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Ethnopharmacology relevance Yi-Shen-Hua-Shi (YSHS) granule is a modern Chinese patent drug for treating chronic glomerulonephritis (CGN). It is derived from a traditional Chinese medicine formula Sheng-Yang-Yi-Wei decoction that is used to treat CGN in ancient China. Pharmacological activities of YSHS granule have not been reported. In this work, we investigated the anti-CGN effects and TGFβ signaling-related mechanism of action of this herbal drug. Materials and methods The rat model of CGN was established by injection of cationization-bovine serum albumin (C-BSA) for five weeks. After finishing C-BSA injection, drugs were intragastrically administered to the rats once daily for four weeks. Clinical signs were recorded daily. Serum and urine biochemical parameters were analyzed by respective kits. Protein levels were examined by Western blotting. Pathological changes of renal tissues were evaluated by HE and Masson's trichrome staining. Results and conclusions No significant differences in clinical signs and body weights were found among normal, model and drug treatment groups. Proteinuria; albuminuria; increased urine volume; elevated urea nitrogen, creatinine, total cholesterol and triglyceride levels in sera; decreased serum total protein and albumin; as well as renal pathological damage and fibrosis were observed in CGN model rats. YSHS granule ameliorated all the abnormal behavioral and biochemical changes in the model rats. Mechanical investigations showed that YSHS granule down-regulated proteins levels of TGFβ1, phospho-Smad2/3 (Thr 8) and Smad4 in rat renal tissues. In conclusion, YSHS granule demonstrates therapeutic effects in a rat model of CGN, and inhibition of the TGFβ/Smad signaling pathway is involved in the mechanism of action of the granule. This study provides a pharmacological basis for the use of modern YSHS granule and ancient Sheng-Yang-Yi-Wei decoction in treating CGN.

Supplemental material, Online supplementary file 1, for Chemical Profile Assessment and Potential Bioactive Component Screening of a Chinese Patent Herbal Drug Yi-Shen-Hua-Shi Granule by Chan Yuen-Cheung, Zhao Jing, Hu Qin, Guo Hui, Liu Yu-Xi, Wu Jia-Ying, Zheng Zhao-Guang and Yu Zhi-Ling in Natural Product Communications

Background Herba Siegesbeckiae (HS), the dried aerial parts of Siegesbeckia orientalis L., S. pubescens Makino, or S. glabrescens Makino, is traditionally used for treating chronic diseases in China. However, there is no information about the chronic toxicity of HS. The objective of this study is to evaluate the 24-week oral dosing toxicities of HS aqueous extract (HSE) in rats. Methods S. orientalis-originated HS was reflux-extracted with distilled water. Sprague–Dawley rats were randomly divided into four groups, with 10 males and 10 females in each group. The rats were intragastrically administered with HSE at 5, 1.67 and 0.56 g/kg (experimental groups) or an equal volume of distilled water (control group), 6 days a week, for 24 weeks. The high dose of HSE (5 g/kg) was its maximum tolerated dose. Body weight was recorded every 2 days during the experimental period. Chemical, hematological and histopathological parameters, as well as organ weights, were measured at the end of the experiment. Results Decreased body weight gain; increased liver and lung relative weights; histopathological alterations in liver and lung tissues; elevated serum levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase were found after HSE treatments. In liver tissues, HSE treatment upregulated levels of three pro-inflammatory cytokines: IL-6, IL-1β and TNF-α. In lung tissues, HSE treatment caused oxidative stress and activated mitogen-activated protein kinases (MAPKs). Conclusion Long-term oral administration of HSE caused toxicities in rats evidenced by decreased body weight gain, as well as liver and lung damage. Treatment-induced oxidative stress, inflammation and MAPK activation are involved in HSE’s toxicities. Caution should be taken when using HS to treat chronic diseases.

Additional file 2: Figure S3. Original blot images of Western blot. Protein levels of p38, phospho-p38 (Thr180/Tyr182), JNK, phospho-JNK (Thr183/Tyr185), ERK and phospho-ERK (Thr202/Tyr204) and α-Actinin were shown. The bands were shown on different films because of different exposure time.

Additional file 1: Figure S1. An HPLC method developed for quality control of HSE. (a) HPLC chromatograms of the chemical marker kirenol (5.0 μg/mL) (upper panel) and HSE (lower panel). HPLC analysis was performed on an Agilent 1260 system equipped with a Diode-array detector. Separations were performed on an AlltimaTM C-18 analytical column (250 mm × 4.6 mm I.D., 5 μm) and an Alltima C-18 guard-column (12.5 mm × 4.6 mm I.D., 5 μm) maintained at 25 °C. Isocratic elution was performed with a mobile phase of A (0.1% phosphate acid solution, analytical grade, RCI Labscan Limited) and B (ACN, HPLC grade, RCI Labscan Limited) (70:30, v/v). The flow rate was maintained at 0.35 mL/min, and sample injection volume was 5 μL in each test. Since kirenol has a prominent absorption around 215 nm in the UV spectrum, 215 nm was chosen as the reference wavelength. (b) Contents of kirenol in HSE. Figure S2. Body weight changes of rats treated once with 5 g/kg of HSE. Rats were randomly divided into two groups: control group and 5 g/kg of HSE group, each 5 males and 5 females. Rats were i.g. administered with distilled water (control group) or 5 g/kg of HSE (5 g/kg of HSE group), and observed for 14 days. (a) Body weight of male rats. (b) Body weight of female rats. Values are expressed as mean ± SEM (n = 5). Table S1. Absolute and relative organ weights of rats in the 24-week oral dosing toxicity test. Table S2. Urinalyses of rats in the 24-week oral dosing toxicity test. Table S3. Hematological parameters of rats in the 24-week oral dosing toxicity test. Table S4. Serum assay parameters of rats in the 24-week oral dosing toxicity test.

