Components of Goutengsan in Rat Plasma by Microdialysis Sampling and Its Protection on Aß1-42-Induced PC12 Cells Injury.

Goutengsan, a Chinese herbal formula, potential protection on Alzheimer's disease (AD) has been less reported. In current study, we investigated the protection of Goutengsan on Aß1-42-induced pheochromocytoma-derived cells (PC12). Furthermore, the components from Goutengsan in rat plasma were identified by microdialysis (MD) for in vivo sampling. Meanwhile, the protection of components identified was also verified. At last, we found that Goutengsan has a potential protective effect on Aß1-42-induced PC12 cells via reducing cells damage and increasing cells vitality as well as six components (pachymic acid, liquiritin, rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine) which may be effective components. This study helps to understand the treatment of Goutengsan for AD and would facilitate the clinical and further studies for this formula.

Anti-inflammatory Effects of Phyllanthus emblica L on Benzopyrene-Induced Precancerous Lung Lesion by Regulating the IL-1ß/miR-101/Lin28B Signaling Pathway.

BACKGROUND: Phyllanthus emblica L (PEL), a well-known medical plant, has been used in Asian countries for a long time. Increasing evidence suggests that it can prevent the tumorigenesis of cancer associated with nonresolving inflammation. However, the possible anti-inflammatory mechanism responsible for preventing tumorigenesis of precancerous lung lesions is not well elucidated. MATERIALS AND METHODS: Male A/J mice were randomly divided into 5 groups with 10 mice in each group: (1) blank group (saline), (2) benzo(a)pyrene [B(a)P] group, (3) and (4) B(a)P + PEL (5 g/kg/d, 10 g/kg/d, administered by gavage), (5) B(a)P + celecoxib (30 mg/kg/d, administered by gavage). Nodes on the lung surface were observed and calculated. The levels of macrophage inflammatory protein (MIP-2), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1ß were detected by enzyme-linked immunosorbent assay (ELISA) kits. Cyclo-oxygenase-2 (COX-2), hypoxia-inducible factor-1 (HIF-α), IL-1ß, miR-101, and Lin28B protein levels were evaluated by immunohistochemistry and Western blotting. RESULTS: PEL extract treatment significantly reduced the number of nodes on the lung surface and attenuated B(a)P-induced levels of proinflammatory cytokines MIP-2, TNF-α, IL-6, and IL-1ß in lung tissue. The protein expressions of COX-2 and HIF-α were significantly decreased by the treatment of PEL. In addition, both PEL extract and celecoxib markedly upregulate the expression of miR-101 while downregulating IL-1ß and Lin28B levels. CONCLUSION: Our study indicated that treatment with PEL extract can not only protect the lung from inflammatory injury but effectively prevent precancerous lung lesions through regulating the IL-1ß/miR-i101/Lin28B signaling pathway.

Terpene glycoside component from Moutan Cortex ameliorates diabetic nephropathy by regulating endoplasmic reticulum stress-related inflammatory responses.

ETHNOPHARMACOLOGICAL RELEVANCE: Multiple lines of evidences have suggested that endoplasmic reticulum (ER) stress-related inflammatory responses play a critical role in the pathogenesis of diabetic nephropathy (DN). Moutan Cortex (MC), the root bark of Paeonia suffruticosa Andr., is a well-known traditional Chinese medicine (TCM), which has been used clinically for treating inflammatory diseases in China. The findings from our previous research suggested that terpene glycoside (TG) component of MC possessed favorable anti-inflammatory properties in curing DN. However, the underlying mechanisms of MC-TG for treating DN are still unknown. AIM OF THE STUDY: To explore the role of ER stress-related inflammatory responses in the progression of DN, and to investigate the underlying protective mechanisms of MC-TG in kidney damage. MATERIALS AND METHODS: DN rats and advanced glycation end-products (AGEs) induced HBZY-1 cell dysfunction were established to evaluate the protective effect of MC-TG on ameliorating renal injury. Evaluation of pathological lesions was performed by Masson staining and transmission electron microscopy (TEM). Interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), glucose regulated protein 78 (GRP78/Bip), as well as spliced X box binding protein 1(XBP-1(s)) levels in rat serum were detected by an enzyme-linked immunosorbent assay (ELISA). Furthermore, western blotting (WB) was applied to detect the protein expressions including IL-6, MCP-1, intercellular cell adhesion molecule-1 (ICAM-1), GRP78/Bip, XBP-1 (s), phosphorylated inositol-requiring enzyme-1α (p-IRE1α), cleaved activating transcription factor 6 (ATF6), phosphorylated PKR-like endoplasmic reticulum kinase (p-PERK), and phosphorylated nuclear factor κB p65 (p-NF-κB p65) in vivo and in vitro. Immunohistochemistry (IHC) was carried out to determine the phosphorylation of IRE1α and NF-κB p65 in kidney tissues. RESULTS: Pretreatment with MC-TG could markedly improve renal insufficiency and pathologic changes. It could down-regulate ER stress-related factors GRP78/Bip, XBP-1(s) levels, and also reduce the pro-inflammatory molecules IL-6, MCP-1, and ICAM-1 expressions. Furthermore, a significant decrease in phosphorylation of IRE1α and NF-κB p65 by the treatment of MC-TG. CONCLUSIONS: These findings indicated that MC-TG ameliorated ER stress-related inflammation in the pathogenesis of DN, wherein the protective mechanism might be associated with the inhibition of IRE1/NF-κB activation. Thus, MC-TG might be a potential therapeutic candidate for the prevention and treatment of DN.

