mechanism of treating primary liver cancer (PLC) remains to be revealed. This study aims to clarify the molecules and mechanisms of CB in treating PLC. MATERIALS AND METHODS: After oral bioavailability (OB) and drug-likeness (DL) screening, 15 small molecules were identified as the potential ingredients against PLC. Following this, related targets network constructions and pathways were applied to clarify the mechanism of CB in treating PLC. An in vitro experiment was carried out to identify the function of CB in treating PLC. RESULTS: Eleven compounds of CB were identified that play an anti-PLC role, including oleanolic acid, ergosterol, ursolic acid, etc. The potential targets which were observed include IL6, MAPK-8, VEGFA, Caspase-3, etc. Further analysis showed that the mechanism of CB in the treatment of PLC involved apoptosis-related pathways and immune-related pathways. CONCLUSION: In summary, the current study combines network pharmacology and in vitro experiments to reveal the mechanism of CB against PLC. We concluded that 11 ingredients of CB have an anti-PLC effect. Furthermore, CB plays a key role in treating PLC mainly by apoptosisrelated pathways and immune-related pathways. Our experiment verifies that CB promotes the apoptosis of SMMC-7721.

In vitro cultured calculus bovis attenuates cerebral ischaemia-reperfusion injury by inhibiting neuronal apoptosis and protecting mitochondrial function in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: In vitro cultured calculus bovis (ICCB), which is produced based on the formation mechanism of bovine gallstones, is used to replace the natural bezoar. It has been used in the clinic to treat brain diseases, including stroke, Alzheimer's disease and depression. AIM OF STUDY: ICCB is used to treat encephalopathy in the clinic. We explored the effects of ICCB on cerebral ischaemia-reperfusion injury (CIRI) and the potential associated mechanisms. MATERIALS AND METHODS: Rats were subjected to middle cerebral artery occlusion for 90 min, followed by 24 h of reperfusion, after being given different concentrations of ICCB once a day for 3 days. Subsequently, the neurological scores, brain oedema and volume of cerebral infarction were measured, and the histopathological changes in the cortex neurons were observed by haematoxylin and eosin staining (H&E). Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Ultrastructural changes in the mitochondria of the cortex were assessed by transmission electron microscopy (TEM). The apoptosis-related proteins Bax, Bcl-2, caspase-9, caspase-3, Mito-Cyt C and Cyto-Cyt C were detected by Western blotting. RESULTS: Compared with those in the control group, the neurological scores, the volumes of cerebral infarction, and the brain water contents were significantly decreased in the ICCB groups at doses of 50 and 100 mg/kg. The ICCB treatment effectively decreased the neuronal apoptosis resulting from the CIRI-induced neuron injury. In addition, the histopathological damage and the mitochondria ultrastructure injury were partially improved in the CIRI rats after ICCB treatment. Western blotting analysis indicated that ICCB significantly decreased the expression of Bax, caspase-9, caspase-3 and Cyto-Cyt C protein levels while increasing the expression of Bcl-2 and Mito-Cyt C protein levels. CONCLUSION: The ICCB protected against CIRI by suppressing the mitochondria-mediated apoptotic signalling pathway.

Calculus bovis: A review of the traditional usages, origin, chemistry, pharmacological activities and toxicology.

ETHNOPHARMACOLOGICAL RELEVANCE: Calculus bovis (C. bovis), a widespread known traditional animal drug in China and Japan, has been widely used for a long time to treat various diseases, including high fever, convulsion and stroke. The aim of the present paper is to comprehensively review knowledge about C. bovis in terms of traditional usages, origin, chemical constituents, pharmacological activities and toxicology to seek an applicable substitute for NCB and provide potential new strategies utilizing C. bovis. Additionally, directions and perspectives for future investigations regarding C. bovis are also discussed. MATERIALS AND METHODS: In this paper, the traditional usages, origin, chemical constituents, pharmacology, and toxicology of C. bovis are comprehensively and systematically summarized by searching scientific databases, including Web of Science, PubMed, ScienceDirect, Springer, CNKI, Baidu Scholar and others. Additionally, some classic books of Chinese herbal medicine, academic papers authored by individuals with MSc and PhD degrees, local government reports as well as the state of local drug standards are also retrieved. RESULTS: Currently, C. bovis mainly derives from four sources: natural Calculus bovis (NCB), Calculus bovis sativus (CBS), Cultured calculus bovis (CCB) and Calculus bovis artifactus (CBA). Owing to their different formation processes, the chemical constituents of the four kinds of C. bovis show certain differences. Additionally, over 44 chemical constituents have been isolated and identified from C. bovis, mainly including bile pigments, bile acids, cholesterols and amino acids. Further investigations have revealed a wide range of pharmacological effects of C. bovis, with effects on the nervous system, cardiovascular system, respiratory system, digestive system, immune system and others. Furthermore, NCB and CBA show hypotoxicity, but high concentrations of bilirubin can cause neurotoxicity and hearing impairment. Additionally, pharmacokinetic data for C. bovis are still lacking. CONCLUSION: CBS contains analogous types and amounts of constituents and exerts similar therapeutic effects to NCB. Thus, CBS might be used as a sustainable substitute for NCB. Furthermore, the configuration and concentration of bile acids and bilirubin in C. bovis are responsible for the difference in pharmacological effects in the four types C. bovis. Further studies should focus on the structure-function relationship of bile acids and bilirubin in C. bovis by employing pharmacokinetics.

