Liuwei Dihuang Pills Enhance Osteogenic Differentiation in MC3T3-E1 Cells through the Activation of the Wnt/ß-Catenin Signaling Pathway.

OBJECTIVE: The therapeutic efficacy and molecular mechanisms of traditional Chinese medicines (TCMs), such as Liuwei Dihuang pills (LWDH pills), in treating osteoporosis (OP) remain an area of active research and interest in modern medicine. This study investigated the mechanistic underpinnings of LWDH pills in the treatment of OP based on network pharmacology, bioinformatics, and in vitro experiments. METHODS: The active ingredients and targets of LWDH pills were retrieved through the TCMSP database. OP-related targets were identified using the CTD, GeneCards, and DisGeNET databases. The STRING platform was employed to construct a protein-protein interaction (PPI) network, and core targets for LWDH pills in treating OP were identified. The GO functional and KEGG pathway enrichment analyses for potential targets were performed using the R package "clusterProfiler". A "drug-target" network diagram was created using Cytoscape 3.7.1 software. The viability of MC3T3-E1 cells was evaluated using the CCK-8 method after treatment with various concentrations (1.25%, 2.5%, 5%, and 10%) of LWDH pill-medicated serum for 24, 48, and 72 h. Following a 48 h treatment of MC3T3-E1 cells with LWDH pill-medicated serum, the protein levels of collagen Ⅰ, RUNX2, Wnt3, and ß-catenin were quantified using the Western blot analysis, and the activity of alkaline phosphatase (ALP) was measured. RESULTS: A total of 197 putative targets for LWDH pills for OP treatment were pinpointed, from which 20 core targets were singled out, including TP53, JUN, TNF, CTNNB1 (ß-catenin), and GSK3B. The putative targets were predominantly involved in signaling pathways such as the Wnt signaling pathway, the MAPK signaling pathway, and the PI3K-Akt signaling pathway. The intervention with LWDH pill-medicated serum for 24, 48, and 72 h did not result in any notable alterations in the cell viability of MC3T3-E1 cells relative to the control group (all p > 0.05). Significant upregulation in protein levels of collagen Ⅰ, RUNX2, Wnt3, and ß-catenin in MC3T3-E1 cells was observed in response to the treatment with 2.5%, 5%, and 10% of LWDH pill-medicated serum in comparison to that with the 10% rabbit serum group (all p < 0.05). Furthermore, the intervention with LWDH pill-medicated serum resulted in the formation of red calcified nodules in MC3T3-E1 cells, as indicated by ARS staining. CONCLUSIONS: LWDH pills may upregulate the Wnt/ß-catenin signaling pathway to elevate the expression of osteogenic differentiation proteins, including collagen Ⅰ and RUNX2, and to increase the ALP activity in MC3T3-E1 cells for the treatment of OP.

[Mechanism of Liuwei Dihuang Pills in treatment of mice with diminished ovarian reserve based on proteomics].

