Physalin B reduces Aß secretion through down-regulation of BACE1 expression by activating FoxO1 and inhibiting STAT3 phosphorylation.

Physalin B (PB), one of the major active steroidal constituents of Solanaceae Physalis plants, has a wide variety of biological activities. We found that PB significantly down-regulated ß-amyloid (Aß) secretion in N2a/APPsw cells. However, the underlying mechanisms are not well understood. In the current study, we investigated the changes in key enzymes involved in ß-amyloid precursor protein (APP) metabolism and other APP metabolites by treating N2a/APPsw cells with PB at different concentrations. The results indicated that PB reduced Aß secretion, which was caused by down-regulation of ß-secretase (BACE1) expression, as indicated at both the protein and mRNA levels. Further research revealed that PB regulated BACE1 expression by inducing the activation of forkhead box O1 (FoxO1) and inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). In addition, the effect of PB on BACE1 expression and Aß secretion was reversed by treatment with FoxO1 siRNA and STAT3 antagonist S3I-201. In conclusion, these data demonstrated that PB can effectively down-regulate the expression of BACE1 to reduce Aßsecretion by activating the expression of FoxO1 and inhibiting the phosphorylation of STAT3.

[Mechanism of MBL inhibiting the LPS-induced DC maturation].

The study was aimed to investigate the mechanism of mannan-binding lectin (MBL) on bacterial lipopolysaccharide (LPS)-induced human peripheral blood monocyte-derived dendritic cell (DC) maturation. The monocytes were prepared from the peripheral blood of healthy adult volunteers. The immature dendritic cells (imDC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4. FACS was used to investigate the interaction of MBL with imDC and the impact of MBL on LPS binding to imDC. ELISA and Western blot was used to analyze the interaction of MBL with soluble TLR4 ectodomain protein (sTLR4); Western blot was used to detect LPS-induced NF-κB translocation in imDC. The results showed that MBL could directly bind to imDC in the presence of calcium. sTLR4 protein or LPS could competitively inhibit the binding of MBL to imDC. ELISA and Western blot showed that MBL could evidently bind to sTLR4 protein in a concentration-dependent manner. FACS showed that MBL could competitively inhibit the binding of LPS to imDC by binding to imDC directly. Western blot showed that MBL decreased LPS-induced NF-κB translocation in imDC. It is concluded that MBL may competitively inhibit the binding of LPS to imDC by binding to TLR4 expressed on imDC, resulted in inhibition of LPS-induced DC maturation, suggesting that MBL can regulate DC maturation through ligand-binding. This study provides the good foundation to clarify the mechanism of MBL inhibiting the LPS-induced DC maturation.

[Effect of Astragalus injection on the signal conduction of neonatal postasphyxial-serum induced human renal tubular cell injury].

OBJECTIVE: To investigate, from cytoprotein and molecular levels, the action mechanism of astragalus injection (ASI) on the signal conduction of human renal tubular cells (HK-2) injury induced by neonatal postasphyxial-serum (NPS), whether it is through activating the nuclear factor kappaB (NF-kappaB) signaling pathway. METHODS: Taking HK-2 as the target cell and the 20% NPS as the attacking factor, the experiment was conducted by dividing the target cells into two groups before attacking, the blank control group and the ASI pretreated group. The nuclear translocation of NF-kappaB was detected by confocal microscopy with indirect immunofluorescence stain, and the amount of NF-kappaB inhibitor subunit (I-kappaBalpha) was detected by Western blot before attacking. The detections were repeated at various time points in the experiment, i.e., 15 min, 1 h and 2 h after attacking, respectively. RESULTS: Before attacking, the nuclear translocation of NF-kappaB and the amount of I-kappaBalpha were not different in the two groups. But the former increased and the latter decreased significantly in the ASI group at all the time points after attacking with the topmost changes presented at 1 h after attacking, and significantly different to those in the control group at corresponding time CONCLUSION: ASI pretreatment could inhibit the activation of NF-kappaB induced by postasphyxial-serum.

[Interventional effects of emodin on transforming growth factor-beta1/integrin-linked kinase signal way in interleukin-1beta-induced transdifferentiation of rat tubular epithelial-myofibroblasts].

