Qilongtian ameliorate bleomycin-induced pulmonary fibrosis in mice via inhibiting IL-17 signal pathway.

Pulmonary fibrosis (PF) is a special type of pulmonary parenchymal disease, with chronic, progressive, fibrosis, and high mortality. There is a lack of safe, effective, and affordable treatment methods. Qilongtian (QLT) is a traditional Chinese prescription that is composed of Panax notoginseng, Earthworm, and Rhodiola, and shows the remarkable clinical curative effect of PF. However, the mechanism of QLT remains to be clarified. Therefore, we studied the effectivity of QLT in treating Bleomycin (BLM) induced PF mice. 36 C57BL/6 J mice were randomized into the control group, the model group, the low-, medium- and high-dose QLT group, and Pirfenidone group. After establishing a model of pulmonary fibrosis in mice, the control and model groups were infused with a normal saline solution, and the delivery group was infused with QLT. Pulmonary function in the mice from each group was detected. Pulmonary tissue morphologies and collagen deposition were stained by HE and Masson. The content of hydroxyproline (HYP) was detected by alkaline hydrolysis and the mRNA and protein expression of related genes in pulmonary tissues were detected by using q-PCR, ELISA, and Western blot. Our studies have shown that QLT significantly reduced the inflammatory injury, hydroxy-proline content, and collagen deposition of pulmonary tissue in BLM-induced PF mice and down-regulated the cytokine related to inflammation and fibrosis and PF expression on the mRNA and protein level in PF mice. To identify the mechanism of QLT, the Transcriptome was measured and the IL-17 signal pathway was screened out for further research. Further studies indicated that QLT reduced the mRNAs and protein levels of interleukin 17 (IL-17), c-c motif chemokine ligand 12 (CCL12), c-x-c motif chemokine ligand 5 (CXCL5), fos-like antigen 1 (FOSL1), matrix metalloproteinase-9 (MMP9), and amphiregulin (AREG), which are inflammation and fibrosis-related genes in the IL-17 signal pathway. The results indicated that the potential mechanism for QLT in the prevention of PF progression was by inhibiting inflammation resulting in the IL-17 signal pathway. Our study provides the novel scientific basis of QLT and represents new therapeutics for PF in clinical.

Qi-Long-Tian capsule alleviates pulmonary fibrosis development by modulating inflammatory response and gut microbiota.