This study aims to investigate the efficacy and possible mechanism of Liuwei Dihuang Pills in the treatment of diminished ovarian reserve(DOR) by using proteomic techniques. Firstly, cyclophosphamide(60 mg·kg~(-1)) combined with busulfan(6 mg·kg~(-1)) was injected intraperitoneally to establish the mouse model of DOR. After drug injection, the mice were continuously observed and the success of modeling was evaluated by the disturbance of the estrous cycle. After successful modeling, the mice were administrated with the suspension of Liuwei Dihuang Pills by gavage for 28 days. At the end of the gavage, four female mice were selected and caged together with males at a ratio of 2∶1 for the determination of the pregnancy rate. Blood and ovary samples were collected from the remaining mice on the next day after the end of gavage. Hematoxylin-eosin(HE) staining and transmission electron microscopy(TEM) were then employed to observe the morphological and ultrastructural changes in the ovaries. The serum levels of hormones and oxidation indicators were measured by enzyme-linked immunosorbent assay. Quantitative proteomics techniques were used to compare the ovarian protein expression before and after modeling and before and after the intervention with Liuwei Dihuang Pills. The results showed that Liuwei Dihuang Pills regulated the estrous cycle of DOR mice, elevated the serum levels of hormones and anti-oxidation indicators, promoted follicle development, protected the mitochondrial morphology of ovarian granulosa cells, and increased the litter size and survival of DOR mice. Furthermore, Liuwei Dihuang Pills negatively regulated the expression of 12 differentially expressed proteins associated with DOR, which were mainly involved in lipid catabolism, inflammatory response, immune regulation, and coenzyme biosynthesis. These differentially expressed proteins were significantly enriched in sphingolipid metabolism, arachidonic acid metabolism, ribosomes, ferroptosis, and cGMP-PKG signaling pathway. In summary, the occurrence of DOR and the treatment of DOR with Liuwei Dihuang Pills are associated with multiple biological pathways, mainly including oxidative stress response, inflammatory response, and immune regulation. "Mitochondria-oxidative stress-apoptosis" is the key to the treatment of DOR by Liuwei Dihuang Pills. YY1 and CYP4F3 may be the key upstream targets that trigger mitochondrial dysfunction and ROS accumulation, and the metabolism of arachidonic acid is the main signaling pathway of drug action.

Mechanism of Liuwei Dihuang Pills in treatment of mice with diminished ovarian reserve based on proteomics / 中国中药杂志

This study aims to investigate the efficacy and possible mechanism of Liuwei Dihuang Pills in the treatment of diminished ovarian reserve(DOR) by using proteomic techniques. Firstly, cyclophosphamide(60 mg·kg~(-1)) combined with busulfan(6 mg·kg~(-1)) was injected intraperitoneally to establish the mouse model of DOR. After drug injection, the mice were continuously observed and the success of modeling was evaluated by the disturbance of the estrous cycle. After successful modeling, the mice were administrated with the suspension of Liuwei Dihuang Pills by gavage for 28 days. At the end of the gavage, four female mice were selected and caged together with males at a ratio of 2∶1 for the determination of the pregnancy rate. Blood and ovary samples were collected from the remaining mice on the next day after the end of gavage. Hematoxylin-eosin(HE) staining and transmission electron microscopy(TEM) were then employed to observe the morphological and ultrastructural changes in the ovaries. The serum levels of hormones and oxidation indicators were measured by enzyme-linked immunosorbent assay. Quantitative proteomics techniques were used to compare the ovarian protein expression before and after modeling and before and after the intervention with Liuwei Dihuang Pills. The results showed that Liuwei Dihuang Pills regulated the estrous cycle of DOR mice, elevated the serum levels of hormones and anti-oxidation indicators, promoted follicle development, protected the mitochondrial morphology of ovarian granulosa cells, and increased the litter size and survival of DOR mice. Furthermore, Liuwei Dihuang Pills negatively regulated the expression of 12 differentially expressed proteins associated with DOR, which were mainly involved in lipid catabolism, inflammatory response, immune regulation, and coenzyme biosynthesis. These differentially expressed proteins were significantly enriched in sphingolipid metabolism, arachidonic acid metabolism, ribosomes, ferroptosis, and cGMP-PKG signaling pathway. In summary, the occurrence of DOR and the treatment of DOR with Liuwei Dihuang Pills are associated with multiple biological pathways, mainly including oxidative stress response, inflammatory response, and immune regulation. "Mitochondria-oxidative stress-apoptosis" is the key to the treatment of DOR by Liuwei Dihuang Pills. YY1 and CYP4F3 may be the key upstream targets that trigger mitochondrial dysfunction and ROS accumulation, and the metabolism of arachidonic acid is the main signaling pathway of drug action.

[Liuwei Dihuang Pills alleviates postmenopausal osteoporosis and fatigue in rats by inhibiting the epigenetic regulatory molecule BRD4 pathway].

