A study on molecular mechanism of Xihuang pill in the treatment of glioblastoma based on network pharmacology and validation in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Xihuang pill has been utilized to treat cancer for more than three hundred years in China. The molecular mechanisms of Xihuang pill in treating glioblastoma remains unclear. AIM OF THE STUDY: This study aimed to explore the core molecular mechanisms of Xihuang pill in treating glioblastoma by an integrative pharmacology-based investigation. MATERIALS AND METHODS: The main active compounds of Xihuang pill were identified from TCMSP, BATMAN-TCM, TCMID and CNKI. Glioblastoma-related therapeutic targets were retrieved from GeneCards and UniProt. Subsequently, a protein-protein interaction (PPI) network analysis was constructed using STRING. GO and KEGG enrichment were performed to analyze the intersection targets between the active compounds of Xihuang pill and glioblastoma. Based on the above analysis, we built a CTP network. The in vitro and in vivo experiments were further performed to validate the crucial molecular targets of Xihuang pill for the treatment of glioblastoma. RESULTS: A total of sixty active compounds of Xihuang pill and ten potential targets related to glioblastoma were found. Based on topological analysis, fourteen ingredients were selected as the main active compounds, and MY11 might be the most important metabolite in Xihuang pill. PI3K/Akt signaling pathway and receptor tyrosine kinases were considered as crucial targets for Xihuang pill against glioblastoma through KEGG enrichment and CTP analysis. The present experiments indicated that Xihuang pill suppressed the activation of PI3K/Akt/mTOR signaling pathway in glioblastoma cells and mouse xenografts via modulating the expression of PTEN and Rheb proteins, the interaction between TSC2 and Rheb, and the production of PIP3. Meanwhile, after glioblastoma cells treatment with Xihuang pil, the release of IL-1ß, INF-γ was increased and the production of IL-10, TGF-ß1 was decreased in glioblastoma cells after incubated with Xihuang pill. In addition, the activation of the upstream positive modulators of PI3K/Akt/mTOR pathway including PDGF/PDGFR and FGF/FGFR signaling were down-regulated in glioblastoma cells and mouse xenografts after treatment with Xihuang pill. CONCLUSION: Taken together, Xihuang pill inhibiting glioblastoma cell growth might be partly through down-regulating the activation of PDGF/PDGFR or FGF/FGFR-PI3K/Akt/mTOR signaling axis and improving immuno-suppressive micro-environment of glioblastoma.

[Simulated processing with vinegar changes chemical structures of main terpenoids in Euphorbiae Ebracteolatae Radix].

This study aims to explore the chemical structure transformation mechanisms of the main terpenoids in the effective fraction of Euphorbiae Ebracteolatae Radix(EER) during the processing with vinegar. The terpenoids including ent-11α-hydroxyabicta-8(14),13(15)-dien-16,12-olide(HAO), jolkinolide B(JNB), fischeria A(FA), and eupractenoid A(EA) were heated at 160 ℃ with 6% acetic acid for 40 min, and then LC-MS/MS was employed to analyze the structural transformation rules of the terpenoids. Further, we analyzed the changes in the relative content of the four compounds and their transformation products in raw and vinegar processed EER to verify the transformation rules during the simulated processing with vinegar. In addition, JNB and FA were processed with single heating, heating with water or heating with acetic acid. We then employed HPLC to compare the content of these two terpenoids and their transformation products before and after processing, so as to investigate the effect of different processing methods on chemical structure transformation. The results showed that the lactone ring of the abietane-type diterpenoids HAO and JNB and the norditerpene lactone FA were opened by heating with acetic acid. When there were hydroxyl groups in the structures, terpenoids were esterized to esters and oxidized to form carbonyl groups. When there was epoxy ring in the structures, ring opening reaction was easy to occur. During the heating with acetic acid, the heterodimeric diterpenoid EA underwent the cleavage of ether bond to produce the rosane-type diterpenoid euphebracteolatin A(EHTA) and another abietane-type diterpenoid. The changes in the relative content of terpenoids and their transformation products in raw and vinegar-processed EER were basically consistent with those of simulated processing of components with vinegar. The HPLC results revealed that the effect of different simulated processing methods on structural transformation varied. Heating with acid can change JNB and FA into new components. Heating with water can also promote the structural transformation, with the efficiency obviously lower than that of heating with acid. Direct heating had no influence on the structure of JNB, while it significantly reduced the relative content of FA. The components treated with direct heating did not produce the products like those of the heating with acid. These results indicated that vinegar plays a key role in the structural transformation of diterpenoids during the processing of EER with vinegar. The structural transformation of diterpenoids in EER during the processing with vinegar may be the material basis for vinegar processed EER to reduce toxicity and preserve effect.

