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OBJECTIVE@#Jiedu Recipe (JR), a Chinese herbal remedy, has been shown to prolong overall survival time and decrease recurrence and metastasis rates in patients with hepatocellular carcinoma (HCC). This work investigated the mechanism of JR in HCC treatment.@*METHODS@#The chemical constituents of JR were detected using liquid chromatography-mass spectrometry. The potential anti-HCC mechanism of JR was screened using network pharmacology and messenger ribonucleic acid (mRNA) microarray chip assay, followed by experimental validation in human HCC cells (SMMC-7721 and Huh7) in vitro and a nude mouse subcutaneous transplantation model of HCC in vivo. HCC cell characteristics of proliferation, migration and invasion under hypoxic setting were investigated using thiazolyl blue tetrazolium bromide, wound healing and Transwell assays, respectively. Image-iT™ Hypoxia Reagent was added to reveal hypoxic conditions. Stem cell sphere formation assay was used to detect the stemness. Epithelial-mesenchymal transition (EMT) markers like E-cadherin, vimentin and α-smooth muscle actin, and pluripotent transcription factors including nanog homeobox, octamer-binding transcription factor 4, and sex-determining region Y box protein 2 were analyzed using Western blotting and real-time polymerase chain reaction. Western blot was performed to ascertain the anti-HCC effect of JR under hypoxia involving the Wnt/β-catenin pathway.@*RESULTS@#According to network pharmacology and mRNA microarray chip analysis, JR may potentially act on hypoxia and inhibit the Wnt/β-catenin pathway. In vitro and in vivo experiments showed that JR significantly decreased hypoxia, and suppressed HCC cell features of proliferation, migration and invasion; furthermore, the hypoxia-induced increases in EMT and stemness marker expression in HCC cells were inhibited by JR. Results based on the co-administration of JR and an agonist (LiCl) or inhibitor (IWR-1-endo) verified that JR suppressed HCC cancer stem-like properties under hypoxia by blocking the Wnt/β-catenin pathway.@*CONCLUSION@#JR exerts potent anti-HCC effects by inhibiting cancer stemness via abating the Wnt/β-catenin pathway under hypoxic conditions. Please cite this article as: Guo BJ, Ruan Y, Wang YJ, Xiao CL, Zhong ZP, Cheng BB, Du J, Li B, Gu W, Yin ZF. Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia. J Integr Med. 2023; 21(5): 474-486.
الموضوعات
Animals , Mice , Humans , Carcinoma, Hepatocellular/genetics , beta Catenin/pharmacology , Liver Neoplasms/genetics , Drugs, Chinese Herbal/therapeutic use , RNA, Messenger/therapeutic use , Cell Line, Tumor , Cell Proliferation , Cell Movement , Gene Expression Regulation, Neoplasticالملخص
Colorectal cancer (CRC) is the third most lethal cancer and leading cause of cancer mortality worldwide. A key driver of CRC development is colon inflammatory responses especially in patients with inflammatory bowl disease (IBD). It has been proved that Panax notoginseng saponins (PNS) have anti-inflammatory, anti-oxidant and anti-tumor effects. The chemopreventive and immunomodulatory functions of PNS on colitis-associated colorectal cancer (CAC) have not been evaluated.This present study was designed to study the potential protective effects of PNS on AOM/DSS-induced CAC mice to explore the possible mechanism of PNS against CAC. Our study showed that PNS significantly alleviated colitis severity and prevented the occurrence of CAC. Functional assays revealed that PNS relieved immunosuppression of Treg cells in the CAC microenvironment by inhibiting the expression of IDO1 mediated directly by signal transducer and activator of transcription 1 (STAT1) rather than phosphorylated STAT1. Ultimately, Rh1, one of the PNS metabolites, exhibited the best inhibitory effect on IDO1 enzyme activity. Our study showed that PNS exerted significant chemopreventive function and immunomodulatory properties on CAC. It could reduce macrophages accumulation and Treg cells differentiation to reshape the immune microenvironment of CAC. These findings provided a promising approach for CAC intervention.