OBJECTIVE: To explore the role of epigenetic signal molecule bromodomain protein 4 (BRD4) in mediating the therapeutic effect of Liuwei Dihuang (LWDH) Pills on postmenopausal osteoporosis (PMOP) and fatigue. METHODS: Thirty rat models of PMOP induced by bilateral ovariectomy were randomized equally into two groups for treatment with normal saline (model group) or LWDH Pills (385.7 mg/kg), with another 15 sham-operated rats as the sham operation group. After 12 weeks of treatment, femoral samples were taken to determine the bone density and BRD4 protein expression. The weight-bearing exhaustive swimming time of the rat models was recorded, and serum levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured using enzyme-linked immunosorbent assay. In cultured primary osteoblasts the changes in the expressions of BRD4, MAPK and NF-κB proteins were detected by immunofluorescence staining following treatment with LWDH Pills. RESULTS: The rat models of PMOP showed significantly up-regulated expression of BRD4 protein in the femoral tissue (P < 0.05), which was obviously lowered by treatment with LWDH Pills. The rats treated with LWDH Pills also showed significant improvement of fatigue. Immunofluorescent staining of the osteoblasts showed that treatment with LWDH Pills significantly decreased the protein expressions of BRD4, MAPK and NF-κB. Analysis of the GSE56116 dataset revealed that that patients with kidney-yin deficiency had significantly higher BRD4 expression than those in the kidney-yang-deficiency group and non-kidney-deficiency group (P < 0.05). The upregulation of BRD4 expression involved multiple signaling pathways including neural ligand receptor response, cytoskeleton rearrangement, cytokine interaction, and granulocyte colony-stimulating factor chemotaxis pathways. CONCLUSION: LWDH can alleviate PMOP and fatigue by decreasing BRD4 signaling pathway, suggesting that potential of BRD4 as a promising therapeutic target for PMOP.

Liuwei Dihuang Pills Inhibit Podocyte Injury and Alleviate IgA Nephropathy by Directly Altering Mesangial Cell-Derived Exosome Function and Secretion.

Background: Immunoglobulin A nephropathy (IgAN) is the most common glomerular disease worldwide. Its pathological features include IgA immune complex deposition, accompanied by mesangial cell proliferation and mesangial matrix expansion. This study was conducted to investigate the effects of Liuwei Dihuang pills (LWDHW) on IgAN in mice and human podocytes, as well as to determine their underlying mechanisms of action. Methods: For in vitro experiments, podocytes were exposed to the human mesangial cell culture medium supernatant of glomerular cells treated with aggregated IgA1 (aIgA1) and LWDHW-containing serum. Cell viability and the proportion of positive cells were evaluated using CCK-8 and flow apoptosis kits, respectively. The cells were collected for western blot analysis. Twenty-four mice with IgAN induced by oral bovine serum albumin administration combined with tail vein injection of staphylococcal enterotoxin B were randomly divided into four groups of six mice each: untreated model group, model + LWDHW group, model + rapamycin group, and model + LWDHW + rapamycin group. The normal control group contained six mice. The red blood cell count in the urine, urine protein, blood urea nitrogen, serum creatinine, and IgA deposition were determined, and TUNEL and western blotting were performed in the mouse kidney tissues. Results: In vitro experiments showed that LWDHW promoted autophagy by regulating the PI3K/Akt/mTOR signalling pathway and improved the damage to podocytes caused by the aIgA1-treated mesangial cell supernatant. This study demonstrates the effectiveness of LWDHW for treating IgAN. In the animal experiments, LWDHW significantly reduced the urine red blood cell count, serum creatinine and urea nitrogen contents, and 24 h urinary protein function and improved IgA deposition in the kidney tissues, glomerular volume, glomerular cell proliferation and polysaccharide deposition, and glomerular cell apoptosis. The pills also reversed the changes in the LC3II/I ratio and p62 content in the kidney tissues. The combination of LWDHW and rapamycin showed stronger inhibitory effects compared to those of LWDHW or rapamycin alone. Conclusion: LWDHW may improve regulation of the PI3K-Akt-mTOR pathway and inhibit autophagy in podocytes, as well as alleviate IgA nephropathy by directly altering mesangial cell exosomes.

