[Effects of Ziyin Liangxue Formula Combined with Prednisone on Immune Function and ST2/IL-33 Pathway in Mice with Immune Thrombocytopenia].

OBJECTIVE: To investigate the effects of Ziyin Liangxue formula combined with prednisone on immune function and the ST2/IL-33 pathway in mice with immune thrombocytopenia. METHODS: In 40 BALB/c mice, 32 were constructed as immune thrombocytopenia mouse models by antiplatelet serum injection. After successful modeling, the mice were randomly divided into model group, Ziyin Liangxue formula group (0.2 ml/10 g), prednisone group (0.2 ml/10 g), and Ziyin Liangxue formula + prednisone group (0.2 ml/10 g), 8 mice in each group, and the other 8 mice were set as control group. The drugs were administered by gavage at the dose, and the model group and control group were given equal amounts of saline by gavage once a day for 2 weeks of continuous intervention. Blood samples and spleen tissues were collected, the peripheral platelet count was measured by automatic hematology analyzer, the pathological changes in spleen tissue was observed by HE staining, the levels of serum transforming growth factor (TGF)-ß, interleukin (IL)-17, and peripheral blood thrombopoietin (TPO) were detected by enzyme-linked immunosorbent assay (ELISA), the expression of IL-33, sST2, and ST2 in spleen tissue was detected by Western blot, and the cell counts of peripheral blood Th17 and Treg were detected by flow cytometry. RESULTS: Compared with the control group, the number of platelets, the level of TPO, TGF-ß, and Treg cells were significantly decreased (P <0.05), while the level of IL-17, Th17 cells, and the expression of IL-33, sST2, and ST2 protein were significantly increased in the model group (P <0.01). Compared with the model group, the number of platelets, the level of TPO, TGF-ß, and Treg cells were significantly increased (P <0.05), while the level of IL-17, Th17 cells, and the expression of IL-33, sST2, and ST2 protein were significantly decreased in the Ziyin Liangxue formula + prednisone group (P <0.01). CONCLUSION: Ziyin Liangxue formula + prednisone can effectively regulate Th17/Treg balance, thus effectively improve immune thrombocytopenia, and the mechanism may be related to the regulation of ST2/IL-33 signaling pathway.

[Effect of P-coumaric Acid on Apoptosis of Multiple Myeloma Cells Based on Oxidative Stress].

OBJECTIVE: To investigate the effect of p-coumaric acid on apoptosis of multiple myeloma cells and its related mechanism. METHODS: Multiple myeloma cell line MM.1s cells were selected and treated with different concentrations of p-coumaric acid (0, 0.4, 0.8, 1.6, 3.2 mmol/L), and the inhibition rate and half inhibition concentration (IC50) were detected by CCK-8 method. Then MM.1s cells were treated with 1/2 IC50, IC50, 2 IC50 and transfected with ov-Nrf-2 and ov-Nrf-2+IC50. The apoptosis, ROS fluorescence intensity and mitochondrial membrane potential of MM.1s cells were detected by flow cytometry, and the relative expressions of cellular Nrf-2 and HO-1 protein were detected by Western blot. RESULTS: P-coumaric acid inhibited the proliferation of MM.1s cells in a dose-dependent manner(r =0.997) with an IC50 value of 2.754 mmol/L. Compared with the control group, apoptosis and ROS fluorescence intensity of MM.1s cells were significantly increased in the 1/2 IC50 group, IC50 group, 2 IC50 group and ov-Nrf-2+IC50 group (P <0.01), the expressions of Nrf-2, HO-1 protein in the IC50 group and 2 IC50 group were significantly decreased (P <0.05). Compared with the IC50 group, the cells apoptosis and ROS fluorescence intensity were significantly decreased (P <0.01), and the expressions of Nrf-2 and HO-1 protein were significantly increased in the ov-Nrf-2+IC50 group (P <0.01). CONCLUSION: P-coumaric acid can inhibit the proliferation of MM.1s cells and may target the Nrf-2/HO-1 signaling pathway to affect oxidative stress in MM cells thereby inducing their apoptosis.