الموضوعات
Animals , Humans , Mice , Colitis/drug therapy , Colitis-Associated Neoplasms/drug therapy , Macrophages , Panax notoginseng , Saponins/therapeutic use , Tumor Microenvironmentالملخص
ObjectiveA feedforward control model for dry granulation of polysaccharide components was established to guide the adjustment and optimization of critical process parameters (CPPs) in the design space, so as to reduce the impact of fluctuations in raw materials properties on the quality of medicines. MethodTaking Astragali Radix extract powder as the model drug, the design space of dry granulation CPPs was determined by Box-Behnken design. Astragali Radix mixed powder with different powder properties were prepared by mixture design, the variance inflation factor (VIF) was used to diagnose the multicollinearity of the powder properties, and principal component analysis (PCA) was used to extract the characteristic data of the model. Radial basis function neural network (RBFNN) was used to establish a feedforward control model for reflecting the relationship between the powder properties of polysaccharide components, dry granulation CPPs and one-time molding rate. ResultThe design space for dry granulation CPPs of polysaccharide components was 16-35 Hz for feeding speed, 10-23 Hz for roller speed, and 10-46 kg·cm-2 for roller pressure. The established RBFNN feedforward control model had a good predictive effect on the one-time molding rate of dry granulation of polysaccharide components, which could be used to guide the adjustment and optimization of CPPs in the design space, the relative error was 0.38%-6.73%, and the average relative error was 3.42%. ConclusionThe established feedforward control model can well reflect the relationship between the powder properties of the polysaccharide components, the dry granulation CPPs and the one-time molding rate of the granules, which can be used to guide the adjustment and optimization of CPPs in the design space, reduce the impact of material property fluctuation on product quality, and provide ideas for promoting the quality of traditional Chinese medicine from passive control to active control.
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Objective:To observe the improving effect of Danggui Shaoyaosan on diminished ovarian reserve (DOR) in rats triggered by Tripterygia wilfordii polyglycoside tablet combined with stress, and to explore the role of transforming growth factor-<italic>β</italic><sub>1 </sub>(TGF-<italic>β</italic><sub>1</sub>)/Smads signaling pathway in such improvement. Method:Forty-eight female SD rats with normal sexual cycle were selected and randomly divided into a normal group (<italic>n</italic>=8) and a modeling group (<italic>n</italic>=40), and the ones in the modeling group were given Tripterygium wilfordii polyglycoside tablets (50 mg·kg<sup>-1</sup>) combined with random stress for 15 d. After successful modeling, the rats were randomized into the model group, low-, medium-, and high-dose (3.96, 7.92, 15.84 g·kg<sup>-1</sup>) Danggui Shaoyaosan groups, and estradiol valerate group (0.09 mg·kg<sup>-1</sup>), with eight in each group. Under the premise of stress exposure, they were separately gavaged with the normal saline, low-, medium- and high-dose Danggui Shaoyaosan, and estradiol valerate for 15 successive days. The estrous cycle of rats in each group was observed daily. After intervention, the rats were sacrificed and the ovarian visceral index was calculated. The pathological changes in ovarian tissues were observed by hematoxylin eosin (HE) staining. The protein expression levels of TGF-<italic>β</italic><sub>1</sub> and TGF-<italic>β</italic><sub>1 </sub>receptor (TGF-<italic>β</italic><sub>1</sub>R) in the ovarian tissues of rats were measured by immunohistochemistry (IHC), and the mRNA expression levels of Smad2, Smad3, and Smad7 in the ovarian tissues by real-time polymerase chain reaction (Real-time PCR). Result:Compared with the normal group, the model group exhibited disordered estrus cycle (<italic>P</italic><0.05), reduced visceral index (<italic>P</italic><0.01), and down-regulated TGF-<italic>β</italic><sub>1</sub> and TGF-<italic>β</italic><sub>1</sub>R protein and Smad2 and Smad3 mRNA expression in the ovarian tissues (<italic>P</italic><0.01), and up-regulated Smad7 mRNA expression (<italic>P</italic><0.01). Compared with the model group, Danggui Shaoyaosan at the low, medium, and high doses and estradiol valerate improved the estrus cycle of rats to varying degrees (<italic>P</italic><0.05) and increased the visceral index, with better effects observed in the medium-group and high-dose Danggui Shaoyaosan groups (<italic>P</italic><0.05,<italic>P</italic><0.01). Besides, the protein expression levels of TGF-<italic>β</italic><sub>1</sub> and TGF-<italic>β</italic><sub>1</sub>R and the mRNA expression levels of Smad2 and Smad3 in the ovarian tissues were elevated to varying degrees (<italic>P</italic><0.01), and the Smad7 mRNA expression declined (<italic>P</italic><0.01). The improvements in TGF-<italic>β</italic><sub>1</sub> and TGF-<italic>β</italic><sub>1</sub>R protein expression of the medium-dose Danggui Shaoyaosan group and estradiol valerate group were more obvious. Conclusion:Danggui Shaoyaosan significantly improves ovarian reserve in DOR rats, which is closely related to the regulation of TGF-<italic>β</italic><sub>1</sub>/Smads signaling pathway.