[Comparative proteomic profiling of hippocampi in mice treated with Jingui Shenqi Pills and Liuwei Dihuang Pills].

The effects of Jingui Shenqi Pills(Jingui) and Liuwei Dihuang Pills(Liuwei) which respectively tonify kidney Yang and kidney Yin on brain function have attracted great attention, while the differences of protein expression regulated by Jingui and Liuwei remain to be studied. This study explored the difference of protein expression profiles in the hippocampi of mice orally administrated with the two drugs for 7 days. The protein expression was quantified using LC-MS/MS. The results showed that among the 5 860 proteins tested, 151, 282 and 75 proteins responded to Jingui alone, Liuwei alone, and both drugs, respectively. The ratio of up-regulated proteins to down-regulated proteins was 1.627 in Jingui group while only 0.56 in Liuwei group. The proteins up-regulated by Jingui were mainly involved in membrane transport, synaptic vesicle cycle, serotonergic synapse, dopaminergic synapse and so on, suggesting that Jingui may play a role in promoting the transport of neurotransmitter in the nervous system. The proteins down-regulated by Liuwei were mainly involved in membrane transport, synapse, ion transport(potassium and sodium transport), neurotransmitter transport, innate and acquired immune responses, complement activation, inflammatory response, etc. In particular, Liuwei showed obvious down-regulation effect on the members of solute carrier(SLC) superfamily, which suggested that Liuwei had potential inhibitory effect on membrane excitation and transport. Finally, consistent results were obtained in the normal mouse and the mouse model with corticosterone-induced depressive-like behavior. This study provides an experimental basis for understanding the effect of Jingui and Liuwei on brain function from protein network.

Comparative proteomic profiling of hippocampi in mice treated with Jingui Shenqi Pills and Liuwei Dihuang Pills / 中国中药杂志

The effects of Jingui Shenqi Pills(Jingui) and Liuwei Dihuang Pills(Liuwei) which respectively tonify kidney Yang and kidney Yin on brain function have attracted great attention, while the differences of protein expression regulated by Jingui and Liuwei remain to be studied. This study explored the difference of protein expression profiles in the hippocampi of mice orally administrated with the two drugs for 7 days. The protein expression was quantified using LC-MS/MS. The results showed that among the 5 860 proteins tested, 151, 282 and 75 proteins responded to Jingui alone, Liuwei alone, and both drugs, respectively. The ratio of up-regulated proteins to down-regulated proteins was 1.627 in Jingui group while only 0.56 in Liuwei group. The proteins up-regulated by Jingui were mainly involved in membrane transport, synaptic vesicle cycle, serotonergic synapse, dopaminergic synapse and so on, suggesting that Jingui may play a role in promoting the transport of neurotransmitter in the nervous system. The proteins down-regulated by Liuwei were mainly involved in membrane transport, synapse, ion transport(potassium and sodium transport), neurotransmitter transport, innate and acquired immune responses, complement activation, inflammatory response, etc. In particular, Liuwei showed obvious down-regulation effect on the members of solute carrier(SLC) superfamily, which suggested that Liuwei had potential inhibitory effect on membrane excitation and transport. Finally, consistent results were obtained in the normal mouse and the mouse model with corticosterone-induced depressive-like behavior. This study provides an experimental basis for understanding the effect of Jingui and Liuwei on brain function from protein network.

[Evaluation and analysis of high-quality products of Liuwei Dihuang Pills].