Effect of WSP, a Chinese Herbal Formula, on Th17/Treg Ratio and HBeAg Seroconversion in Telbivudine-Treated HBeAg-Positive Chronic Hepatitis B Patients with High Baseline ALT Levels (20-30 Times the ULN).

Chronic hepatitis B (CHB) is a global health problem. Clinically, many patients have baseline alanine aminotransferase (ALT) levels above 20 times the upper limit of normal (ULN), but there are few reports about these patients. The prospective randomized placebo-controlled clinical study was designed to investigate the effect of WSP, a Chinese herbal formula, on telbivudine- (LDT-) treated HBeAg-positive CHB patients with high baseline ALT levels (20-30 times the ULN) and kidney-yang deficiency syndrome. Eligible patients were randomized to receive LDT 600 mg/day in combination with WSP (treatment group) or placebo granules (control group) 16.28 g/day for 52 weeks. The results showed that HBeAg seroconversion (SC) rate (44.1%) in the treatment group (n=34) was significantly superior to that (20.6%) in the control group (n=34) at 52 weeks (P < 0.05). Meanwhile, WSP could promote HBV DNA negative conversion (85.3% versus 61.8%, P < 0.05) and ALT normalization (94.1% versus 76.5%, P < 0.05) compared with the placebo. There were no drug-related serious adverse events. During the treatment, the peripheral blood Th17/Treg ratio first increased and then decreased in the treatment group and reached the peak at 12 weeks (P < 0.05). At 12, 24, 36, and 52 weeks, Th17/Treg ratio in the treatment group was better than those in the control group (all P < 0.05). In addition, the patients (n=22) with HBeAg SC had higher Th17/Treg ratio than the patients (n=46) without SC at 12 weeks (0.68±0.26 versus 0.43±0.18, P < 0.001). In conclusion, WSP could safely enhance HBeAg SC and promote HBV DNA negative conversion and ALT normalization in LDT-treated HBeAg-positive CHB patients with high baseline ALT levels (20-30 times the ULN) and kidney-yang deficiency syndrome. Th17/Treg ratio was not only related to the mechanisms of WSP but also a good predictor of 52-week HBeAg SC.

Protection of tauroursodeoxycholic acid on high glucose-induced human retinal microvascular endothelial cells dysfunction and streptozotocin-induced diabetic retinopathy rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tauroursodeoxycholic acid (TUDCA), one of the main ingredients from bear gall which hold "Clearing heat and detoxification, Removing liver fire for improving eyesight" functions, is formed by the conjugation of ursodeoxycholic acid (UDCA) with taurine. However, the limited information of TUDCA on protecting diabetic retinopathy (DR) has been known. The present study was conducted to evaluate the protection of TUDCA on high glucose-induced human retinal microvascular endothelial cells (HRMECs) dysfunction and streptozotocin (STZ)-induced diabetic retinopathy (DR) rats and the possible mechanism underlying was also explored. MATERIALS AND METHODS: The proliferation of high glucose-induced HRMECs was determined by MTT assay. DR rats' model was established by an administration of high-glucose-fat diet and an intraperitoneal injection of STZ (30mg/kg). The cell supernatant and rats' serum were collected for the assays of NO content by ELISA kits. Retinas were stained with hematoxylin and eosin (HE) to observe pathological changes. Immunohistochemical assay was applied to examine the protein expression of ICAM-1, NOS, NF-κB p65 and VEGF in rat retinas. Furthermore, western blot analysis was carried out to examine the protein expression of ICAM-1, NOS, NF-κB p65 and VEGF in high glucose-induced HRMECs. RESULTS: After treating with TUDCA, high glucose-induced HRMECs proliferation could be significantly inhibited. TUDCA (5.0µM, 25.0µM and 125.0µM) could decrease NO content in high glucose-induced HRMECs. Furthermore, TUDCA (500mg/kg/d and 250mg/kg/d) also decrease NO content in serum of DR rats. Additionally, both immunocytochemistry analysis and western blot analysis showed that the over-expression of ICAM-1, NOS, NF-κB p65 and VEGF were significantly decreased by TUDCA. CONCLUSION: The data indicated that TUDCA could ameliorate DR by decreasing NO content and down-regulating the protein expression of ICAM-1, NOS, NF-κB p65 and VEGF. Thus, our experimental results suggested that TUDCA might be a potential drug for the prevention and treatment of DR.