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Objective:This paper constructs a generalized regression neural network (GRNN) model to predict the disintegration time of traditional Chinese medicine (TCM) tablets. Method:Taking Astragali Radix as a model drug, the mixed Astragali Radix powders with different powder properties were prepared by mixing Astragali Radix extract powders with microcrystalline cellulose and lactose, which were made to Astragali Radix tablets by direct compression method. The powder properties of mixed Astragali Radix powders and the disintegration time of Astragali Radix tablets were determined, respectively. The correlation between the original data was eliminated by principal component analysis (PCA). The principal component factors were used as the input layer of the GRNN model, and the disintegration time was used as the output layer for network training. Finally, the verification group data was used to predict the disintegration time, and the network prediction accuracy was calculated by comparing with the actual value. Result:Three principal component factors were obtained through PCA by analyzing the original nine variables that were correlated with each other (Hausner ratio, true density, tap density, compression degree, angle of repose, bulk density, porosity, water content and total dissolved solids), which reduced the complexity of the network. The prediction value of the disintegration time based on this prediction method was in good agreement with the actual value, the error of disintegration time was 0.01-1.34 min and the average relative error was 3.16%. Conclusion:Based on the GRNN mathematical model, the physical properties of Astragali Radix extract powders can be used to accurately predict the disintegration time of Astragali Radix tablets, which provides a reference for studying the disintegration time of TCM tablets.
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This paper aims to construct a Bayesian(BN) fault diagnosis model of traditional Chinese medicine dry granulation based on the failure model and effect analysis(FMEA), effectively control risk factors and ensure the quality of granules.Firstly, the risk ana-lysis of dry granulation process was carried out with FMEA, and the selected medium and high risk factors were taken as node variables to establish corresponding BN network with causality.According to the mathematical reasoning method of probability theory, the model was accurately inferred and verified by Netica, and the granule nonconformance was used as the evidence for reversed reasoning to determine the most likely cause of the failure that affected the granule quality.The BN fault diagnosis model of traditional Chinese medicine dry gra-nulation was established based on the medium and high risk factors of process, prescription and equipment screened out by FMEA, such as roller pressure, raw material viscosity, clearance between rollers in the paper.The fault diagnosis of traditional Chinese medicine dry granulation process was then carried out according to the model, and the posterior probability of each node under the premise of nonconforming granule quality was obtained.This method could provide strong support for operators to quickly eliminate faults and make decisions, so as to improve the efficiency and accuracy for fault diagnosis and prediction, with innovation in its application.
الموضوعات
Bayes Theorem , Medicine, Chinese Traditional , Probabilityالملخص
To discuss the characteristics of symptoms improvement based on the follow-up evaluation of Eustachian tube balloon dilation medium to long-term efficacy in patients with symptomatic Eustachian tube dysfunction (SETD). Patients from 2015 to 2017 were followed up after Eustachian tube balloon dilation (with the sense of aural fullness, or tinnitus and hearing ambiguity). All participants had been done ETDQ-7 before surgery and were re-evaluated with ETDQ-7 in follow-up. The improvement of overall and individual symptoms scores in ETDQ-7, the effects of gender and the difference of scores at different stages (12-18 months, 18-24 months and 24-30 months) after the operation were analyzed. There were 29 patients, including 16 males and 13 females, whose age ranged from 20 to 62 years old. The medium to long-term score of ETDQ-7 significantly declined after surgery (27.0±7.9 . 14.1±7.5, 0.05). Among all symptoms, symptoms like "blockage feeling in ear or being like under the water, constriction feeling" , "sound of blisters or explosions in the ear" decreased obviously (0.05). Comparing different stages after surgery, the scores of ETDQ-7 existed no difference (0.05). And the difference of gender showed no significant influence on surgery effects. The subjective symptoms of patients with Eustachian tube dysfunction diagnosed with SETD can be significantly improved in the medium to long-term follow-up after Eustachian tube balloon dilation, and the degree of improvement is not linearly related to the postoperative time.
