New indication of Chuankezhi injection for steroid-resistant focal segmental glomerulosclerosis and its mechanism of action.

Background: Chuankezhi (CKZ) injection is a traditional Chinese medicine (TCM) injection extracted from Chinese herbs Epimedium sagittatum (Yin Yang Huo) and Morinda officinalis (Bai Ji Tian). Studies have shown that CKZ has a positive effect on improving diabetic nephropathy and regulating immune function. Focal segmental glomerulosclerosis (FSGS) is a kind of refractory nephropathy, which has been confirmed as closely associated with immunity. Whether CKZ is effective against FSGS and how it works warrant further study. This study aimed to verify the efficacy of CKZ in rats with steroid-resistant (SR) FSGS and explore its mechanism of action. Methods: We established an SR FSGS model in male Sprague Dawley (SD) rats by injecting adriamycin into the tail vein. Based on group intervention and comparison, the primary efficacy parameters of FSGS were observed, including general condition, 24-hour urine protein, serum albumin, cholesterol, triglyceride, and renal pathological changes. Network pharmacological analysis and molecular docking were used to predict the mechanism of action of CKZ. Finally, we used quantitative polymerase chain reaction (qPCR) and western blot (WB) to detect messenger RNA (mRNA) expression and protein phosphorylation at specific targets in rat kidney tissue to validate the predicted results. Results: Intramuscular injection of CKZ had a dose-dependent effect in SR FSGS model rats, including lowering urine protein, increasing serum albumin, lowering cholesterol and triglyceride, and treating pathological lesions in the kidney. Network pharmacological analysis and Molecular docking revealed that 5 active components (Icariin, Icariside II, Epimedin C, Icaritin, and Noricaritin) might be the critical components. The findings also revealed that Akt was perhaps the critical target gene, the PI3K-Akt signaling pathway was perhaps the critical pathway, and reversible protein phosphorylation was probably the critical biological process. The qPCR and WB analyses showed that CKZ significantly increased the relative mRNA expression and protein phosphorylation of PI3K and Akt, respectively. Conclusions: This study showed that intramuscular injection of CKZ has a significant therapeutic effect in SR FSGS rats, which may be associated with the activation of PI3K-Akt signaling by CKZ.

Integrated RNA-sequencing and network pharmacology approach reveals the protection of Yiqi Huoxue formula against idiopathic pulmonary fibrosis by interfering with core transcription factors.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a refractory disease. Therefore, developing effective therapies for IPF is the need of the hour. PURPOSE: Yiqi Huoxue Formula (YQHX) is an herbal formula comprising three herbal medicines: Ligusticum chuanxiong Hort. (Chuanxiong Rhizoma, CR), Panax notoginseng (Burk.) F. H. Chen (Notoginseng Radix Et Rhizoma, NR) and Panax ginseng C. A. Mey. (Ginseng Radix Et Rhizoma, GR). This study aims to determine the anti-pulmonary fibrosis effect of YQHX and explore its mechanism of action. STUDY: Design and Methods: The chemical components in the GR, CR and NR extracts were identified by High Performance Liquid Chromatography. A TGF-ß1-induced myofibroblast cell model was used to test the anti-fibrosis effect of GR, CR, NR and YQHX. RNA-sequencing was used to identify the differentially expressed genes (DEGs) after YQHX treatment. Subsequently, gene enrichment analysis and key transcription factors (TFs) prediction for YQHX-regulated DEGs was performed. The active constituents of GR, CR and NR were obtained from the Traditional Chinese Medicine Database and Analysis Platform. Targets of the active constituents were predicted using the similarity ensemble approach search server and Swiss Target Prediction tool. YQHX-targeted key TFs that transcribed the DEGs were screened out. Then, the effect of YQHX on the bleomycin-induced pulmonary fibrosis mouse model was studied. Finally, one of the predicted TFs, STAT3, was selected to validate the prediction accuracy. RESULTS: Seven, two, and five compounds were identified in the GR, CR, and NR extracts, respectively. YQHX and its constituents-GR, CR and NR-inhibited the expression of fibrotic markers, including α -SMA and fibronectin, indicating that YQHX inhibited TGF-ß1-induced myofibroblast activation. RNA-sequencing identified 291 genes that were up-regulated in the TGF-ß1 group but down-regulated after YQHX treatment. In total, 55 key TFs that transcribed YQHX-regulated targets were predicted. A regulatory network of 24 active ingredients and 232 corresponding targets for YQHX was established. Among YQHX's predicted targets, 20 were TFs. On overlapping YQHX-targeted TFs and DEGs' key TFs, six key TFs, including HIF1A, STAT6, STAT3, PPARA, DDIT3 and AR, were identified as the targets of YQHX. Additionally, YQHX alleviated bleomycin-induced pulmonary fibrosis in a mouse model by inhibiting the phosphorylation of STAT3 in the lungs of pulmonary fibrosis mice. CONCLUSIONS: This study provides pharmacological support for the use of YQHX in the treatment of IPF. The potential mechanism of action of YQHX is speculated to involve the modulation of core TFs and inhibition of pathogenetic gene expressions in IPF.

Chemiluminescence quenching microarrays for high throughput screening of antioxidants and its application in evaluating herbal extracts and pure compounds.

In this paper, a novel high throughput screening method for antioxidants was described. The screening process was completed on a 5 cm × 5 cm silica gel 60 plate. Luminol was used as the fluorogenic substrate while hydrogen peroxide employed to excite the chemiluminescence. A dark spot was observed due to quenching effect of antioxidant. A linear model of the integral area of the spot as a function of analyte amount was fit by using vitamin C as positive control. Excellent linearity (R2 = 0.9978) was obtained in the range of 2.31-23.1 µg. The variation coefficients of intra- and inter-chip precisions were below 5% (n = 10) and 10% (n = 6) respectively. The new method was validated by comparing the analysis result of six flavonoids with that from a sequential injection method. The Pearson correlation coefficient was up to 0.98. A set of traditionally used herbal medicines was screened, and Rhei Radix et Rhizoma showed the strongest signal. The major bioactive ingredients were further assigned by flow injection and MS analyses. These results reveal the prospects of the proposed method to supply a promising tool in vitro for high throughput screening and activity evaluation of antioxidants in a fast, low-cost and reliable manner.

Pharmacokinetics, Tissue Distribution, and Metabolism Study of Icariin in Rat.

Icariin is one of the predominant flavonoids contained in Herba Epimedii (Yin-yang-huo in Chinese), a well-known Chinese medicine for the treatment of cancers and immune system diseases. Although Herba Epimedii has been widely used in China and there are so many and various research reports on the herbal drug and its main flavones, very limited data is available on the tissue distribution and biotransformation of icariin. In the present study, a liquid chromatographic method combined with electrospray ionization tandem mass spectrometry was developed to quantify the concentration of icariin in rat plasma and various tissues collected at different time points after oral administration of the total flavonoid extract of Herba Epimedii at a dose of 0.69 g/kg (corresponding to 42 mg/g icariin). Biological samples were processed by simple protein precipitation. Genistein was chosen as internal standard. The method was successfully applied to plasma pharmacokinetic and tissue distribution studies of icariin in rat. As a result, it was worth noting that the tissue distribution characteristics of icariin exhibited a significant gender difference. Moreover, in vivo metabolism of icariin was also investigated. A total of 11 potential metabolites were found in rat feces collected in different time periods after oral and intramuscular administration of icariin. In vivo metabolic pathways were involved in hydrolysis, demethylation, oxidation, and conjugation. The preclinical data would be useful for fully understanding in vivo disposition of this compound and interpreting the mechanism of its biological response.