Beneficial Effects of a Flavonoid Fraction of Herba Epimedii on Bone Metabolism in Ovariectomized Rats.

A flavonoid fraction of Herba Epimedii, including eight flavonoid glycoside compounds, epimedoside A, ikarisoside F, baohuoside II, sagittatoside A, sagittatoside B, 7-O-rhamnosyl icariside II, 2"-O-rhamnosyl icariside II, and baohuoside I, was isolated and prepared from the leaves of Herba Epimedii. This study was conducted to assess the potential effect of the flavonoid fraction of Herba Epimedii on osteoporosis in ovariectomized rats. Rats received repeated administration of a vehicle (ovariectomized), the flavonoid fraction of Herba Epimedii (7.5, 15, 30 mg/kg/d), and ipriflavone (200 mg/kg/d) once a day for 8 weeks, beginning 4 weeks after ovariectomization. Then, the bone turnover markers, bone biomechanical properties, trabecular architecture, and related protein expressions were evaluated by biochemical assay kits, mechanical testing, microcomputed tomography, immunohistochemical evaluation, and Western blot analysis. Treatment with the flavonoid fraction of Herba Epimedii (15, 30 mg/kg/d) and ipriflavone (200 mg/kg/d) significantly increased bone strength while dramatically inhibiting the serum alkaline phosphatase and tartrate-resistant acid phosphatase levels in ovariectomized rats. Furthermore, the flavonoid fraction of Herba Epimedii also increased osteoprotegerin protein expression and reduced the receptor activator of nuclear factor-κB ligand protein expression compared with ovariectomized rats. In addition, the microcomputed tomography results showed that the flavonoid fraction of Herba Epimedii treatment significantly improved trabecular bone mineral density and restored the bone microarchitecture in ovariectomized rats. Therefore, our results indicated that the flavonoid fraction of Herba Epimedii might be beneficial for improving postmenopausal osteoporosis and should be considered as a promising candidate for treating postmenopausal osteoporosis.

[Effect of Epimedii Herba alcohol extract on inhibition of lung tumor growth and immunomodulatory].

To observe the effect of Epimedii Herba alcohol extract (HE) on tumor growth of lung cancer by establishing the model of Lewis tumor-bearing mice, ELISA method was used to detect the levels of TNF-α, IL-10, IL-17, IL-2 in serum. Ki67 and P53 protein expression was detected in lung cancer tissues by using Western blot assay method and immunohistochemical assay method. The experimental results showed that HE has certain inhibitory effect on Lewis lung cancer tumor growth, and it can reduce the levels of TNF-α, IL-10 and IL-17 in serum, improve the level of IL-2,significantly decrease the expression of Ki67, and significantly increase P53 expression. HE has obvious inhibitory effect against lung cancer, and has the ability to improve immune regulating effect. This study reveals the anti-lung cancer effect of HE may be related to its ability of improving immunity, thus provides the basis for further research on anti-lung cancer effect of HE.

[Comparison of chemical compositions in Moutan Cortex, Paeoniae Rubra Radix and Paeoniae Alba Radix based on "component structure" theory].

To analyze and compare the chemical compositions of Moutan Cortex, Paeoniae Rubra Radix and Paeoniae Alba Radix based on "component structure" theory. Thirteen batches of Moutan Cortex, 14 batches of Paeoniae Rubra Radix from different origins and 10 batches of Paeoniae Alba Radix from different origins were analyzed by HPLC-DAD method. Hierarchical cluster analysis and principal component analysis were used for analysis. The significant differences of principal component from Moutan Cortex, Paeoniae Rubra Radix and Paeoniae Alba Radix were investigated by using F test. HPLC fingerprints were established for 13 batches of Moutan Cortex, 14 batches of Paeoniae Rubra Radix and 10 batches of Paeoniae Alba Radix, and 7 glycosides and phenolic acids components were identified. Comparative study of Moutan Coetex, Paeoniae Rubra Radix and Paeoniae Alba Radix was conducted according to the results of hierarchical cluster analysis, principal component analysis and "component structure" theory. Moutan Cortex, Paeoniae Rubra Radix and Paeoniae Alba Radix have significant differences in mass fraction of major chemical components and their ratios, leading to different curative effects.