Liuwei Dihuang Pills is a typical traditional Chinese medicine with "the same product made by many manufacturers". The quality and price of products made in various factories was different obviously. In this study, the quality differences of Liuwei Dihuang Pills were evaluated over multi-dimensions and throughout the whole production cycle involving raw materials, production process, quality control and post-marketing re-studies based on the "Chinese patent medicine evaluation standard with quality at the core" established earlier by our research group. The research results showed that the product quality of various manufacturers was significantly different, and the product quality was positively correlated with the market shares of enterprises, indicating that enterprises with more market shares paid more attention to product quality; and the sales determined the concern degree of enterprises on products, which was in line with general cognition. During the single-item evaluation of Liuwei Dihuang Pills, the score of raw material selection was relatively low, and the enterprises paid less attention to the initial raw materials. The whole production process was better, and the national compulsory implementation of "Production Quality Management Standard"(GMP) had a positive effect in improving product quality. Quality control could basically guarantee product quality. Intelligent manufacturing promoted by the government was beneficial to ensure product uniformity. The score rate of "quality evaluation" item was basically qualified, which indicated that the quality control level of Liuwei Dihuang Pills was acceptable as a whole, but there was still room for improvement. "Re-evaluation and Brand Construction" had the lowest scores, reflecting that enterprises did not pay enough attention to in-depth study and re-evaluation of "the same product made by many manufacturers". The evaluation results were in line with expectations, provided a reference example for the evaluation of high-quality Chinese patent medicine, a basis for the high-quality and good price of Chinese patent medicine, scientific data for improving medical insurance bidding, and thus facilitated promoting the healthy development of the traditional Chinese medicine industry.

Liuwei Dihuang Pills alleviate the polycystic ovary syndrome with improved insulin sensitivity through PI3K/Akt signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Polycystic ovary syndrome (PCOS) is a complex gynecological endocrine disease commonly occurred in women of childbearing age. The main hallmark of PCOS includes elevated androgen production and insulin resistance (IR). Liuwei Dihuang Pills (LWDH Pills), a commonly prescribed traditional Chinese medicine (TCM) is widely used as a tonic prescription to treat diabetes, female menopause syndrome and other symptoms with'Kidney-Yin' deficiency. It has been reported the effects LWDH pills on PI3K/Akt signaling pathway in T2DM treatment. Recent studies have also indicated that the treatment of menopausal syndrome may be associated with the ovarian sexual hormone levels regulated by LWDH pills to alleviate female infertility. However, its potential benefits on PCOS have not been fully elucidated. AIM OF THE STUDY: The primary aim of this study was to investigate the alterations of PI3K/Akt pathway in polycystic ovary syndrome-insulin resistance (PCOS-IR) progression induced by letrozole combined with high fat diet (HFD) and then to explore the detailed mechanism of LWDH Pills to alleviate PCOS. MATERIALS AND METHODS: The female Sprague-Dawley rats were continuously treated with letrozole (p.o administration at 1 mg kg-1·day-1) and HFD for 21 days to establish the PCOS-IR model. Concurrently, metformin (200 mg kg-1·day-1) or LWDH Pills was orally administrated (1.2 or 3.6 g kg-1·day-1) to intervene disease progression. The ovarian pathology was evaluated by HE (hematoxylin-eosin) staining. The serum sexual hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, testosterone, progesterone and fasting insulin (FINS) were determined by radioimmunoassay. The protein expressions of IRS-1, PI3Kp85α, Akt and FoxO1a were analyzed by western blotting, while the mRNA levels of follicle-stimulating hormone receptor (FSHR) and Cyp19a1 in ovarian tissue were measured by qPCR. RESULTS: The upregulated phosphorylation of IRS-1 (S307), down-regulated phosphorylation of PI3Kp85α, Akt, and FoxO1a were significantly reversed by LWDH Pills (3.6 g kg-1·day-1) in PCOS-IR rats with up-regulated mRNA levels of FSHR and Cyp19a1 in ovary. Also, the index of insulin resistance was gradually adjusted to normal by LWDH Pills. The serum levels of FSH, estradiol, progesterone levels were significantly raised while LH, testosterone were reduced. The ovarian polycystic changes were alleviated while the atresia follicles were reduced. CONCLUSION: LWDH Pills therapy obviously improved the ovarian polycystic pathogenesis and regained the development of follicles via upregulating Cyp19a1, alleviated insulin resistance through acting on PI3K/Akt signaling pathway. These findings have provided scientific evidence for LWDH Pills to treat PCOS.