Pulmonary fibrosis (PF) is a chronic, progressive, and fibrotic interstitial lung disease with a high mortality rate. Qi-Long-Tian (QLT) capsule is an herbal formula with great potential for antifibrotic effects, consisting of San Qi (Notoginseng Radix et Rhizoma), Di Long [Pheretima aspergillum (E. Perrier)], and Hong Jingtian (Rhodiolae Crenulatae Radix et Rhizoma), and has been used in clinical practice for many years. To explore the relationship between the effects of Qi-Long-Tian capsule and gut microbiota of PF mice, pulmonary fibrosis model were established by tracheal drip injection of bleomycin. Thirty-six mice were randomly divided into 6 groups: control group (control), model group (model), QLT capsule low dose group (QL), QLT capsule medium dose group (QM), QLT capsule high dose group (QH), and pirfenidone group (PFD). After 21 days of treatment, after pulmonary function tests, the lung tissues, serums, and enterobacterial samples were collected for further analysis. HE staining and Masson's staining were used to detect changes as the main indicators of PF in each group, and the expression of hydroxyproline (HYP) related to collagen metabolism was detected by and alkaline hydrolysis method. qRT-PCR and ELISA were used to detect the mRNA and protein expressions of pro-inflammatory factors include interleukin 1ß (IL-1ß), interleukin 6 (IL-6), transforming growth factor ß1 (TGF-ß1), tumor necrosis factor α (TNF-α) in lung tissues and serums, and the inflammation-mediating factors include tight junction protein (ZO-1, Claudin, Occludin). ELISA was used to detect the protein expressions of secretory immunoglobulin A (sIgA), short-chain fatty acids (SCFAs), and lipopolysaccharide (LPS) in colonic tissues. 16sRNA gene sequencing was used to detect changes in the abundance and diversity of intestinal flora in the control, model, and QM groups, to search for differential genera, and analyze the correlation with inflammatory factors. QLT capsule effectively improved the status of pulmonary fibrosis and reduced HYP. In addition, QLT capsule significantly reduced the abnormal levels of pro-inflammatory factors, including IL-1ß, IL-6, TNF-α, and TGF-ß in lung tissue and serum, while improving the levels of pro-inflammatory related factors ZO-1, Claudin, Occludin, sIgA, SCFAs, and reducing LPS in the colon. The comparison between the alpha diversity and beta diversity in enterobacteria suggested that the composition of the gut flora in the control, model, and QLT capsule groups were different. QLT capsule significantly increased the relative abundance of Bacteroidia (which might limit the onset of inflammation) and decreased the relative abundance of Clostridia (which might promote inflammation). In addition, these two enterobacteria were closely associated with pro-inflammatory-related indicators and pro-inflammatory factors in PF. All these results suggest that QLT capsule intervenes in pulmonary fibrosis by regulating the differential genera of intestinal flora, increasing immunoglobulin secretion, repairing the intestinal mucosal barrier, reducing LPS entry into the blood, and decreasing inflammatory factor secretion in the serum, which in turn alleviates pulmonary inflammation. This study clarifies the therapeutic mechanism of QLT capsule in PF and provides a theoretical basis for it. It provides a theoretical basis for its further clinical application.

Identification of LEA, a podocalyxin-like glycoprotein, as a predictor for the progression of colorectal cancer.

Large external antigen (LEA) is considered as a colorectal cancer (CRC)-associated antigen, which was found via mAb ND-1 generated using hybridoma technology, but its molecular features remain unknown. To facilitate the clinical application of LEA, we identified LEA as a podocalyxin-like protein 1 (PODXL) with molecular weight of approximately 230 kDa, a hyperglycosylated protein, using immunoprecipitation and mass spectrometry in combination, and verified that ND-1-recognized epitope is on the terminal sialic acid of LEA. Correlation analysis between LEA and PODXL in molecular weight, immunological cross-reactivity, and gene expression dependence supported the PODXL identity of the LEA. Moreover, we assessed the clinical significance of the LEA in 89 pairs of primary CRC tissues and adjacent nontumor colorectal tissues using ND-1 by quantum dot-based immunohistochemistry (QD-IHC). High LEA expression was correlated significantly with T stage (P = 0.010). Patients with high LEA expression showed significantly poorer prognosis than those with LEA low expression (P = 0.007). Multivariate analysis indicated LEA expression as an independent predictor. Furthermore, the comparative analysis showed that mAb ND-1-based IHC analysis toward sugar residue of PODXL has higher sensitivity and specificity to evaluate the LEA/PODXL expression than mAb 3D3-based method toward core protein of PODXL in CRC cell lines and clinical samples. In addition, we first found that LEA/PODXL can be secreted in exosomes from cancer cells and CRC patient peripheral blood. Our results demonstrate that LEA is an independent predictor for CRC progression and has the potential to be applied for clinical setting with high sensitivity, high specificity, and noninvasive access.

Anticancer Effects of Sinocrassulosides VI/VII from Silene viscidula on HeLa Cells.

