Kuijieling decoction suppresses NLRP3-Mediated pyroptosis to alleviate inflammation and experimental colitis in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) is an autoimmune disease. Although the mortality rate of UC is not very high, it has a considerable morbidity rate and an unsatisfactory cure rate. Without effective treatment, UC is likely to develop into colon cancer. Kuijieling (KJL) is an effective empirical formula to treat UC in the clinical setting, and it has been proven to have curative effects against UC. AIM OF THE STUDY: In a previous study, we demonstrated that KJL could suppress NOD-like receptor protein 3 (NLRP3) to reduce inflammatory cytokines and alleviate UC. In this study, we investigated the mechanism of KJL in more detail, from the perspective of pyroptosis. MATERIALS AND METHODS: We established a dextran sulfate sodium-induced UC mouse model and RAW264.7 cells to measure different indicators with different experimental methods. The efficiency of KJL was evaluated by measuring the length and unit weight of mouse colons, and assessment of pathological injury was performed using HE staining. We detected different expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, gasdermin-D C-terminal domain (GSDMD-C), gasdermin-D N-terminal domain (GSDMD-N), IL-1ß, and IL-18 in colon tissues and cells using RT-qPCR and western blotting. Immunohistochemistry was used for tissues and immunofluorescence for cells to confirm protein expression. IL-1ß and IL-18 were measured with enzyme-linked immunosorbent assay in serum, tissue, and cell culture supernatant. MiR-223 was detected using RT-qPCR. RESULTS: After administration of KJL suspension, colon damage in KJL groups was milder than in model groups. ASC, caspase-1, IL-1ß, and IL-18 mRNA levels in colon tissue were decreased to different degrees in the KJL groups. Protein expression of NLRP3, caspase-1, GSDMD-N, IL-1ß, and IL-18 in vivo decreased significantly in the KJL groups. In addition, Mir-223 level decreased in colon tissue of the KJL groups. In vitro, NLRP3, ASC, caspase-1, GSDMD-N, IL-1ß, and IL-18 levels decreased to varying degrees, at both mRNA and protein levels. Mir-223 was lower in the KJL groups than in the model group. Furthermore, KJL was shown to regulate the level of miR-223, which returned to normal after its expression was inhibited or promoted, and the levels of associated indicators also returned to normal after transfection. CONCLUSIONS: KJL is able to inhibit pyroptosis to alleviate UC, but these suppression effects were not mediated through miR-223 regulation.

Kuijieling-Containing Serum Regulates Th17 and Treg Cell Differentiation by Inhibiting STAT3 Signaling In Vitro.

OBJECT: To investigate the effect of Kuijieling (KJL) on the balance between T helper 17 (Th17) and regulatory T (Treg) cells in peripheral blood mononuclear cells (PBMC) in vitro and explore the underlying mechanism. MATERIALS AND METHODS: PBMCs isolated from rats were stimulated with transforming growth factor-ß, interleukin (IL)-6, and IL-23 to induce the imbalance of Th17 and Treg cells and were treated with 10, 5, or 2.5% KJL-containing serum. The proportion of Th17 or Treg cells in CD4+ T cells was analyzed by flow cytometry, the concentrations of IL-17, IL-21, and IL-10 were assayed by ELISA, mRNA expressions of retinoic acid-related orphan receptor γt (RORγt), forkhead box protein 3 (Foxp3), and signal transducer and activator of transcription 3 (STAT3) were quantified by PCR, and phosphorylated STAT3 (p-STAT3) was analyzed by flow cytometry. RESULTS: KJL-containing serum decreased the proportion of Th17 cells and increased the proportion of Treg cells in CD4+ T cells, decreased the concentration of IL-17 and IL-21, enhanced the level of IL-10 in the cell culture supernatant, promoted the expression of Foxp3, and inhibited the levels of RORγt, STAT3, and p-STAT3. CONCLUSION: KJL suppresses the STAT3 pathway to remedy the imbalance between Th17 and Treg cells.

Kuijieling regulates the differentiation of Treg and Th17 cells to ameliorate experimental colitis in rats.

