[Combination of shikonin and gefitinib reverses drug resistance in human non-small cell lung cancer and its mechanism].

The occurrence and development of tumors are associated with the cell energy metabolism. Inhibiting energy metabolism of lung cancer cells is an important strategy to overcome drug resistance. Based on the cellular energy metabolism pathway, this study observed the effect of combination of shikonin(SKN) and gefitinib(GFB) on the drug resistance in non-small cell lung cancer and explored the underlying mechanism. The human non-small cell lung cancer line HCC827/GR resistant to gefitinib was used as the cell model in vitro. The CCK-8 assay and flow cytometry were employed to investigate the cell viability and apoptosis, respectively. The high performance liquid chromatography was employed to measure the intracellular accumulation of GFB. A Seahorse XFe96 Analyzer was used to detect the changes of cellular energy metabolism. Western blot was employed to determine the expression of the proteins involved in the drug resistance. The tumor-bearing nude mouse model was used to verify the efficacy of SKN+GFB in overcoming drug resistance in vivo. The results showed that SKN+GFB significantly reduced the IC_(50) of GFB on HCC827/GR cells, with the combination index of 0.628, indicating that the combination of the two drugs had a synergistic effect and promoted cell apoptosis. SKN increased the intracellular accumulation of GFB. SKN+GFB lowered the oxygen consumption rate(OCR) and glycolytic proton efflux rate(GlycoPER) in cell energy metabolism, and down-regulated the overexpression of PKM2, p-EGFR, P-gp, and HIF-1α in drug resistance. The results of reversing drug resistance test in vivo showed that GFB or SKN alone had no significant antitumor effect, while the combination at different doses induced the apoptosis of the tumor tissue and inhibited the expression of PKM2 and P-gp, demonstrating a significant antitumor effect. Moreover, the tumor inhibition rate in the high-dose combination group reached 64.01%. In summary, SKN+GFB may interfere with the energy metabolism to limit the function of HCC827/GR cells, thus reversing the GFB resistance in non-small cell lung cancer.

[Discussion on influence factors, mechanism and traditional Chinese medicine pathogenesis of idiosyncratic drug-induced liver injury].

Idiosyncratic drug-induced liver injury (IDILI) is a kind of unique adverse drug reaction with relative high morbidity compared with other idiosyncratic diseases. Its occurrence, however, has nothing to do with pharmacological effects and clinical dosage of drugs administered, and only a small number of susceptible individuals will suffer from it. Especially to deserve to be mentioned, the proportion of TCM-induced IDILI showed an ascending trend year by year. So in this article, the author has reviewed some facts related with TCM-induced IDILI, including the predisposing causes and occurrence mechanism, and tries to provide reference for the prevention, diagnosis and treatment of TCM-induced IDILI through the analysis of characteristics and research status of TCM-induced IDILI and exploration of the internal relationship between Chinese medicine constitution type and IDILI.

[Individualized clinical treatment from the prospective of hepatotoxicity of non-toxic traditional Chinese medicine].

Traditional Chinese medicine has a long history in clinical application, and been proved to be safe and effective. In recent years, the toxicity and side-effects caused by the western medicine have been attracted much attention. As a result, increasing people have shifted their attention to traditional Chinese medicine. Nonetheless, due to the natural origin of traditional Chinese medicine and the lack of basic knowledge about them, many people mistakenly consider the absolute safety of traditional Chinese medicine, except for well-known toxic ones, such as arsenic. However, according to the clinical practices and recent studies, great importance shall be attached to the toxicity of non-toxic traditional Chinese medicine, in particular the hepatotoxicity. Relevant studies indicated that the toxicity of non-toxic traditional Chinese medicine is closely correlated with individual gene polymorphism and constitution. By discussing the causes and mechanisms of the hepatotoxicity induced by non-toxic traditional Chinese medicine in clinical practices, we wrote this article with the aim to provide new ideas for individualized clinical therapy of traditional Chinese medicine and give guidance for rational and safe use of traditional Chinese medicine.

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.

[Prevention effect of Ligusticum chuanxiong extraction against oxidative stress injury induced by myocardial ischemia through activation of Nrf2 signaling pathway].