[Effect of different parts of Pinelliae Rhizoma Decoction against airway inflammation and analysis of effective components].

This study investigated the material basis and mechanism of Pinelliae Rhizoma Decoction in the treatment of airway inflammation. The cigarette smoke combined with lipopolysaccharide(LPS) was used to induce an airway inflammation model in mice. The expression levels of IL-6 and IL-8 in the bronchoalveolar lavage fluid(BALF) and the phosphorylation levels of p38 and IκB in the lungs of mice were taken as indexes to screen the effective extracts by system solvent extraction from Pinelliae Rhizoma Decoction(dichloromethane extract, ethyl acetate extract, n-butanol extract, etc.). Meanwhile, the human bronchial epithelial(16-HBE) cell model of cigarette smoke extract(CSE)-induced injury was established, and the mRNA expression levels of IL-6 and IL-8 and the phosphorylation levels of p38 and IκB proteins were also taken as indexes to evaluate the anti-inflammatory effect of different extracts of Pinelliae Rhizoma Decoction. The results showed that Pinelliae Rhizoma Decoction significantly antagonized airway inflammation in mice by down-regulating the expression levels of IL-6 and IL-8 in mice with airway inflammation and 16-HBE cells with CSE-induced injury and inhibiting the phosphorylation levels of p38 and IκB. The dichloromethane and ethyl acetate extracts of Pinelliae Rhizoma Decoction showed significant anti-inflammatory effects, while such effects of other extracts were not prominent. Furthermore, the database of Pinelliae Rhizoma composition was constructed, and the components in effective extracts were analyzed by HPLC-TOF-MS and Nano-LC-MS/MS. As revealed by the results, the compositions of the two effective extracts were similar with 36 common components. They were combined and then divided into Pinelliae Rhizoma alkaloids(PTAs) and Pinelliae Rhizoma non-alkaloids(PTNAs) by 732 cation-exchange resin. Further in vitro investigation confirmed the significant anti-inflammatory effect of PTNAs, while such effect of PTAs was not manifest. The MS analysis showed 172 peptides and 7 organic acids in PTNAs. The peptide content in PTNAs was 63.5% measured by quantitative analysis of BCA assay, and the organic acid content was 9.92% by potentiometric titration method. The findings of this study suggested that Pinelliae Rhizoma Decoction could antagonize airway inflammation in mice by inhibiting phosphorylation of p38 and IκB and blocking the activation of MAPK and NF-κB signaling pathways, and the effective components were related to the peptides and organic acids in PTNAs. The above results lay a foundation for the research on the mechanism and material basis of Pinelliae Rhizoma in antagonizing airway inflammation.

[Effect of Pinelliae Rhizoma processed with lime and licorice on toxic lectin protein].

