Martynoside rescues 5-fluorouracil-impaired ribosome biogenesis by stabilizing RPL27A.

Martynoside (MAR), a bioactive component in several well-known tonic traditional Chinese herbs, exhibits pro-hematopoietic activity during 5-fluorouracil (5-FU) treatment. However, the molecular target and the mechanism of MAR are poorly understood. Here, by adopting the mRNA display with a library of even-distribution (md-LED) method, we systematically examined MAR-protein interactions in vitro and identified the ribosomal protein L27a (RPL27A) as a key cellular target of MAR. Structural and mutational analysis confirmed the specific interaction between MAR and the exon 4,5-encoded region of RPL27A. MAR attenuated 5-FU-induced cytotoxicity in bone marrow nucleated cells, increased RPL27A protein stability, and reduced the ubiquitination of RPL27A at lys92 (K92) and lys94 (K94). Disruption of MAR binding at key residues of RPL27A completely abolished the MAR-induced stabilization. Furthermore, by integrating label-free quantitative ubiquitination proteomics, transcriptomics, and ribosome function assays, we revealed that MAR restored RPL27A protein levels and thus rescued ribosome biogenesis impaired by 5-FU. Specifically, MAR increased mature ribosomal RNA (rRNA) abundance, prevented ribosomal protein degradation, facilitated ribosome assembly, and maintained nucleolar integrity. Collectively, our findings characterize the target of a component of Chinese medicine, reveal the importance of ribosome biogenesis in hematopoiesis, and open up a new direction for improving hematopoiesis by targeting RPL27A.

Ex vivo and in vivo chemoprotective activity and potential mechanism of Martynoside against 5-fluorouracil-induced bone marrow cytotoxicity.

Martynoside (MAR) is a bioactive glycoside of Rehmannia glutinosa, a traditional Chinese herb frequently prescribed for treating chemotherapy-induced pancytopenia. Despite its clinical usage in China for thousands of years, the mechanism of MAR's hematopoietic activity and its impact on chemotherapy-induced antitumor activity are still unclear. Here, we showed that MAR protected ex vivo bone marrow cells from 5-fluorouracil (5-FU)-induced cell death and inflammation response by down-regulating the TNF signaling pathway, in which II1b was the most regulatory gene. Besides, using mouse models with melanoma and colon cancer, we further demonstrated that MAR had protective effects against 5-FU-induced myelosuppression in mice without compromising its antitumor activity. Our results showed that MAR increased the number of bone marrow nucleated cells (BMNCs) and the percentage of leukocyte and granulocytic populations in 5-FU-induced myelosuppressive mice, accompanied by an increase in numbers of circulating white blood cells and platelets. The transcriptome profile of BMNCs further showed that the mode of action of MAR might be associated with the increased survival of BMNCs and the improvement of the bone marrow microenvironment. In summary, we revealed the potential molecular mechanism of MAR to counteract 5-FU-induced bone marrow cytotoxicity both ex vivo and in vivo, and highlighted its potential clinical usage in cancer patients experiencing chemotherapy-induced multi-lineage myelosuppression.

Antitumor effects of different Ganoderma lucidum spore powder in cell- and zebrafish-based bioassays.

OBJECTIVE: Ganoderma lucidum spore (GLS) is gaining recognition as a medicinal part of G. lucidum and has been reported to possess various pharmacological properties, such as antitumor activity. In this work, wall-broken GLS powder (BGLSP) and wall-removed GLS powder (RGLSP), two kinds of GLS powder with different manufacturing techniques, were compared in terms of contents of active constituents and in vivo and in vitro antitumor effects. METHODS: The ultraviolet and visible spectrophotometry method was used to determine the contents of polysaccharides and total triterpenoids in BGLSP and RGLSP. Seventeen individual triterpenoids were further quantified using ultra-high-performance liquid chromatography and quantitative analysis of multi-components by single marker. The antitumor effects of BGLSP and RGLSP were evaluated using in vitro cell viability assay against human gastric carcinoma SGC-7901, lung carcinoma A549 and lymphoma Ramos and further validated by in vivo zebrafish xenograft models with transplanted SGC-7901, A549 and Ramos. RESULTS: The results showed that the contents of polysaccharides, total triterpenoids and individual triterpenoids of RGLSP were significantly higher than those of BGLSP. Although both BGLSP and RGLSP inhibited the three tumor cell lines in vitro in a dose-dependent manner, the inhibitory effects of RGLSP were much better than those of BGLSP. In the in vivo zebrafish assay, RGLSP exhibited more potent inhibitory activities against tumors transplanted into the zebrafish compared with BGLSP, and the inhibition rates of RGLSP reached approximately 78%, 31% and 83% on SGC-7901, A549 and Ramos, respectively. CONCLUSION: The results indicated that the antitumor effects of GLS were positively correlated with the contents of the polysaccharides and triterpenoids and demonstrated that the wall-removing manufacturing technique could significantly improve the levels of active constituents, and thereby enhance the antitumor activity.