Ligusticum chuanxiong is one of the common traditional Chinese medicinal herbs for treating various cardiovascular and cerebrovascular diseases, and a number of previous studies have demonstrated that the extract of L. chuanxiong has strong antioxidative activity. This paper was mainly aimed to investigate the effects and possible mechanisms of L. chuanxiong extraction on oxidative stress induced by myocardial ischemia injury in rats. The rats were subcutaneously injected with isoprenaline hydrochloride to induce myocardial ischemia injury and treated for 2 weeks. Then the cardiac indexes of the rats were recorded. The concentration of malondialdehyde (MDA) and activities of serum creatine kinase (CK), lactate dehydrogenase (LDH), aspartate transaminase (AST), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) were measured by colorimetry. Light microscope was used to observe the morphological changes of myocardium, and the protein expressions of nuclear factor E2 related factor 2 (Nrf2), quinone oxidoreductase (NQO1) and hemeoxygenase-1 (HO-1) in cardiac tissue were evaluated by Western blot. The results showed that L. chuanxiong extraction could decrease cardiac indexes and the values of CK, LDH and AST in blood serum, increase activities of serum SOD and T-AOC, reduce serum MDA concentration, improve myocardium structure after ischemia injury, and up-regulate the protein expressions of Nrf2, NQO1 and HO-1 in cardiac tissue. These findings revealed that the cardioprotective effects of L. chuanxiong extraction may be related to inhibiting oxidative stress through the activation of Nrf2 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.

[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.

[Metabolism of paeoniflorin by rat intestinal flora in vitro].

In order to clarify the effect of intestinal flora on the absorption and metabolism of paeoniflorin in vivo, the metabolism of paeoniflorin by rat intestinal flora was studied under the in vitro anaerobic condition. Paeoniflorin was incubated with rat anaerobic intestinal flora for 48 h, and UPLC was used to detect the changes of paeoniflorin at different incubation time points under the following chromatographic conditions：WelchromTM C18 chromatographic column (4.6 mm×100 mm, 5 µm), with 0.1% formic acid(A)-acetonitrile(B) as the mobile phase for gradient elution. The flow rate was 0.4 mL•min⁻¹, and column temperature was 30 ℃. UPLC-Q-TOF-MS with positive ion mode(ESI ion source) was applied to investigate the structural characterization of metabolic products. The structures of the metabolites were identified by accurate molecular weight, TOF-MS/MS fragmentation information, combined with retention time and literature data review, and the intestinal metabolic rules were then analyzed. After incubation for 24 h, the paeoniflorin was metabolized completely, and the resulting metabolites(albiflorin, albiflorinaglycone, deacylate albiflorin, deacylate albiflorin aglycone and paeonilactone-B) were detected in rat intestinal flora. The metabolic pathway analysis showed that the isolated rat intestinal flora first transformed peoniflorin into albiflorin, and then further metabolized by glucose removal, phenyl group removal, or four-membered ring pyrolysis and rearrangement. Paeoniflorin was gradually transformed into more hydrophobic metabolites with smaller molecular mass, which were better absorbed by the intestinal tract.

[Simultaneous determination of 8 main compounds in Eclipta Herba by UPLC].

To establish an UPLC method for the simultaneous determination of 8 compounds in Eclipta Herba, such as isoquercitrin, luteoloside, demethylwedelolactone, isochlorogenic acid A, isochlorogenic acid C, luteolin, wedelolactone and apigenin. The experiment was performed with a Waters Acquity UPLC BEH C18 (2.1 mm×100 mm, 1.7 µm) column by gradient elution of 0.1% formic acid in water and acetonitrile: 0-4 min,10%-13% A; 4-10 min, 13%-16% A; 10-13 min, 16%-25% A; 13-17 min, 25%-28% A; 17-20 min,28%-40% A;20-25 min,40%-95% A. The flow rate was 0.3 mL•min⁻¹.. The condition of was the colum temperature was maintained at 35 ℃ and the detected wavelength was set at 350 mm. 8 components were separated clearly by this method. Also a good linearity was obtained between the chosen concentration(r≥0.999 0). The measured data showed that the recovery rate range from 96.60%-103.4% (n=6) and their RSD values were 0.86%-2.4%. The method has high recovery rate, good reproducibility and stability. It provides a scientific basis for the identification and quality evaluation of Eclipta Herba.

[Development ideas of fine pharmaceutical materials of traditional Chinese medicine preparations based on active cluster theory].

The pharmaceutical materials are divided into coarse and fine types in the development of traditional Chinese medicine preparations. Fine materials with clear composition, stable content and high quality control conform to the international development trend of traditional Chinese medicine preparations. In this paper, the status of fine materials was analyzed, and the development ideas were tentatively put forward. On the one hand, the study on simple methods and efficient equipment shall be strengthened for the simultaneous separation of multiple components of traditional Chinese medicine; on the other hand, the knowledge for traditional Chinese medicine shall be broadened to further develop the scientific compatibility of monomers under the guidance of the theory of traditional Chinese medicine.