Screening for anti-inflammatory components from Corydalis bungeana Turcz. based on macrophage binding combined with HPLC.

BACKGROUND: Corydalis bungeana Turcz. (CB; family: Corydalis DC.) is an anti-inflammatory medicinal herb used widely in traditional Chinese medicine (TCM) for upper respiratory tract infection, etc., but its anti-inflammatory active molecules are unknown. This study was designed to screen for the anti-inflammatory components from CB based on macrophage binding combined with HPLC. METHODS: Xylene-induced ear edema in mouse and carrageenan-induced hind-paw edema in rats were used to evaluate the anti-inflammatory activity of CB. The macrophage binding with high-performance liquid chromatography (HPLC) analysis and HPLC-MS were established to screen the potential active compounds. ELISA kits were performed to measure the levels of IL-6, IL-10, TNF-α and NO in RAW 264.7 macrophages culture media. RESULTS: The alkaloid extract of CB could inhibit significantly xylene-induced ear edema in mouse and carrageenan-induced hind-paw edema in rats. Two components binded to RAW 264.7 cell were identified as 12-hydroxycorynoline and corynoline. Bioassays demonstrated that these two compounds significantly inhibited LPS-induced IL-6, IL-10, TNF-α and NO levels. CONCLUSIONS: The results suggest that corynoline and 12-hydroxycorynoline contribute to the anti-inflammatory effects of the alkaloid extract of CB. Our findings suggest that these two compounds can be used as candidate for anti-inflammatory drugs.

Effects of icariin on orthodontic tooth movement in rats.

PURPOSE: Orthodontic tooth movement (OTM) is achieved through bone remodeling of the alveolar bone. Icariin, the active ingredient isolated from Herba Epimedii which is a traditional Chinese medicine (TCM) commonly used for osteoporosis treatment in China. The purpose of the study is to explore the effect of icariin on OTM in rats, and analyze the possible mechanism involved. METHODS: 48 rats were selected and divided into 2 groups: the control group and the experimental group. Rats in the experimental group were given 20 mg/kg/day icariin by intragastric administration, while the control group received the same volume solvent. All rats were placed a closed coil spring between their upper first molar and incisor, exerting a force of about 40 g to establish animal models of OTM. As the first molar moved mesially, a space between the first and second molar was created. The rats were sacrificed in batch on the 7th, 14th, 21st and 28th days after orthodontic treatment. The amount of tooth movement was measured, and histomorphometric analysis based on slices from periodontium adjacent to the maxillary first molars were used to observe new bone formation, bone resorption and quantify osteoclasts. KEY RESULTS: Icariin increased OTM (P<0.05) by 65.2%, 35.3%, 11.7% and 16.7% on day 7, 14, 21, 28 respectively compared with the control group. The number of osteoclasts in the icariin group showed a transient but sudden increase and then a persistent decrease. CONCLUSIONS: Icariin could accelerate OTM in rats through promoting bone remodeling of alveolar bone.

[Structural components of Chinese medicine and pharmacology network: systematical overall regulation on pathological network].