Profiling the mid-adult cecal microbiota associated with host healthy by using herbal formula Kang ShuaiLao Pian treated mid-adult mice.

With the occurrence of aging process, decreased neuron dopamine, disrupted brown adipose tissue (BAT) remodeling and decreased butyrate level all reflect a weak host healthy in certain degree. Nevertheless, the signs of mid-adult gut microbiota, and its association with host healthy are not well understood. In current study, we deemed to illustrate the associations of age, neuron dopamine, BAT remodeling, butyrate and gut microbiota with the aid of traditional herbal formula Kang Shuai Lao Pian (KSLP), which is known for its anti-aging effect. Here, ELISA was performed to detect the production of brain dopamine, the mass of inguinal white adipose tissue versus interscapular brown adipose tissue (iWAT/iBAT) was calculated and considered as a sign of BAT remodeling, 16S rRNA gene sequencing was used to the detection of gut microbiota profiling and gas chromatography was used to measure the butyrate level in mice feces. Our results indicated mid-adult mice already present distinctive gut microbiota profiling compared with young mice, concomitant with which are the lower brain dopamine level and disrupted brown adipose remodeling. KSLP treatment improved the host healthy and regulated gut microbiota with enriched Firmicutes at the expense of Bacteroidetes, particularly increased the relative abundance of bacteria functionally related to dopamine and butyrate productions, which suggest KSLP treatment constructs a healthier gut environment. In conclusion, modulation of gut microbiota and butyrate may connectively regulate dopamine production and BAT remodeling through gut-brain axis and gut-metabolism axis.

RNA-sequencing based bone marrow cell transcriptome analysis reveals the potential mechanisms of E'jiao against blood-deficiency in mice.

As a health-care food and traditional Chinese medicine, E'jiao, from the skin of Equus animus L, has been used to nourish blood in China for more than 2000 years. In modern medicine, there are also evidences indicate it has a beneficial effect on chemotherapy-caused blood deficiency. However, its mechanism of action for blood invigoration remains unclear. In the present study, we investigated the hematopoietic effect of E'jiao in 5-Fluorouracil-treated mice. In addition to the counting of bone marrow nucleated cells (BMNCs), flow cytometry was used to detect the population of hematopoietic stem cells (HSCs), and colony-forming unit (CFU) was used to assay the differentiation ability of hematopoietic progenitor cells (HPCs). Gene expression profiles of bone marrow cells were obtained from RNA sequencing (RNA-seq) and differentially expressed genes (DEGs) were analyzed with an emphasis on hematopoiesis-related pathways. The results show that E'jiao promotes the proliferation of both BMNCs and HSCs, as well as the differentiation of HPCs. By providing a hematopoiesis-related molecular regulatory network of E'jiao, we point out that the mechanism of E'jiao is associated with pathways including ECM-receptor interaction, Wnt signaling pathway, PI3K-Akt signaling pathway, TGF-beta signaling pathway, Hematopoietic cell lineage and Osteoclast differentiation, in which Ibsp, Col1a1, Col1a2, Notum, Sost, Dkk1, Irx5, Irx3 and Dcn are the key regulatory molecules. These findings provide valuable molecular basis for the mechanism of action of E'jiao.

Protective effect of Jie-Geng-Tang against Staphylococcus aureus induced acute lung injury in mice and discovery of its effective constituents.