Natural products are becoming increasingly important in chemoprevention and for cancer therapy. Silene viscidula (S. viscidula), a traditional Chinese herb, has long been used as an anti-inflammatory and neuroleptic agent. However, the anticancer activity of S. viscidula has remained unclear. In this study, 16 compounds were extracted from S. viscidula. Among those compounds, sinocrassulosides VI/VII, an inseparable isomer mixture, possess the strongest inhibitory activity on HeLa cells with the IC50 value of 2.37 µM. Mechanism studies found that sinocrassulosides VI/VII downregulated the expression of cyclin D1 and decreased retinoblastoma (Rb) phosphorylation, which arrested HeLa cells in the G1 phase. Also, sinocrassulosides VI/VII could induce senescence via the upregulation of p16 and a significant increase of ß-galactosidase (ß-gal) staining. Our results suggest that sinocrassulosides VI/VII may be a new therapeutic potential agent for cervical cancer. In addition, we explored the structure-activity relationships of three groups of the configurational isomer with similar chemical structure from S. viscidula. We first demonstrated that the length of the ester chains linked to the carboxyl group of the glucuronic acid residue could affect the potent cytotoxicity. This finding will open new avenues for developing effective anticancer compounds by modifying the components derived from plants in nature.

Establishment of a gastric cancer subline with high metastatic potential using a novel microfluidic system.

Metastasis is an important hallmark of malignant tumors. In this study, we developed a microfluidic system to screen highly metastatic sublines via differential resolution of cell invasiveness. The system was composed of a PDMS-glass device connected with a syringe pump and a Petri dish. To facilitate the selection process, a long-term cell invasion driving force based on a chemotactic factor gradient was created using the Petri dish-based liquid supply pattern, and the invasive cells were collected for round-by-round selection via an open region in the chip. Using the system, we established an SGC-7901/B2 subline from the human gastric cancer SGC-7901 cell line by only two rounds of selection. In vitro assays showed that the SGC-7901/B2 cells were superior to the parental cells in proliferation and invasiveness. Furthermore, an in vivo tumorigenicity assay demonstrated that compared with the parental cells, the subline had stronger spontaneous metastatic and proliferative capability, which led to a shorter survival duration. Additionally, the protein expression differences including E-cadherin and Smad3 between the subline and parental cells were revealed. In conclusion, this microfluidic system is a highly effective tool for selecting highly metastatic sublines, and SGC-7901/B2 cells could serve as a potential model for tumor metastasis research.

Quantitative detection of the tumor-associated antigen large external antigen in colorectal cancer tissues and cells using quantum dot probe.

The large external antigen (LEA) is a cell surface glycoprotein that has been proven to be highly expressed in colorectal cancer (CRC) as a tumor-associated antigen. To evaluate and validate the relationship between LEA expression and clinical characteristics of CRC with high efficiency, LEA expression levels were detected in 85 tissue blocks from CRC patients by quantum dot-based immunohistochemistry (QD-IHC) combined with imaging quantitative analysis using quantum dots with a 605 nm emission wavelength (QD605) conjugated to an ND-1 monoclonal antibody against LEA as a probe. Conventional IHC was performed in parallel for comparison. Both QD-IHC and conventional IHC showed that LEA was specifically expressed in CRC, but not in non-CRC tissues, and high LEA expression was significantly associated with a more advanced T-stage (P<0.05), indicating that LEA is likely to serve as a CRC prognostic marker. Compared with conventional IHC, receiver operating characteristic analysis revealed that QD-IHC possessed higher sensitivity, resulting in an increased positive detection rate of CRC, from 70.1% to 89.6%. In addition, a simpler operation, objective analysis of results, and excellent repeatability make QD-IHC an attractive alternative to conventional IHC in clinical practice. Furthermore, to explore whether the QD probes can be utilized to quantitatively detect living cells or single cells, quantum dot-based immunocytochemistry (QD-ICC) combined with imaging quantitative analysis was developed to evaluate LEA expression in several CRC cell lines. It was demonstrated that QD-ICC could also predict the correlation between LEA expression and the T-stage characteristics of the cell lines, which was confirmed by flow cytometry. The results of this study indicate that QD-ICC has the potential to noninvasively detect rare circulating tumor cells in clinical samples in real clinical applications.