Identification of Calculus Bovis and its mixed varieties by ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) combined with the principal component analysis (PCA) method.

The method of ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) was established and combined with principal component analysis (PCA) to identify natural Calculus Bovis, in vitro cultured Calculus Bovis and artificial Calculus Bovis. PCA, which was particularly powerful in dealing with multicollinearity and variables that outnumber the samples, was used to analyze the UHPLC-MS data of the processed samples, and potential markers were analyzed and described based on orthogonal partial least-squares discriminant analysis. According to the results in this study, the approach of combining UHPLC-QTOF-MS with PCA was proven to be credible and could be used to identify Calculus Bovis from in vitro cultured Calculus Bovis and artificial Calculus Bovis and to determine if there is Calculus Bovis in patented Chinese medicines that should contained Calculus Bovis medicinal materials.

[Mechanism of Calculus Bovis Sativus in inhibiting hepatocyte lipid deposition based on serum pharmacology].

The aim of this paper was to investigate the molecular mechanism of Calculus Bovis Sativus( CBS) in alleviating lipid accumulation in vitro by serum pharmacology. The CBS-containing serum of mice was obtained by serum pharmacology method to evaluate its effect on the proliferation of LO2 hepatocytes. The lipid reducing effects of CBS-containing serum through Nrf2 was evaluated by fructose-induced LO2 hepatocyte steatosis model,nuclear factor erythroid 2 related factor 2( Nrf2) agonist oltipraz combined intervention,cell oil red O staining and intracellular triglyceride( TG) content. The effects of CBS-containing serum on lipid peroxidation and hepatocytes apoptosis were evaluated by reactive oxygen species( ROS) and apoptosis assay,respectively. Real-time quantitative polymerase chain reaction( PCR) was used to detect the relative expression of lipid synthesis-related genes and apoptosis-related genes.RESULTS:: showed that CBS drug-containing serum had no significant effect on LO2 hepatocyte proliferation. As compared with the model group,CBS-containing serum could effectively reduce the formation of lipid droplets in fructose-induced LO2 hepatocytes,significantly reduce intracellular TG and ROS levels,and significantly reduce hepatocyte apoptosis rate( P < 0. 05). As compared with the model group,carbohydrate responsive element binding protein( ChREBP),sterol regulatory element binding protein-1 c( SREBP-1 c),fatty acid synthase( FAS),acetyl-CoA carboxylase 1( ACC1),stearoyl-CoA desaturase 1( SCD1),Bax and caspase-3 mRNA levels were significantly reduced in CBS drug-containing serum treatment group( P<0. 05). All of the above effects could be reversed by oltipraz.In conclusion,CBS-containing serum can significantly inhibit the fructose-induced LO2 liver fat deposition,and the mechanism may be related to reducing intracellular ROS level through the Nrf2 pathway and improving intracellular peroxidation state to reduce apoptosis.

[Study on the effect of in vitro cultured calculus bovis in perioperative period after H-UPPP operation].