Development of the disease is the result of several factors involved in biological network changes. The nature of drug intervention is to regulate these pathological changes to the normal range. Advantages of traditional Chinese medicine (TCM) are to integrally and systematically regulate this biological networks and systematic pathology through multi-targets, multi-levels, multi-channels. Structural components TCM provides the controlled and precise basis "substance" for this regulation and also to clarify the "truth" of the nature of the regulation by the network pharmacology. Network pharmacology provides new strategy for the research on mechanism of structural components TCM. This study not only reflects the overall characteristics of the development of the disease, but also fully embodies the essence of TCM for preventing and treating diseases through changing traditional model on "one drug, one gene, one disease". This paper explores systematically the integration essence, features and research strategies of structural components TCM and the network pharmacology, understand the interaction of structural components TCM and body from the perspective of the overall concept of improving or restoring the balance of.biological networks. It is effective measure to reveal the structure of a multi-component for regulating biological networks mechanisms, and also provide new ideas and methods for further scientific research and innovation of structural component TCM.

Inhibition of Tumor Growth and Immunomodulatory Effects of Flavonoids and Scutebarbatines of Scutellaria barbata D. Don in Lewis-Bearing C57BL/6 Mice.

Immunomodulatory effect has been found to be an important therapeutic measure for immune responses against cancer. In this study, we evaluated the inhibition of Scutellaria barbata D. Don (SB), an anti-inflammatory and an antitumor Chinese herb, including flavonoids and scutebarbatines on tumor growth and its immunomodulatory effects in vivo. HPLC and LC/MS/MS methods were conducted for the analysis of flavonoids and scutebarbatines in SB. Lewis-bearing C57BL/6 mice model was established and tumor volume was evaluated by high frequency color ultrasound experiment. ELISA and western blot analysis were performed for the determination of immunomodulatory factors. SB treatment at the dose of 10, 6.67, and 3.33 g crude drug/kg/d significantly inhibited tumor growth of Lewis-bearing C57BL/6 mice with the inhibition rates of 44.41 ± 5.44%, 33.56 ± 4.85%, and 27.57 ± 4.96%, respectively. More importantly, the spleen and thymus indexes were increased remarkably by SB treatment. SB could decrease IL-17, IL-10, FOXP3, TGF-ß1, RORγt, and IL-6 levels whereas it could increase remarkably IL-2 and IFN-γ levels. Our results demonstrated that SB could inhibit tumor growth in vivo through regulating immune function in tumor-bearing mice and suggested that the immunomodulatory function of SB had a potential therapeutic effect in lung cancer.

[Research development of the chemical material basis of Alisma orientalis and its toxicity].

Alisma orientalis is a traditional herb medicine commonly used in clinical. With the increasing report of its toxicity in clinical, the renal toxicity of Alisma orientalis has got gradually attention. This paper systematically reviews the research on the chemical material basis of Alisma orientalis including its chemical composition and toxicity of ingredients; and also declares its toxic ingredients and targets according to Network toxicology. Based on the controversy on renal toxicity of Alisma orientalis, we analyzed the possible reasons that may be associated with renal toxicity. It might be associated with the differences of the material basis composition and regulatory toxicology network, differences in employed processing technology, the metabolic function leading to accumulation of compounds, dosage and duration of the experiment and compatibility. The review provides possible reference and ideas for the quality control and rational use of Alisma orientalis.

Comparison on hypoglycemic and antioxidant activities of the fresh and dried Portulaca oleracea L. in insulin-resistant HepG2 cells and streptozotocin-induced C57BL/6J diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Fresh Portulaca oleracea L. (family: Portulacaceae; POL) has been used as a folk medicine for the treatment of diabetes mellitus for a long time. More bioactive components with higher activity could be retained in fresh medicinal herbs compared to the dried ones. The present study was conducted to compare different antidiabetic activity between fresh and dried POL, including hypoglycemic and antioxidant activities both in vivo and in vitro. Furthermore, in order to explore which components were responsible for the antidiabetic activity, the difference on chemical components between fresh and dried POL was analyzed and compared. MATERIALS AND METHODS: Insulin-resistant HepG2 cells induced by insulin were used to evaluate the promoting effect of the fresh and dried POL on glucose utilization in vitro. Streptozotocin (STZ)-induced C57BL/6J diabetic mice were used to compare the differences on hypoglycemic and antioxidant activities of fresh and dried POL, including the fasting blood glucose, glucose tolerance, serum insulin level, malondialdehyde (MDA) level and superoxide dismutase (SOD) activity in vivo. UPLC/Q-TOF-MS method was performed to analyze the difference of antidiabetic components between fresh and dried POL. RESULTS: Compared with the dried POL extract, the fresh POL extract significantly increased the consumption of extracellular glucose in insulin-resistant HepG2 cells (P<0.05). In STZ-induced C57BL/6J diabetic mice, both fresh and dried extracts decreased markedly the fasting blood glucose (FBG) levels, and improved significantly oral glucose tolerance test (OGTT), as well as enhanced significantly insulin secretion and antioxidative activities (P<0.05; P<0.01). Furthermore, the fresh extract showed stronger antidiabetic activity (P<0.05). The UPLC/Q-TOF-MS analysis results also revealed that the relative contents of polyphenols and alkaloids in the fresh herbs were more abundant than those in the dried POL. CONCLUSION: Our results indicated that both fresh and dried POL possessed antidiabetic activities, besides stronger activity was observed in the fresh herb. These findings provided evidence for the application and development of fresh POL in the treatment of diabetes mellitus.