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Objective:To carry out the risk assessment on the factors in the process of granulation fluidized bed of traditional Chinese medicine(TCM) by using failure model and effect analysis(FMEA) and Bayesian network(BN), in order to effectively control risk factors and improve product quality. Method:The risk analysis of the fluidized bed granulation process was carried out by FMEA and the selected medium risk and high risk factors were taken as the main control points, the corresponding BN was established. The sensitivity analysis was used to screen out the main risk factors affecting particle fluidity, particle size uniformity, solubility and product cleanliness, the occurrence probability of each risk factor was determined by the evidence of unqualified particle quality, finally, taking fluidized bed granulation process of Sanye tablets as an example, the FMEA and BN were combined into the risk assessment process to verify the effectiveness and reliability of the method. Result:Based on the middle and high risk points of fluidized bed process, particle size of raw materials, moisture content and hygroscopicity of raw materials, dosage, concentration and addition amount of binder, cleaning degree and integrity of collection bag, and nozzle position, which were selected by FMEA, a fluidized bed granulation risk network with causality was constructed. Among them, hygroscopicity of raw materials, concentration and addition amount of binder, inlet temperature and atomization pressure were high probability risk factors, and the probability of occurrence were 55%, 63%, 59%and 58%, respectively. According to the Bayesian risk relationship network which controlled Sanye tablets fluidized bed granulation analysis results showed that the P values of inlet temperature, atomization pressure and concentration of binder were 0.003 4, 0.032 6 and 0.041 8, respectively in the regression model of influencing factors and particle size uniformity, indicating that there was a significant correlation between the three factors and the particle quality, which was basically consistent with the conclusion obtained by FMEA-BN method. Conclusion:The combination of FMEA and BN for visualized risk assessment of fluidized bed granulation helps to effectively control the risk factors in the granulation process, reduce product quality risks and provide strong support for the improvement of granulation process of TCM.
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Objective:To investigate the compatible stability of Xingnaojing injection in combination with 9 common medicines, and to provide a reference for clinical application of this injection. Method:According to the clinical application, Xingnaojing injection was mixed with 9 common medicines and placed in the room under dark and light conditions for 6 h. The appearance of compatible solutions was observed, and the HPLC fingerprint was analyzed by similarity evaluation and principal component analysis(PCA). Result:There were no significant changes in the appearance of compatibility of Xingnaojing injection and 9 common medicines, including piracetam and sodium chloride injection, sodium chloride injection and others. The similarities of fingerprint among compatibility of Xingnaojing injection and 9 common medicines were >0.98 at 0 h of compatibility, 6 h of placement and 6 h of illumination. The results of PCA showed that 9 groups of compatible solutions were clustered into 2 categories, the compatibility of Xingnaojing injection and 8 groups including piracetam and sodium chloride injection clustered into one category, and the relative peak areas of the characteristic components of Xingnaojing injection did not change significantly after compatibility, the compatibility of Xingnaojing injection and Danshen Chuanxiongqin injection clustered into another category, the relative peak areas of some characteristic components of Xingnaojing injection increased after compatibility of 0 h and 6 h,and it was more obvious after 6 h of illumination. Conclusion:The compatibility of Xingnaojing injection and 8 common medicines including piracetam and sodium chloride injection has good stability, while the compatibility has stability problems after Xingnaojing injection mixed with Danshen Chuanxiongqin injection. It is suggested that clinical attention should be paid to their compatibility and rational combination of medicines.
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This paper constructs a prediction model of material attribute-tensile strength based on principal component analysis-radial basis neural network( PCA-RBF),in order to predict the formability of traditional Chinese medicine tablets. Firstly,design Expert8. 0 software was used to design the dosage of different types of extracts,the mixture of traditional Chinese medicine with different physical properties was obtained,the powder properties of each extract and the tensile strength of tablets were determined,the correlation of the original input layer data was eliminated by PCA,the new variables unrelated to each other were trained as the input data of RBF neural network,and the tensile strength of the tablets was predicted. The experimental results showed that the PCA-RBF model had a good predictive effect on the tensile strength of the tablet,the minimum relative error was 0. 25%,the maximum relative error was2. 21%,and the average error was 1. 35%,which had a high fitting degree and better network prediction accuracy. This study initially constructed a prediction model of material properties-tensile strength of Chinese herbal tablets based on PCA-RBF,which provided a reference for the establishment of effective quality control methods for traditional Chinese medicine preparations.