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.

[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.

[Study on nephrotoxicity of ethanol extract from Alismatis Rhizoma in rats and its molecular mechanism].

Female rats were fed with ethanol extraction of Alismatis Rhizoma for 6 months to study its nephrotoxicity and molecular mechanism. HPLC was used to determine the components in ethanol extraction of Alismatis Rhizoma. An assessment of renal pathology was determined by HE staining. Meanwhile Western blot, immunohistochemical assay and q-PCR were used to assess the protein expression and mRNA level of Kim-1, clusterin, LCN2, osteopontin, ceruloplasmin and TIMP1 in rat kidney. Eight components were identified in ethanol extraction of Alismatis Rhizoma. Tubule-interstitial inflammation, renal tubular epithelial cell exfoliation and morphological changes of cell were observed in rat kidney. Comparing with control blank group, the protein expression of clusterin, Kim-1, LCN2, osteopontin and TIMP1 in rat kidney was significantly increased while the protein expression of ceruloplasmin was significantly decreased. The mRNA level of Kim-1, TIMP1, osteopontin, clusterin and LCN2 was significantly increased while the mRNA level of ceruloplasmin was significantly decreased. In this study, it was inferred that there is chronic toxicity in kidney by using high dosage of ethanol extraction of Alismatis Rhizoma for a long time. And the underlying molecular mechanism was related to regulate the protein expression of ceruloplasmin, clusterin, Kim-1, LCN2, osteopontin and TIMP1.

[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.

[Evaluation of nephrotoxicity induced by total terpenoids from Alismatis Rhizoma on HK-2 cells in vitro and its induction of apoptosis].

To evaluate the nephrotoxicity of total terpenoids from Alismatis Rhizoma on human kidney proximal tubular cells (HK-2), explore the iraction in inducing apoptosis of HK-2 cells, and provide reference for the research of controversial nephrotoxicity of total terpenoids from Alismatis Rhizoma, HK-2 cells were used and cells viability was measured by MTT colorimetric method. An assessment of cells apoptosis was also conducted by using flow cytometry. Meanwhile western blot assay was used to detect the protein expressions of caspase-3, Bcl-2, Bcl-xl, Kim-1, clusterin and TFF-3. At last, q-PCR was used to detect the mRNA expressions of caspase-3, Bcl-2, Bcl-xl, Kim-1, clusterin and TFF-3. The flow cytometry results showed that cells apoptosis rate was (37.48±1.76)%, (26.91±1.91)% and (25.61±2.05)% respectively after treating with total terpenoids (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹). Western blot results showed that Bcl-2 and Bcl-xl protein levels were significantly decreased after treating with total terpenoids (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹), while the protein expression of caspase-3 was significantly increased. q-PCR results were the same with western blot results, that mRNA expressions of Bcl-2 and Bcl-xl were significantly decreased while mRNA expression of caspase-3 was significantly increased after treating with total terpenoids (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹). Western blot results and q-PCR results showed that both mRNA and protein expressions of Kim-1, clusterin and TFF-3 were significantly increased after treating with total terpenoids from Alismatis Rhizoma (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹). HK-2 cells in vitro evaluation results showed that, total terpenoids from Alismatis Rhizoma may have nephrotoxicity effect, but further study is still needed for verification; meanwhile, they could induce HK-2 cells apoptosis, providing basis for nephrotoxicity study and safe application of Alismatis Rhizoma.

Development of innovative structural components of Chinese medicine by multidisciplinary crossing.

"Prescription embodied in Preparation", Chinese medicine preparation, aims to study the specific form of Chinese medicine from raw materials to preparation for acting on patients directly. Its development has gone through three stages according to the characteristics of raw materials pretreatment, including "direct smash and initial extraction for Chinese materia medica", "Extensive extraction and preliminarily impurity for Chinese materia medica" and "Refining and purification for Chinese materia medica". With the development of new technologies and new theories, Chinese medicine preparation emerged in a new stage: structural components of Chinese medicine, with the characteristics of definited material basis, clear mechanisms, determined ADME/T properties, reasonable drug release system designs and scientific productions quality controls. This requires multidisciplinary to solve systemly the problems of Chinese medicine preparation. In this article, we reviewed the development of Chinese medicine preparation in different times, and analyzed the development and the characteristics of Chinese medicine preparation; and mainly focused on a fact that multidisciplinary promoted the study and development of Chinese medicine preparation, especially in structural components of Chinese medicine. It provides development direction and theoretical basis for Chinese medicine preparation.

[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.