BACKGROUND: Regulatory T (Treg) cells and T helper 17 (Th17) cells play crucial roles in ulcerative colitis (UC). Kuijieling (KJL) is an effective Chinese medicine formula for treating UC in clinic. Kuijieling has shown remedy effect on the imbalance between Treg and Th17 cells. This study aimed to further reveal the exact underlying mechanism of how Kuijieling regulates the differentiation of Treg and Th17 cells in the treatment of UC. METHODS: Colitis was induced by trinitrobenzene sulfonic acid in rats and treated by KJL. Pathological injury was evaluated by HE staining and pathological score. Transforming growth factor-ß1 (TGF-ß1), interleukin(IL)-2, IL-6, IL-10, IL-17, IL-23 and IL-21 in plasma were assayed by ELISA. Forkhead box P3 (Foxp3), signal transducer and activator of transcription (STAT) 5 expressed in colon mucosa were measured by western blot. Immunohistochemistry was employed for quantifying retinoic acid-related orphan receptor γt (RORγt) and STAT3 in colon. RT-PCR was used to analyze the expression of IL-2, IL-17, IL-23, IL-21 mRNA in colon. RESULTS: After the administration of KJL, pathological injury in colon mucosa was reduced and histological score was decreased, transforming growth factor-ß1 (TGF-ß1), interleukin(IL)-2, IL-10 in blood and Foxp3, STAT5, IL-2 in colon increased significantly, IL-6, IL-23, IL-17, IL-21 in blood and RORγt, STAT3, IL-23, IL-17, IL-21 in colon decreased. Our result showed that KJL regulates the related cytokines and transcription factors to promote Treg cells and suppress Th17 cells. CONCLUSION: KJL restores the balance between Treg and Th17 cells through regulating the differentiation of them, therefore contributes to the treatment of UC.

Spatiotemporal distributions of nutrients in the downstream from Gezhouba Dam in Yangtze River, China.

INTRODUCTION: From December 2008 to November 2009, an investigation of water quality was performed in the 70-km long downstream from Gezhouba Dam in Yangtze River. METHODS: Twelve sites in all were chosen. Nine parameters of water quality including the total phosphorus, the total nitrogen, chlorophyll a (Chl.a), nitrite, nitrate, ammonia, water temperature, DO, and pH were monitored almost monthly. The multivariate statistical technique (cluster analysis) and the nonparametric method (Kruskal-Wallis Test and Spearman's rank correlation) were applied to evaluate the spatiotemporal variations of water quality data sets. RESULTS AND DISCUSSION: According to the Chinese environmental quality standards for surface water (GB3838-2002), the water quality in the river section investigated can attain to the III water standards except total nitrogen. Further analysis indicated that there were no significant spatial differences in these parameters of water quality, but the sampling date had a significant effect. The temporal variation of water quality can be related to the discharge of Gezhouba Dam and moreover be affected by the reservoir regulation. During the discharge, the discharge increased the concentration of total phosphorus and then decreased the N:P ratio, which helps to the occurrence of algae blooms. The high consternation of phosphorus and the low N:P ratio show that the water body can be in the process of eutrophication during the discharge of Gezhouba Dam. In fact, Chl.a had begun to rise in the same period.

Present status and changes of the phytoplankton community after invasion of Neosalanx taihuensis since 1982 in a deep oligotrophic plateau lake, Lake Fuxian in the subtropical China.

Phytoplankton assemblages in the subtrophical oligotrophic Lake Fuxian, the second deepest lake in China, were investigated monthly from September 2002 to August 2003. A total of 113 species belonging to seven phyla were identified, among them, a filamentous green alga, Mougeotia sp., dominated almost throughout the study period and comprised most of the total phytoplankton biomass. Mougeotia sp. has made a substantial development during the past decades: it was absent in 1957, only occasionally present in 1983, increased substantially in 1993, and became predominant in 2002-2003. It is likely that natural invasion of the Taihu Lake noodlefish (Neosalanx taihuensis) has led to a change of dominant herbivorous zooplankton from small to large calanoid, which has increased grazing pressure on small edible algae, and thus has indirectly favored the development of the inedible filamentous Mougeotia sp.

The low TN:TP ratio, a cause or a result of Microcystis blooms?

An enclosure experiment in the shallow, subtropical Lake Donghu, China, was performed in the summer of 2001 to examine the effect of TN:TP (total phosphorus) ratios and P-reduction on the occurrence of Microcysitis blooms. The treatments were performed with enough amounts of N but with different amounts of P in the water column and sediment. Microcystis blooms occurred in the enclosures either with an initial TN:TP <29 or TN:TP>29 where the nutrients (N, P) were high enough. Microcysitis blooms never occurred in the treatments with low P concentration in spite of the presence of sufficient N. The P-rich sediments served as an important source for the P supply in the water column, and such a process was activated greatly by the outburst of Microcystis blooms which pumped up selectively P from the sediments and thus decreased the TN:TP ratios. Therefore, the low TN:TP ratio is not a cause but rather a result of Microcystis blooms.