الموضوعات
Medicine, Chinese Traditional , Neural Networks, Computer , Powders , Tablets , Technology, Pharmaceutical , Tensile Strengthالملخص
Objective To analyze and compare the clinical features of fulminant type 1 diabetes mellitus (FT1DM). Methods The clinical data of 135 cases of FT1DM in recent 10 years were collected. The 135 patients included those admitted in the First Medical Center of Chinese PLA General Hospital and those reported in published papers with sample number ≥10 cases. Of which the clinical data of 20 adult patients admitted in Chinese PLA General Hospital were compared with that of 87 adult patients in non-special population diagnosed by other 6 domestic hospitals. All the patients were divided into youth group with age <18 years and adult group with age ≥18 years, the clinical features of the two groups were then compared. Last, by combination of consulted literature, the data of all the Chinese adult patients recruited in present study (include the perinatal patients) were compared with that of Japanese FT1DM patients. Results The clinical features of adult patients in Chinese PLA General Hospital were similar to that of adult patients in non-special population diagnosed by other 6 domestic hospitals: The age of onset ranged from 29.9 to 35.9 years, body mass index (BMI) was from 19.9 kg/m2 to 23.2 kg/m2, the course of disease ranged from 2 to 5 days. Severe hyperglycemia occurred at onset of disease, with the average HbA1c <7%. Adolescents accounted for only 13.3% of all FT1DM patients (18/135). Compared to the youth group, the adult group had more severe symptoms and more critical state of illness; the digestive tract symptoms such as nausea and vomiting were more common (P=0.026), blood glucose level at onset was higher (P=0.013), HbA1c was lower (P=0.002), acidosis was more serious (P=0.034), and serum alanine aminotransferase (ALT), creatinine and blood potassium levels increased more significantly, but the proportion of precursor infection in youth group was higher (P=0.028). In addition, no honeymoon period was found in the adult group, but 3 cases were found in the youth group (3/18, 16.7%). Compared with Japanese adult FT1DM cases, patients in China tended to be younger, lower rates of hyperglycemia, flu-like symptoms and blood glucose level, but digestive tract symptoms were more common at the same time. No significant differences existed between the two groups in BMI, course of disease, HbA1c, fasting C-peptide level, blood pH value, blood potassium level and the proportion of increased serum amylase. Conclusions No significant difference exists in clinical characteristics of adult FT1DM cases in non-special population among different regions in China. However, the symptoms are more severe and the condition is more critical in adult patients than in youth patients. Comparing the Chinese and Japanese FT1DM patients, some differences may exist in the age of onset, blood glucose level and clinical manifestations.
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Apelin is a novel endogenous active peptide. The aim of this study is to investigate whether apelin in the paraventricular nucleus (PVN) can improve the cardiac function in rats subjected to thoracic surgery trauma, and whether it is involved in the protective effect of electro-acupuncture (EA). Sprague-Dawley rats were randomly divided into non-stressed group (control), thoracic surgical trauma stressed group (trauma) and bilateral Neiguan EA applied on thoracic surgical trauma stressed group (trauma + EA-PC 6). The mRNA expressions of apelin receptor (APJR) and apelin in the PVN were detected by real time-PCR. The exogenous apelin-13 (6 mmol/L, 0.1 μL) was microinjected into the rat PVN in the thoracic trauma group, and the effects of apelin-13 on the blood pressure (BP), heart rate (HR) and the discharge of rostral ventrolateral medulla (RVLM) neurons were observed through the simultaneous recording technology by polygraph. The results showed that the APJR mRNA expression was significantly decreased in the rats of trauma group as compared with that in the control group (P < 0.05), and a decline trend of apelin mRNA expression was also observed. EA application at bilateral Neiguan acupoints partially recovered the decline of APJR and apelin mRNA expression by the treatment of thoracic trauma. Both mean arterial pressure and HR in the thoracic surgical trauma group were significantly increased by the microinjection of exogenous apelin-13 into the PVN (P < 0.05), and the single-unit discharge rate of RVLM neurons also had an increasing trend. These results suggest that apelin in the PVN can improve the cardiac function of thoracic surgical trauma rats, and may be involved in the protective effects of EA.
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Soy isoflavones exert a wide variety of biological activities, such as antioxidant, anti-inflammatory and anti-cancer properties. Nuclear factor erythroid 2-related factor 2 (Nrf2), a bZip transcription factor, plays a key role in soy isoflavones induced protection against oxidative stress and cancer. To obtain more effective isofavones, a series of 7,4'-bis-(3-amino-2-hydroxypropoxy), 7 or 4'-(3-amino-2-hydroxypropoxy) isoflavone derivatives have been synthesized as potential antitumor agents and Nrf2/ARE (antioxidant response element) activators. The cytotoxicity of these compounds in human cancer cell lines MDA-MB-231, HT-29, HCT116, HepG2 and 7402 was tested by MTT assay. In this study, the cytotoxicity of compound 3b exhibited highest cytotoxic activity and at the safety dose range, it also strongly up-regulated antioxidant response element (ARE)-luciferase reporter activity. In addition, compound 3b induced Nrf2 nuclear translocation and upregulated its downstream target genes NQO-1 and HO-1 at protein level. Taken together, our results suggest that compound 3b could be a potential agent for cancer themotherapy or cancer chemoprevention.