ETHNOPHARMACOLOGICAL RELEVANCE: Jie-Geng-Tang (JGT), a famous traditional Chinese medicine prescription, consists of Platycodonis Radix and Glycyrrhizae Radix et Rhizoma. According to traditional medicinal theory, JGT exerts various effects, including apocenosis, detoxifying, moisturizing the lung and relieving sore throat. It is often used to treat throat inflammation and lung diseases. AIM OF THE STUDY: To determine the protective effect of JGT on Staphylococcus aureus (S. aureus)-induced acute lung injury (ALI) in mice and to identify the compounds in the prescription that may be responsible for antibacterial activity. MATERIALS AND METHODS: The protective effect of JGT was assessed using S. aureus-induced ALI mice (i.g., 2.7 g/kg/day). Bacterial burden, pathological morphology, cytokine levels of TNF-α, IL-1ß, KC, and MIP-2 were evaluated in the lung and bronchoalveolar lavage fluid at 24 h post-infection, respectively. Twenty three compounds in the prescription were evaluated for their minimum inhibitory concentration (MIC) in vitro by means of microbroth dilution method against S. aureus. The antibacterial effects in vitro of licochalcone A and isoliquiritigenin were also investigated by transmission electron microscopy. In vivo antibacterial activities of licochalcone A and isoliquiritigenin were evaluated by survival rates, bacterial burden, and pathological morphology of lung tissues on S. aureus-induced ALI in mice (i.p., 160 mg/kg/day). RESULTS: Pretreatment with JGT significantly improved the pathological morphology of lung tissues on S. aureus-induced ALI in mice, accompanied with the reduced bacterial burden in the lungs and inhibited expression of inflammatory cytokine levels at 24 h post-infection. Five compounds, namely licochalcone A, licoisoflavone B, glyasperin A, isoliquiritigenin, and licochalcone B from Jie-Geng-Tang displayed good antibacterial activities against S. aureus (MIC < 128 µg/mL). Furthermore, applications of licochalcone A and isoliquiritigenin resulted in the increased survival rates, reduced bacterial burden in the lungs, and improved pathological morphology of lung tissues in S. aureus infected mice. CONCLUSION: The study demonstrated that Jie-Geng-Tang presented protective role of acute lung injury, which supported its traditional use for the treatment of lung diseases. Licochalcone A, isoliquiritigenin, licoisoflavone B, glyasperin A, and licochalcone B might contribute to the antibacterial activity of JGT on S. aureus-induced acute lung injury. The anti-S. aureus activity of licoisoflavone B, glyasperin A, and licochalcone B in vitro, as well as the anti-S. aureus activity of licochalcone A in vivo, were first reported in this study.

Pharmacological and transcriptome profiling analyses of Fufang E'jiao Jiang during chemotherapy-induced myelosuppression in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang E'jiao Jiang (FEJ), a famous traditional Chinese medicine formula from "Liangyi Ointment", consists of five crude drugs, Colla corii asini, Radix Ginseng Rubra, Radix Rehmanniae Preparata, Codonopsis pilosula, and Crataegus pinnatifida Bge. It has pronounced functions of qi-nourishing and blood-activating. Recently, it has been widely used in China as a medication against myelosuppression in cancer treatment. AIM OF THE STUDY: We aimed to investigate the complex mode of action and underlying mechanisms of Fufang E'jiao Jiang (FEJ) regarding its hematopoietic effect. MAIN METHODS: Mice were divided into 5 groups of control, model, high dose FEJ (HFEJ), medium dose FEJ (MFEJ) and low dose FEJ (LFEJ). After 10 days from the administration, bone marrow cells (BMCs) were extracted for nucleated cells counts, flow cytometry analysis of hematopoietic stem cells (HSCs) population, as well as hematopoietic progenitor cells (HPCs) colony-forming unit (CFU) assay. A portion of bone marrow nucleated cells (BMNCs) of MFEJ group were prepared for RNA sequencing (RNA-Seq). The transcriptome data were analyzed based on the differentially expressed genes (DEGs). The molecular mechanisms of FEJ were deducted based on the biological processes and protein-protein interaction (PPI) network. RESULTS: FEJ could significantly increase the percentage of HSCs and the quantities of BFU-E and CFU-GM in BMSCs. FEJ could stimulate the proliferation of HSC and the differentiation of HPC to all lineages, which may thereby accelerate the recovery of hematopoietic function in myelosuppressive mice. By providing transcriptome profile we highlighted several genes and biological processes that might be applicable for FEJ to treat chemotherapy-induced myelosuppression. GO analysis showed that the co-expressed DEGs in FEJ vs model and model vs control group were involved in biological processes including ossification, osteoblast differentiation, bone mineralization and bone development. The KEGG pathway analysis pointed out ECM-receptor interaction and PI3K-AKT signaling pathway as the most relevant pathways to the function of FEJ on myelosuppression. PPI network showed MMP2 and COL1A1 were the relatively large nodes. CONCLUSION: FEJ has the hematopoietic effect in chemotherapy-induced myelosuppression mice. It might be achieved by improving the proliferative capacity of HSCs and the differentiation ability of HPCs. The molecular mode of action of FEJ might be the improvement of the bone marrow microenvironment via ECM-receptor interaction, the promoted proliferation of HSC through regulation of PI3K-AKT signaling pathway, and the involvement of osteoblasts and osteoclasts. MMP2 and COL1A1 appear to be the key relevant regulatory molecules. These results provide significant insight into the hematopoietic effects of FEJ in myelosuppression and point out novel targets for future validating analyses.