The present study was aimed to investigate the effect of lime and licorice processing of Pinelliae Rhizoma on its toxic lectin protein and clarify the scientific detoxification connotation of lime and licorice processing of Pinelliae Rhizoma. Western blot was used to semi-quantitatively analyze the contents of lectin in Pinelliae Rhizoma and Pinelliae Rhizoma Praeparatum. Raw products and lectin were treated by soaking in licorice juice, lime solution or mixture solution of these two to investigate the different processing time on the content of toxic lectin protein. SDS-PAGE gel electrophoresis was used to analyze the changes of lectin protein bands in the solution and precipitates before and after processing. MALDI-TOF technology was used to qualitatively analyze and compare the protein molecular weight before and after processing. The results showed that the contents of lectin in Pinelliae Rhizoma and Pinelliae Rhizoma Praeparatum were 5.01% and 0.04% respectively, indicating that processing could significantly reduce the content of active lectin in raw products. The results also showed that the content of lectin in raw drugs decreased significantly after soaking in lime solution for one day or in licorice juice for three day, and the effect was greatest in mixture solution. Qualitative analysis showed that after being treated by soaking in lime solution, the lectin protein was decomposed into small peptide segments, while after being treated by soaking in licorice juice, the lectin protein was denatured and precipitated. The structure of lectin protein in Pinelliae Rhizoma was broken after being processed with licorice juice and lime solution, which significantly reduced the content of toxic lectinprotein. This is one of the detoxification mechanisms of Pinelliae Rhizoma processing.

[Effect of processing on toxic components lectin from four kinds of Araceae toxic medicines].

The study aimed to investigate the effect of processing on lectin protein in four toxic Chinese medicines tubers of Pinellia ternata,P. pedatisecta,Arisema heterophyllum and Typhonium giganteum. Western blot was used to semi-quantitatively analyze the content of lectin in the four kinds of toxic Chinese medicines and their different processed products. Raw products and lectin were treated by heating or soaking in ginger juice or alum solution. The effects of different excipients and the heating methods on lectin proteins were investigated. The results showed that the content of lectin in raw products of P. pedatisecta,P. ternata,A. heterophyllum,and T. giganteum were 7. 3%,4. 9%,2. 7%,2. 3%,respectively. And the content of lectin in Pinelliae Rhizoma praeparatum cum alumine was 0. 027%. Lectin was not detected in the Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine,Arisaematis Rhizma Praeparatum and Typhonii Rhizoma Praeparatum,which indicated that processing could significantly reduce the content of active lectin in raw products. The results also showed that with the prolongation of soaking and heating time,the content of lectin in raw products decreased gradually,while the content was almost unchanged when soaked in ginger juice alone. The effects of different excipients and heating on lectin were the same as those on raw products. Therefore,the method with alum soaking and heating can reduce the content of active lectin,which is the key to reduce the toxicity of toxic Chinese medicines. In this paper,Western blot was used to study the content of toxic protein in Araceae toxic Chinese medicines as an evaluation method of the processing degree.

[Effects of toxic fractions of Euphorbiae Ebracteolatae Radix on toxicity of mice intestinal tract and colon aquaporins expression level before and after vinegar processing].

To investigate the toxicity changes of Euphorbiae Ebracteolatae Radix (EER) before and after vinegar processing, toxic diterpenoids were concentrated with chloroform as extraction solvent from EER. Then the residue was extracted for non-chloroform extract with 95% ethanol and water after extraction with chloroform. The chloroform extraction of vinegar processed EER was prepared with the same method. The mice received the drug by oral administration. Moisture content in mice feces, duodenum and colon tissue, aquaporin AQP1, AQP3, AQP4 protein expression levels were assayed as the indexes to investigate the toxicity variation of chloroform fraction, non-chloroform fraction, as well as intestinal tract toxicity before and after vinegar processing of EER. The results showed that the chloroform fraction extracted from EER could significantly increase the moisture content in mice feces, duodenum and colon, and decrease AQP1 protein expression level, increase AQP3 and AQP4 protein expression levels in the colon. The intestinal toxicity of the chloroform extract was significantly higher than that of non-chloroform extract. The moisture content in mice feces, duodenum and colon was significantly decreased, and the AQPs protein expression tended to be normal in the colon after vinegar processing. The results showed that the chloroform fraction extracted from EER could lead to diarrhea, intestinal edema, and the intestinal toxicity action was associated with interfering AQPs protein expression and promoting intestinal fluid transport disorder in mice. Vinegar-processing could reduce intestinal toxicity of EER, so vinegar processing was considered to be the scientific processing method of EER.