Objective:TThe aim of this study is to investigate the effect of in vitro cultured Calculus Bovis on the inflammation of oropharynx and body in patients with OSA during the perioperative period of H-UPPP.Method:Eighty patients with OSA and H-UPPP indications were enrolled. The patients were divided into experimental group and control group by random number table, 40 cases in each group. The experimental group was given in vitro cultured Calculus Bovis, while the control group was not given bovine bezoar in vitro. The postoperative oropharyngeal pain, time to resume normal diet, local edema, concentration of IL-1ß, IL-8 and TNF-α in saliva, and concentration of IL-1ß, IL-8 and TNF-α in blood were compared between the two groups. Result:The pain of oropharynx in the experimental group was lighter than that in the control group on the 3rd, 5th and 7th day after operation (P<0.05), but there was no significant difference in the pain of oropharynx between the two groups on the 1st day after operation(P>0.05); the time of restoring normal diet in the experimental group was shorter than that in the control group (P<0.05); the edema of oropharynx in the experimental group was lighter than that in the control group on the 5th and 7th day after operation (P<0.05).The levels of IL-1ß, IL-8 and TNF-α in saliva were lower than those in control group on the 3rd, 5th and 7th day after operation (P<0.05), and the levels of IL-1ß, IL-8 and TNF-α in blood on the 5th and 7th day after operation were lower than those in control group (P<0.05).Conclusion:In vitro perioperative period of H-UPPP can improve the postoperative sore throat and local edema of oropharynx, shorten the time of normal diet and reduce the expression of related inflammatory factors in oropharynx and blood.

Calculus Bovis Sativus Improves Bile Acid Homeostasis via Farnesoid X Receptor-Mediated Signaling in Rats With Estrogen-Induced Cholestasis.

Cholestatic diseases are characterized by toxic bile acid (BA) accumulation, and abnormal BA composition, which subsequently lead to liver injury. Biochemical synthetic Calculus Bovis Sativus (CBS) is derived from natural Calculus Bovis, a traditional Chinese medicine, which has been used to treat hepatic diseases for thousands of years. Although it has been shown that CBS administration to 17α-ethinylestradiol (EE)-induced cholestatic rats improves bile flow and liver injury, the involved underlying mechanism is largely unknown. In this study, we showed that CBS administration to EE-induced cholestatic rats significantly decreased serum and hepatic BA levels and reversed hepatic BA composition. DNA microarray analysis suggested that the critical pathways enriched by CBS treatment were bile secretion and primary BA synthesis. These findings led us to focus on the effects of CBS on regulating BA homeostasis, including BA transport, synthesis and metabolism. CBS enhanced hepatic BA secretion by inducing efflux transporter expression and inhibiting uptake transporter expression. Moreover, CBS reduced BA synthesis by repressing the expression of BA synthetic enzymes, CYP7A1 and CYP8B1, and increased BA metabolism by inducing the expression of metabolic enzymes, CYP3A2, CYP2B10, and SULT2A1. Mechanistic studies indicated that CBS increased protein expression and nuclear translocation of hepatic and intestinal farnesoid X receptor (FXR) to regulate the expression of these transporters and enzymes. We further demonstrated that beneficial effects of CBS administration on EE-induced cholestatic rats were significantly blocked by guggulsterone, a FXR antagonist. Therefore, CBS improved BA homeostasis through FXR-mediated signaling in estrogen-induced cholestatic rats. Together, these findings suggested that CBS might be a novel and potentially effective drug for the treatment of cholestasis.

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 ERß 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 ICI182780. Additionally, EE significantly induced he- patic ERα expression, which was reversed by ICI182780 or CBS (150 mg/kg) treatment, suggesting that CBS 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.

Investigation of the synergistic effects of haloperidol combined with Calculus Bovis Sativus in treating MK-801-induced schizophrenia in rats.

Clinical studies that focused on treating schizophrenia showed that Calculus Bovis Sativus (CBS), a substitute of Calculus Bovis, when used in combination with haloperidol could significantly lower the dosage of haloperidol compared with treatment with haloperidol alone, whereas efficacy was maintained. The aim of this study was to investigate the synergetic anti-schizophrenia effects in rats using CBS in combination with haloperidol. An open field test was conducted to verify the pharmacodynamic effects of a combination treatment of CBS and haloperidol on MK-801-induced schizophrenic rats. Rat plasma concentrations of intragastric haloperidol and intravenous haloperidol were determined after oral administration of a single dose or 1-week of pretreatment with CBS (50 mg/kg). The pharmacodynamic data showed a significant decrease in locomotor activity and an increase in the percentage of the central distance when haloperidol was concomitantly administered with CBS compared with haloperidol administration alone. The AUC0-∞ and Cmax of haloperidol in the orally coadministered groups were significantly higher compared with the oral treatment with haloperidol alone. In conclusion, oral coadministration of CBS with haloperidol resulted in a synergistic effect in rats. The enhanced oral bioavailability of haloperidol when combined with CBS might be attributed to the interaction between them.