OBJECTIVE: To study the effects of transforming growth factor-beta1/integrin-linked kinase (TGF-beta1/ILK) signal way in interleukin-1beta (IL-1beta)-induced rat tubular epithelial-myofibroblast transdifferentiation (TEMT), and to investigate whether emodin inhibits IL-1beta-induced TEMT through the TGF-beta1/ILK signal way-dependent mechanism. METHODS: Normal rat kidney epithelial cell line (NRK52E) was used in this study. NRK52E cells were divided into blank control group, emodin control group, IL-1beta-induced group, emodin-inhibited group, SB431542 (TGF-beta 1 type I receptor blocker)-inhibited group, emodin plus SB431542-inhibited group, emodin-pretreated group and emodin-reversed group. After 48-hour culture, morphological changes of the NRK52E cells were observed by an inverted phase contrast microscope. The expressions of alpha-smooth muscle actin (alpha-SMA) and E-cadherin were detected by two-color immunohistochemical staining, while the expressions of TGF-beta1 and ILK were detected by one-color immunohistochemical staining. We also performed the imaging analysis to quantitatively analyze the result of the immunohistochemical staining. The secretion of fibronectin (FN) was analyzed by enzyme-linked immunosorbent assay. RESULTS: Compared with the blank control group, IL-1beta might induce TEMT, which was showed in increasing expression of alpha-SMA, increasing secreting of FN and decreasing expression of E-cadherin, and at the same time the expressions of TGF-beta1 and ILK were enhanced (P<0.05). Emodin might inhibit all of those changes induced by IL-1beta (P<0.05). When TGF-beta1 signal way was intercepted, IL-1beta induced-TEMT was suppressed and the expression of ILK was decreased, however, there was no significant difference in expression of TGF-beta1 between the SB431542 group and the IL-1beta-induced group. Compared with emodin-inhibited group, emodin-pretreatment could not prevent IL-1beta induced-TEMT in a certain extent, but emodin could not revert IL-1beta-induced TEMT. Spearman correlation analysis showed that TGF-beta1 expression had positive correlation with expressions of alpha-SMA, FN, ILK and negative correlation with E-cadherin expression, and the expression of ILK was positively correlated with the expressions of alpha-SMA and FN and negatively correlated with E-cadherin expression. CONCLUSION: IL-1beta induces TEMT partly depending on TGF-beta1/ILK signal way, partly via which emodin inhibits the TEMT induced by IL-1beta.

[Role and mechanism of Salvia miltiorrhiza Bunge in alleviating injury induced by hypoxia/reoxygenation in HK-2 cells].

OBJECTIVE: Salvia miltorrhiza Bunge (SMB) is a traditional Chinese herb, which is considered to promote blood flow and remove blood stasis. This study examined whether SMB can alleviate injury induced by hypoxia/reoxygenation (H/R) in human kidney proximal tubular cells-2 (HK-2 cells). METHODS: There were 3 experimental groups: control, H/R injury and SMB-treated H/R injury. H/R injury of HK-2 cells was induced by first covering the cells with and then removing liquid paraffin wax. Different concentrations of compound SMB solution (0.05%, 0.10%, 0.15% or 0.20%) were administered to the SMB-treated H/R injury group before the hypoxic injury. After 4, 12 and 24 hrs of hypoxia and 4, 12, 24 and 48 hrs of reoxygenation, morphologic changes of HK-2 cells were observed under an inverted microscope. Cell viability was measured by the MTT method. Lactate dehydrogenase (LDH) activity in the culture supernatants was assayed using biochemical methods; TNF-alpha levels were determined by radioimmunoassay (RIA). RESULTS: The number of HK-2 cells was significantly reduced in the H/R injury group after hypoxia, and reached a nadir 24 hrs after hypoxia treatment. Various concentrations of SMB-treated groups showed significantly greater number of HK-2 cells than the H/R injury group. SMB solution (0.10%) produced the best effect. The levels of LDH and TNF-alpha in the H/R injury group were significantly increased, and reached a peak between 24 hrs of hypoxia and 4 hrs of reoxygenation when compared to the control group. Pre-treating with 0.10% SMB resulted in significantly lower levels of LDH and TNF-alpha than in the untreated H/R injury group at various time points of H/R. CONCLUSIONS: SMB has protective effects against H/R injury of HK-2 cells, possibly through inhibition of inflammatory cytokines.