Herba Siegesbeckiae (HS, the dried aerial part of Siegesbeckia orientalis L.) is a commonly used traditional Chinese medicinal herb for treating inflammatory diseases. HS has been reported to exert anti-inflammatory effects by inhibiting the MAPKs and NF-κB pathways, the downstream effectors of TLR4 signalling. This study aims to further investigate the involvement of TLR4 signalling cascades in the effects of an ethanolic extract of HS (HS for short) on inflammatory mediators in murine macrophages. HS was extracted using 50% ethanol. Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were used as the cell model. ELISA was used to detect cytokine/chemokine secretion. Real time-PCR and immunoblotting were used to examine mRNA and protein levels, respectively. We observed that HS dose-dependently inhibited the secretion of PGE2, MCP-1, MIP-1α and RANTES, and down-regulated mRNA levels of iNOS, COX-2, IL-1β, IL-6, TNF-α, mPGES-1, MCP-1, MIP-1α and RANTES in LPS-stimulated RAW264.7 cells. HS did not affect the protein levels of TAK1, TBK1, PI3K, Akt, IKK, c-Jun, c-Fos and IRF3, while, dose-dependently decreased levels of their phosphorylated forms. The protein levels of IRAK1 and IRAK4 were upregulated, while those of TRAF6 and TRAF3 were downregulated by HS. Moreover, the nuclear protein levels of AP-1, NF-κB and IRF3 were dose-dependently decreased by HS. These results indicate that suppression of the IRAK4/MAPKs/AP-1, IRAK4/MAPKs/NF-κB, IRAK4/PI3K/NF-κB and TRAF3/TBK1/IRF3 pathways is associated with the inhibitory effects of HS on inflammatory mediators in LPS-stimulated RAW264.7 cells. This study provides a pharmacological basis for the clinical application of this herb in the treatment of inflammatory disorders.

Background Herba Siegesbeckiae (HS, Xixiancao in Chinese) is a commonly used traditional Chinese medicinal herb for soothing joints. In ancient materia medica books, HS is recorded to be the aerial part of Siegesbeckia pubescens Makino (SP) which is also the only origin of HS in the 1963 edition of the Chinese Pharmacopeia (ChP). The aerial parts of Siegesbeckia orientalis L. (SO) and Siegesbeckia glabrescens Makino (SG) have been included as two additional origins for HS in each edition of ChP since 1977. However, chemical and pharmacological comparisons among these three species have not been conducted. Methods An HPLC with diode array detector (HPLC-DAD) method combined with similarity analysis, hierarchical cluster analysis (HCA) and principal component analysis (PCA) was developed for comparing the fingerprint chromatograms of the three species. The inhibitory effects of the three species on NO production and IL-6 secretion in LPS-stimulated RAW264.7 macrophages were compared. Results Fingerprint chromatograms of the three species showed different profiles, but had 13 common peaks. Results from HCA and PCA of the common peaks demonstrated that all 14 herbal samples of the three species tended to be grouped and separated species dependently. The extents of inhibition on NO production and IL-6 secretion of the three species were different, with SG being the most and SP the least potent. Conclusions Both chemical profiles and inflammatory mediator-inhibitory effects of the three species were different. These findings provide a chemical and pharmacological basis for determining whether the three species can all serve as the origins of HS.

A traditional Chinese medicine (TCM) formula (SL) comprising Sophorae Flos and Lonicerae Japonicae Flos was used for treating melanoma in ancient China. We have previously shown that an ethanolic extract of SL (SLE) possesses anti-melanoma effects and suppresses STAT3 signaling in vitro and in vivo. STAT3 has been linked to the development of melanoma immunosuppressive microenvironment. In this work, we investigated whether SLE inhibits melanoma growth by reprogramming the tumor microenvironment in mouse and co-culture cell models. In B16F10 melanoma-bearing mice, we found that intragastric administration of SLE (1.2 g/kg) dramatically inhibited tumor growth. This observation was associated with the downregulation of protein levels of phospho-STAT3 (Tyr 705) and STAT3-regulated immunosuppressive cytokines, and mRNA levels of STAT3-targeted genes involved in tumor growth and immune evasion. We also observed increased Th, Tc and dendritic cells in the melanomas and spleens in SLE-treated mice compared to that in control mice. In a co-culture system composed of B16F10 cells and mouse primary splenic lymphocytes, it was found that SLE not only inhibited STAT3 activation in B16F10 cells, but also downregulated mRNA levels of STAT3-targeted genes in the splenic lymphocytes. In this co-culture setting, SLE decreased the levels of STAT3-regulated immunosuppressive cytokines, increased the percentages of Th, Tc and dendritic cells as well. Furthermore, effects of SLE on STAT3 phosphorylation, cytokine levels and immune cell subtype percentages were significantly weaker in the B16STAT3C cells (stable cells harboring a constitutively active STAT3 variant STAT3C)/splenic lymphocytes co-culture system than in the B16V cells (cells stably transfected with the empty vector)/splenic lymphocytes co-culture system, indicating that STAT3 over-activation diminishes SLE’s effects. In summary, our findings indicate that reprograming the immune microenvironment, partially mediated by inhibiting STAT3 signaling, contributes to the anti-melanoma mechanisms of SLE. This study provides further pharmacological groundwork for developing SLE as a modern agent for melanoma prevention/treatment, and supports the notion that reprograming immunosuppressive microenvironment is a viable anti-melanoma strategy.