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Humans , Antineoplastic Agents, Phytogenic , Chemistry , Pharmacology , Cell Line, Tumor , Gene Expression , Isoflavones , Chemistry , Pharmacology , Molecular Structure , NF-E2-Related Factor 2 , Genetics , Metabolismالملخص
Chonghe gel originated from the Chinese ancient prescription, can be used for the treatment of diabetic foot. This experiment was to study the transdermal absorbability of paeoniflorin and osthole in Chonghe gel . Franz diffusing cells method was adopted for the in vitro model of rat belly skins. Paeoniflorin and osthole in the receiving liquid, skins and gel were determined by HPLC. The receiving liquid were screened, and Chonghe gel and Chonghe ointment were compared by transdermal absorbability. Result showed that ethanol-normal saline (2: 8) solution was the appropriate receiving liquid. The penetration rates of paeoniflorin and osthole were 78.07, 7.08 microg x cm(-2) x h(-1). respectively. In 24 h, the accumulated penetration rates were (31.51 +/- 1.33)%, (12.38 +/- 1.28)%, respectively. The retention rates of paeoniflorin and osthole in skin were (0.92 +/- 0.45)%, (4.81 +/- 1.03) %, respectively. The retention of osthole in skins was a drug reservoir. Transdermal behavior of effective constituents in Chonghe gel was more efficient than that in ointment. In vitro, the transdermal behavior of paeoniflorin in Chonghe gel was close to a Weibull process, while the behavior of osthole was close to Higuchi process.
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Animals , Male , Rats , Chemistry, Pharmaceutical , Drugs, Chinese Herbal , Chemistry , Metabolism , Ethanol , Chemistry , Gels , Rats, Wistar , Skin , Metabolism , Skin Absorptionالملخص
As an important link during the whole operation process of acupuncture, it is very necessary to launch quantity study closely related to acupuncture finger force in the acupuncture quantity study. After retrieval of related literatures on finger force during acupuncture in recent 20 years, it was found out that although some exploration on acupuncture finger force had been made, it was scattered and had no deep research, which pointed out it was a weak link in the acupuncture quantity study. So study of finger force should be paid attention to in acupuncture-moxibustion field, the level of theoretical and experimental research and development of measuring instrument on acupuncture finger force should be strengthened, the application of instrument should be expanded in teaching and scientific research areas, which could promote the modernization and internationalization of acupuncture and moxibustion better and faster.
الموضوعات
Animals , Humans , Acupuncture Therapy , Methods , Biomechanical Phenomenaالملخص
<p><b>OBJECTIVE</b>To investigate the ways testosterone influences the murine bone marrow-derived macrophages (BMMs) and how testosterone affects the function of BMMs after bound to their membrane surface.</p><p><b>METHODS</b>BMMs were cultured in vitro, their total RNA and proteins isolated, and the expression of intracellular androgen receptor (AR) detected through RT-PCR and Western blotting. The binding site of testosterone (T) to the membrane surface of BMMs was observed by confocal laser scanning microscopy after T-BSA-FITC incubation. Moreover, the intracellular Ca2+ was tested by Fura-2 method, and the influence of ionic currents on BMMs plasma membrane induced by testosterone was examined by the whole cell patch-clamp.</p><p><b>RESULTS</b>RT-PCR and Western blotting failed to detect intracellular ARs in BMMs, but confocal laser scanning microscopy showed testosterone to be bound to the membrane surface of BMMs by impermeable T-BSA-FITC, inducing a rapid rise in the intracellular free Ca2+ concentration ([Ca2+]i) of Fura-2 loaded BMMs, predominantly due to the influx of extracellular Ca2+ through Ni2+ -blockable Ca2+ channels in the plasma membrane. Similarly, the patch-clamp technique revealed T-induced calcium influx in BMMs.</p><p><b>CONCLUSION</b>It is reasonable to assume that the testosterone receptor exists on the plasma membranes, and testosterone act through unconventional plasma membrane receptors, induce Ca2+ influx and a rapid rise in the intracellular Ca2+ concentration, and influence the function of BMMs.</p>