Alleviation of drought stress in Phyllostachys edulis by N and P application.

The aim of this study was to explore whether nutrition supply can improve the drought tolerance of Moso bamboo under dry conditions. One-year-old seedlings were exposed to two soil water content levels [wellwatered, 70 ± 5% soil-relative-water-content (SRWC) and drought stress, 30 ± 5% SRWC] and four combinations of nitrogen (N) and phosphorus (P) supply (low-N, low-P, LNLP; low-N, high-P, LNHP; high-N, high-P, HNHP; and high-N, low-P, HNLP) for four months. Plant growth, photosynthesis, chlorophyll fluorescence, water use efficiency and cell membrane stability were determined. The results showed that drought stress significantly decreased total biomass, net-photosynthesis (Pn), stomatal-conductance (gs), leaf-chlorophyll-content (Chlleaf), PSII-quantum-yield (ΦPSII), maximum-quantum-yield-of-photosynthesis (Fv/Fm), photochemical-quenching-coefficient (qP), leaf-instantaneous-water-use efficiency (WUEi), relative-water-content (RWC), photosynthetic-N-use-efficiency (PNUE), and photosynthetic-P-use-efficiency (PPUE). N and P application was found to be effective in enhancing the concentration of leaf N, gs, and Pn while reducing the production of reactive oxygen species under both water regimes. Under LNHP, HNHP and HNLP treatments, the decreases in total biomass, Pn, Chlleaf and Fv/Fm of drought-stressed were less evident than the decreases under LNLP. The study suggests that nutrient application has the potential to mitigate the drastic effects of water stress on Moso bamboo by improving photosynthetic rate, water-use efficiency, and increasing of membrane integrity.

[Intestinal toxicity of different processed products of Crotonis Fructus and effect of processing on fatty oil and total protein].

To study the effect of different processes of Crotonis Fructus on fatty oil, total protein and intestinal toxicity, three kinds of processed products (heat Crotonis Semen Pulveratum, non-heat Crotonis Semen Pulveratum and diluted Crotonis Semen Pulveratum) were prepared. Mice were orally given Crotonis Fructus. The content of DAO and D-lactic acid in the serum were measured by ELISA to investigate the change of intestinal permeability in mice. Western blot was used to determine the expressions of tight junction proteins (occludin, claudin-1) in different intestinal tract, so as to observe the effect of Crotonis Fructus and its processed products on intestinal epithelial barrier. These results showed that Crotonis Fructus could significantly increase the intestinal permeability and reduce the expression of tight junction proteins in duodenum and jejunum, but with little impact on the ileum and colon. The intestinal permeability and the expression of tight junction proteins became normal after processing. However, the order of the toxicity of Crotonis Semen Pulveratum from high to low was non-heat Crotonis Semen Pulveratum > diluted Crotonis Semen Pulveratum≈4heat Crotonis Semen Pulveratum. According to the results of composition, the composition of fatty oil did not change during the processing, but the content and composition of total protein in Crotonis Semen Pulveratum changed significantly. The order of total protein content from high to low was that non-heat Crotonis Semen Pulveratum > heat Crotonis Semen Pulveratum > diluted Crotonis Semen Pulveratum. The molecular weight distribution of the total protein bands of non-heat Crotonis Semen Pulveratum and diluted Crotonis Semen Pulveratum was consistent, but the composition of total protein of heat Crotonis Semen Pulveratum significantly changed as evidenced by decreased and thin some stripes. This indicated that heating and dilution could reduce the content of total protein, and heating could cause partial protein denaturation and inactivation. In conclusion, both dilution and heating can reduce the toxicity of Crotonis Fructus, but the heating shows a more significant attenuation effect, indicating that heating is the key step in Crotonis Semen Pulveratum preparation.