Hederagenin from the leaves of ivy (Hedera helix L.) induces apoptosis in human LoVo colon cells through the mitochondrial pathway.

BACKGROUND: Colorectal cancer has become one of the leading cause of cancer morbidity and mortality throughout world. Hederagenin, a derivative of oleanolic acid isolated from the leaves of ivy (Hedera helix L.), has been shown to have potential anti-tumor activity. The study was conducted to evaluate whether hederagenin could induce apoptosis of human colon cancer LoVo cells and explore the possible mechanism. METHODS: MTT assay was used for evaluating cell viability while Annexin V-FITC/PI assay and Hoechst 33342 nuclear stainining were used for the determination of apoptosis and mitochondrial membrane potential. DCFH-DA fluorescence staining and flow cytometry were used to measure ROS generation. Real-time PCR and western blot analysis were performed for apoptosis-related protein expressions. RESULTS: MTT assay showed that hederagenin could significantly inhibit the viability of LoVo cells in a concentration-dependent and time-dependent manner by IC50 of 1.39 µM at 24 h and 1.17 µM at 48 h. The apoptosis ratio was significantly increased to 32.46% and 81.78% by the induction of hederagenin (1 and 2 µM) in Annexin V-FITC/PI assay. Hederagenin could also induce the nuclear changes characteristic of apoptosis by Hoechst 33342 nuclear stainining under fluorescence microscopy. DCFH-DA fluorescence staining and flow cytometry showed that hederagenin could increase significantly ROS generation in LoVo cells. Real-time PCR showed that hederagenin induced the up-regulation of Bax and down-regulation of Bcl-2, Bcl-xL and Survivin. Western blotting analysis showed that hederagenin decreased the expressions of apoptosis-associated proteins Bcl-2, procaspase-9, procaspase-3, and polyADP- ribosepolymerase (PARP) were increased, while the expressions of Bax, caspase-3, caspase-9 were increased. However, there was no significant change on caspase-8. CONCLUSIONS: These results indicated that the disruption of mitochondrial membrane potential might contribute to the apoptosis of hederagenin in LoVo cells. Our findings suggested that hederagenin might be a promising therapeutic candidate for human colon cancer.

Analysis of phosphate-accumulating organisms cultivated under different carbon sources with polymerase chain reaction-denaturing gradient gel electrophoresis assay.

To investigate the microbial communities of microorganisms cultivated under different carbon sources, three sequencing batch reactors were operated. They were supplied with sewage, glucose and sodium acetate as carbon sources respectively and showed high phosphorus removal performance. The results of denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction-amplified (PCR) 16S rDNA fragments demonstrated that beta-protebacteria, Actinomyces sp. and gamma-protebacteria only exited in 1 # reactor. The microbiological diversity of 1 # reactor exceeded the other two reactors. Flavobacterium, Bacillales, Actinomyces, Actinobacteridae and uncultured bacteria (AF527584, AF502204, AY592749, AB076862, AJ619051, AF495454 and AY133070) could be detected in the biological phosphorus removal reactors.