Research progress on classical traditional Chinese medicine formula Liuwei Dihuang pills in the treatment of type 2 diabetes.

In traditional Chinese medicine (TCM), type 2 diabetes mellitus (T2DM) is regarded as Xiao Ke disease. Liuwei Dihuang pills (LWP), a classical TCM formula, with the function of nourishing kidney yin, has been used for treating Xiao Ke disease in clinic. In this review, we systematically highlighted recent evidence on LWP and T2DM data from clinical and animal studies, summarized the clinical application, pharmacological mechanism and the active compounds of LWP for the treatment of T2DM. This systematic review will provide an insightful understanding of TCM formulas, pharmacological mechanisms, medicinal-disease interactions, and will lay a foundation for the development of new drug therapy for T2DM.

Evaluation and analysis of high-quality products of Liuwei Dihuang Pills / 中国中药杂志

Liuwei Dihuang Pills is a typical traditional Chinese medicine with "the same product made by many manufacturers". The quality and price of products made in various factories was different obviously. In this study, the quality differences of Liuwei Dihuang Pills were evaluated over multi-dimensions and throughout the whole production cycle involving raw materials, production process, quality control and post-marketing re-studies based on the "Chinese patent medicine evaluation standard with quality at the core" established earlier by our research group. The research results showed that the product quality of various manufacturers was significantly different, and the product quality was positively correlated with the market shares of enterprises, indicating that enterprises with more market shares paid more attention to product quality; and the sales determined the concern degree of enterprises on products, which was in line with general cognition. During the single-item evaluation of Liuwei Dihuang Pills, the score of raw material selection was relatively low, and the enterprises paid less attention to the initial raw materials. The whole production process was better, and the national compulsory implementation of "Production Quality Management Standard"(GMP) had a positive effect in improving product quality. Quality control could basically guarantee product quality. Intelligent manufacturing promoted by the government was beneficial to ensure product uniformity. The score rate of "quality evaluation" item was basically qualified, which indicated that the quality control level of Liuwei Dihuang Pills was acceptable as a whole, but there was still room for improvement. "Re-evaluation and Brand Construction" had the lowest scores, reflecting that enterprises did not pay enough attention to in-depth study and re-evaluation of "the same product made by many manufacturers". The evaluation results were in line with expectations, provided a reference example for the evaluation of high-quality Chinese patent medicine, a basis for the high-quality and good price of Chinese patent medicine, scientific data for improving medical insurance bidding, and thus facilitated promoting the healthy development of the traditional Chinese medicine industry.

[Current situation and consideration of patent protection in classical representative famous prescriptions in China].

Classical Representative Famous Prescription is the valuable cultural heritage of Chinese medicine. In November 2018,the State Council issued the " Intensive Implementation of the National Intellectual Property Strategy in 2018 to Accelerate the Construction of IP Strong Country",explicitly proposing to strengthen the intellectual property protection of Classical Representative Famous Prescription.How about the current situation of intellectual property protection of lassical Representative Famous Prescription in China? We selected Liuwei Dihuang Pills,Shengmai Powder and Guizhi Fuling Pills( three representative drugs on market) from Chinese Pharmacopoeia2015 Volume I issued by Chinese Pharmacopoeia Commission to analyze their patent layout,reflecting its status quo of patent protection as follows: first,in recent years,the number of related patent applications for Classical Representative Famous Prescriptions has declined,which was positively correlated with the drug registration and approval policies in recent years,but the policy dividend has not been reflected in the patent application,which may be related to the long period of pharmaceutical R&D; secondly,the patent applicant in the field of Chinese medicine is mainly based on individuals,but the applicant of Classical Representative Famous Prescription is mainly of enterprises,and in addition,the company applicants have the highest authorization rate; thirdly,the main technologies are to improve preparation method and the dosage form in the research and development of Classical Representative Famous Prescription,but these two types of authorized patents have much difficulty in further application on the market. Therefore,the innovative entities shall look for a new breakthrough in secondary development and utilization of Classical Representative Famous Prescriptions.