Transcriptome Profiling Analysis Reveals the Potential Mechanisms of Three Bioactive Ingredients of Fufang E'jiao Jiang During Chemotherapy-Induced Myelosuppression in Mice.

Although multiple bioactive components have been identified in Fufang E'jiao Jiang (FEJ), their hematopoietic effects and molecular mode of action in vivo are still not fully understood. In the current study, we analyzed the effects of martynoside, R-notoginsenoside R2 (R2), and 20S-ginsenoside Rg2 (Rg2) in a 5-fluorouracil-induced myelosuppression mouse model. Bone marrow nucleated cells (BMNCs) counts, hematopoietic progenitor cell colony-forming unit (CFU) assay, as well as flow cytometry analysis of Lin-/c-kit+/Sca-1+ hematopoietic stem cell (HSC) population were conducted, and bone marrow cells were subjected to RNA sequencing. The transcriptome data were processed based on the differentially expressed genes. The results of the analysis show that each of the three compounds stimulates BMNCs and HSC growth, as well as burst-forming unit-erythroid and colony-forming unit granulocyte-monocyte colony expansion. The most relevant transcriptional changes appeared to be involved in regulation of hematopoietic cell lineage, NF-κB and TNF-α signaling, inhibition of inflammation, and acceleration of hematopoietic cell recovery. Notably, the individual compounds shared similar but specified transcriptome profiles. Taken together, the hematopoietic effects for the three tested compounds of FEJ are confirmed in this myelosuppression mouse model. The transcriptome maps of these effects provide valuable information concerning their underlying mechanisms and provide a framework for the continued study of the complex mode of action of FEJ.

Transcriptomic Study Reveals Recovery of Impaired Astrocytes Contribute to Neuroprotective Effects of Danhong Injection Against Cerebral Ischemia/Reperfusion-Induced Injury.

Danhong Injection (DHI) is widely used in clinics for treating cardiovascular and cerebrovascular diseases in China. However, the mode of action of DHI for neuroprotection remains unclear. In the present study, we deemed to investigate the effects of DHI on a rat model of cerebral ischemia/reperfusion injury (IRI) with an emphasis on its regulated gene profile obtained from microarray assays. Firstly, we showed that a 14-day DHI treatment effectively ameliorated severity of neurological deficits, reduced size of ischemic damage, improved status of oxidation stress, as well as systemic inflammation for IRI rats, along with which was a pronounced reduced cell infiltration in the area of periaqueductal gray matter. Secondly, bioinformatic analyses for the 429 differentially expressed genes (DEGs) regulated by DHI treatment pointed out ECM-receptor interaction, neuroactive ligand-receptor interaction, and endocytosis as the top three biological processes, while Toll-like recptor 4 (TLR4) as the most relavant singaling molecule. Lastly, we provided evidences showing that DHI might directly protect primary astrocytes from oxygen and glucose deprivation/re-oxygenation (OGD/Re) injury, the effects of which was associated with LAMC2 and ADRB3, two DEGs related to the top three biological processes according to transcriptomic analysis. In conlusion, we reported that DHI might work through maintaining the integrity for brain-blood barrier and to regulate TLR4-related signaling pathway to diminish the inflammation, therefore, effectively improved the outcomes of IRI. Our findings suggested that the attenuated astrocytic dysfunction could be a novel mechanism contributing to the neuroprotective effects of DHI against cerebral ischemia/reperfusion-induced damage.

Shp2 Deficiency Impairs the Inflammatory Response Against Haemophilus influenzae by Regulating Macrophage Polarization.

Macrophages can polarize and differentiate to regulate initiation, development, and cessation of inflammation during pulmonary infection with nontypeable Haemophilus influenzae (NTHi). However, the underlying molecular mechanisms driving macrophage phenotypic differentiation are largely unclear. Our study investigated the role of Shp2, a Src homology 2 domain-containing phosphatase, in the regulation of pulmonary inflammation and bacterial clearance. Shp2 levels were increased upon NTHi stimulation. Selective inhibition of Shp2 in mice led to an attenuated inflammatory response by skewing macrophages toward alternatively activated macrophage (M2) polarization. Upon pulmonary NTHi infection, Shp2(-/-) mice, in which the gene encoding Shp2 in monocytes/macrophages was deleted, showed an impaired inflammatory response and decreased antibacterial ability, compared with wild-type controls. In vitro data demonstrated that Shp2 regulated activated macrophage (M1) gene expression via activation of p65-nuclear factor-κB signaling, independent of p38 and extracellular regulated kinase-mitogen-activated proteins kinase signaling pathways. Taken together, our study indicates that Shp2 is required to orchestrate macrophage function and regulate host innate immunity against pulmonary bacterial infection.