Current situation and consideration of patent protection in classical representative famous prescriptions in China / 中国中药杂志

Classical Representative Famous Prescription is the valuable cultural heritage of Chinese medicine. In November 2018,the State Council issued the " Intensive Implementation of the National Intellectual Property Strategy in 2018 to Accelerate the Construction of IP Strong Country",explicitly proposing to strengthen the intellectual property protection of Classical Representative Famous Prescription.How about the current situation of intellectual property protection of lassical Representative Famous Prescription in China? We selected Liuwei Dihuang Pills,Shengmai Powder and Guizhi Fuling Pills( three representative drugs on market) from Chinese Pharmacopoeia2015 Volume I issued by Chinese Pharmacopoeia Commission to analyze their patent layout,reflecting its status quo of patent protection as follows: first,in recent years,the number of related patent applications for Classical Representative Famous Prescriptions has declined,which was positively correlated with the drug registration and approval policies in recent years,but the policy dividend has not been reflected in the patent application,which may be related to the long period of pharmaceutical R&D; secondly,the patent applicant in the field of Chinese medicine is mainly based on individuals,but the applicant of Classical Representative Famous Prescription is mainly of enterprises,and in addition,the company applicants have the highest authorization rate; thirdly,the main technologies are to improve preparation method and the dosage form in the research and development of Classical Representative Famous Prescription,but these two types of authorized patents have much difficulty in further application on the market. Therefore,the innovative entities shall look for a new breakthrough in secondary development and utilization of Classical Representative Famous Prescriptions.

Regulatory Effect of Liuwei Dihuang Pills on Connexin 43 Expression in Hepatocarcinoma CBRH7919 Cells in Vitro / 广州中医药大学学报

Objective To investigate the effect of Liuwei Dihuang Pills (LDP)on the expression of connexin 43 (Cx43) in rat hepatocarcinoma CBRH7919 cells. Methods LDP-containing serum was prepared by serum pharmacological method. The protein expression of Cx43 in CBRH7919 cells was determined by Western blotting method, indirect immuno-fluorescent staining labeled by fluorescein isothiocyanate (FITC)-laser scanning confocal microscope technology and flow cytometry. And the mRNA expression of Cx43 in the hepatoma cells was detected by reverse transcription real-time quantitative polymerase chain reaction(RT-qPCR). Results Compared with the blank serum control group,the protein and mRNA expression levels of Cx43 were increased by volume fraction of 2.5%, 5%, 10% LDP-containing serum (P < 0.05 or P < 0.01). Conclusion LDP play an anti-cancer role through promoting the expression of Cx43 in CBRH7919 cells, altering Cx43 membrane location, and improving the gap junctional intercellular communication(GJIC).

Characteristics of materiome release kinetics and visual evaluation of different types of Liuwei Dihuang Pills / 中草药

Objective To investigate the characteristics of materiome release kinetics of Liuwei Dihuang Pills (LDP), and to evaluate its visualization. Methods According to the 2015 edition of Chinese Pharmacopia, using the evaluation methods of release kinetics of Chinese material medica to determine the materiomics release kinetics and the releasecharacteristic of LDP by the paddle method. Then f2 fit factor was calculated and evaluated on other types of LDP in comparison with water-bindered pills and condensed pills as, which are two most common preparations in the market. Moreover, materiome release spectrum and release increment spectrum were used, hoping to quantify, integrate and visualise the materiome release characteristics of different types of LDP. Results The kinetic characteristics of the LDP accord with the Weibull release model, T50 and Td were calculated, and there are significant differences between different types of pills (P < 0.05) in addition to condensed pills and water pills. Also, some differences and similarities in the materiomics release characteristic by f2 fit factor were found in the study. Materiome release spectrum and release increment spectrum can be used to quantify, integrate and visualise the materiome release characteristics of LDP on different types. Conclusion The materiomics release kinetics can be applied to quantify, integrate and visualise the materiome release characteristic of LDP on different types. Basically in agreement with the saying, “water pills from the facilitation, honey pills take the slow, paste pills from the late of, wax-wrapped pills take the difficult”.

Enhanced Estrogenic Activity of Soybean Isoflavones by Coadministration of Liuwei Dihuang Pills in Ovariectomized Rats.

Soybean isoflavones are beneficial for treating hormone-related diseases. Simultaneous consumption of soybean isoflavones and Liuwei Dihuang pills (LWPs) is effective for treating perimenopausal period syndrome. However, why the combination of isoflavones and LWPs is more effective than ingestion of each component alone remains unknown. Here, we show that enhanced estrogenic activities would appear when the ovariectomized rats were fed with a soybean diet in combination of LWPs treatment. Our further studies explored enhancements of Lactobacillus (19-fold) and Bifidobacterium (12-fold) contents in the intestine of rat and 1.84-fold higher intestinal ß-glucosidase activity in LWPs treatment group compared with the control group. As a result, steady-state concentrations of genistein (1.20-fold), daidzein (1.36-fold), and equol (1.43-fold) in serum were significantly elevated in the combination group compared with the soybean alone group. The results present the first evidence of the mechanism of enhanced estrogenic activity of dietary soybean isoflavones in combination with LWPs. Our study indicates that alterations of gut bacteria after LWPs treatment play a key role in the enhanced estrogenic effect of dietary soybean, suggesting a direct relationship between dietary soybean, LWPs, and gut flora.

Effects of Liuwei Dihuang Pills on the Proliferation and Differentiation of Rat Preadipocytes / 中国中医药信息杂志

Objective To investigate the influence of Liuwei Dihuang pills on proliferation and differentiation of rat preadipocytes. Methods Rat preadipocytes were cultured, the influence of Liuwei Dihuang drug-containing serum on proliferation of rat preadipocytes was detected by MTT method, the influence of Liuwei Dihuang drug-containing serum on expression of GPDH was determined by enzyme tissue chemical method, the effect on lipid accumulation was determined by Oil Red O staining. Results Liuwei Dihuang drug-containing serum stimulated rat preadipocyte proliferation, inhibited GPDH up-regulation during differentiation, inhibited lipid accumulation in cell differentiation with the concentration below 10%. Conclusion Liuwei Dihuang pills stimulated rat preadipocyte proliferation, inhibited rat preadipocyte differentiation.

Effect of Liuwei Dihuang Pills Prepared by different Kinds of Techniques on Hemolysin Content in Mice / 中药新药与临床药理

Objective To compare the pharmacodynamic actions of Liuwei Dihuang Pills(LDP)prepared by four kinds of techniques on hemolysin contents in mice.Methods Sensitized by SRBC,immuno-inhibited mice models were induced by dexamethasone and cyclophosphane respectively.The hemolysin contents in the mice fed with LDP prepared by four kinds of techniques were observed to compare the pharmacodynamic actions.Results In experiment 1 that immuno-inhibited mice models were induced by cyclophosphane,the half hemolysin concentration(HC50)was low in the model group and LDP groups,especially in the LDP groups of techniqueⅡ,Ⅲ and Ⅳ.In experiment 2 that immuno-inhibited mice models were induced by dexamethasone,the difference of HC50 was insignificant between the blank control group,LDP groups of techniqueⅠand concentrated LDP group,the difference also being not significant in LDP groups of techniqueⅠat different dosages.Conclusion The dose of LDP can be reduced according to the experiment,however,the process technique of LDP needs to be improved.For this prescription is indicated for the treatment of yin-deficiency syndrome,dexamethasone can be used as the proposed drug for the establishment of the immuno-inhibited models.To confirm the reasonability of formula revolution,more parameters should be added to observe the pharmacodynamic actions.