Where should siRNAs go: applicable organs for siRNA drugs.

RNA interference mediated by small interfering RNAs (siRNAs) has been exploited for the development of therapeutics. siRNAs can be a powerful therapeutic tool because the working mechanisms of siRNAs are straightforward. siRNAs determine targets based on their sequence and specifically regulate the gene expression of the target gene. However, efficient delivery of siRNAs to the target organ has long been an issue that needs to be solved. Tremendous efforts regarding siRNA delivery have led to significant progress in siRNA drug development, and from 2018 to 2022, a total of five siRNA drugs were approved for the treatment of patients. Although all FDA-approved siRNA drugs target the hepatocytes of the liver, siRNA-based drugs targeting different organs are in clinical trials. In this review, we introduce siRNA drugs in the market and siRNA drug candidates in clinical trials that target cells in multiple organs. The liver, eye, and skin are the preferred organs targeted by siRNAs. Three or more siRNA drug candidates are in phase 2 or 3 clinical trials to suppress gene expression in these preferred organs. On the other hand, the lungs, kidneys, and brain are challenging organs with relatively few clinical trials. We discuss the characteristics of each organ related to the advantages and disadvantages of siRNA drug targeting and strategies to overcome the barriers in delivering siRNAs based on organ-specific siRNA drugs that have progressed to clinical trials.

Microstructural Evolution of a Nanostructured Complex Copper Alloy Processed by Accumulative Roll-Bonding of Oxygen Free Copper and DLP.

The accumulative roll-bonding (ARB) process using different copper alloys of oxygen free copper (OFC) and dioxide low-phosphorous copper (DLPC) was performed up to six cycles at ambient temperature without lubrication. A complex copper alloy sheet'in which OFC and DLPC alloys are stacked alternately each other was successfully fabricated by the ARB process. The microstructural evolution and texture development of the complex copper alloy with proceeding of the ARB were investigated by electron back scatter diffraction (EBSD) measurement. The specimen after 1 cycle showed significantly inhomogeneous microstructure in thickness direction, however, the inhomogeneity decreased gradually with increasing the number of ARB cycles. In addition, the grains became finer with the proceeding of the ARB. Resultantly, after 6 cycles, the specimen exhibited an ultrafine grained structure in which the grains above 65% were surrounded by the high angle grain boundaries above 15 degrees. On the other hand, there was no difference in texture development between OFC and DLPC in almost all specimens. In addition, the texture development did not depend on positions in thickness direction; the rolling texture such as {112}<111> and {011}<211> components developed strongly at all regions.

Autophagy suppresses age-dependent ischemia and reperfusion injury in livers of mice.

BACKGROUND & AIMS: As life expectancy increases, there are greater numbers of patients with liver diseases who require surgery or transplantation. Livers of older patients have significantly less reparative capacity following ischemia and reperfusion (I/R) injury, which occurs during these operations. There are no strategies to reduce the age-dependent I/R injury. We investigated the role of autophagy in the age dependence of sensitivity to I/R injury. METHODS: Hepatocytes and livers from 3- and 26-month-old mice were subjected to in vitro and in vivo I/R, respectively. We analyzed changes in autophagy-related proteins (Atg). Mitochondrial dysfunction was visualized using confocal and intravital multi-photon microscopy of isolated hepatocytes and livers from anesthetized mice, respectively. RESULTS: Immunoblot, autophagic flux, genetic, and imaging analyses all associated the increase in sensitivity to I/R injury with age with decreased autophagy and subsequent mitochondrial dysfunction due to calpain-mediated loss of Atg4B. Overexpression of either Atg4B or Beclin-1 recovered Atg4B, increased autophagy, blocked the onset of the mitochondrial permeability transition, and suppressed cell death after I/R in old hepatocytes. Coimmunoprecipitation analysis of hepatocytes and Atg3-knockout cells showed an interaction between Beclin-1 and Atg3, a protein required for autophagosome formation. Intravital multi-photon imaging revealed that overexpression of Beclin-1 or Atg4B attenuated autophagic defects and mitochondrial dysfunction in livers of older mice after I/R. CONCLUSIONS: Loss of Atg4B in livers of old mice increases their sensitivity to I/R injury. Increasing autophagy might ameliorate liver damage and restore mitochondrial function after I/R.

The inhibition of inflammatory molecule expression on 3T3-L1 adipocytes by berberine is not mediated by leptin signaling.

In our previous study, we have shown that berberine has both anti-adipogenic and anti-inflammatory effects on 3T3-L1 adipocytes, and the anti-adipogenic effect is due to the down-regulation of adipogenic enzymes and transcription factors. Here we focused more on anti-inflammatory effect of berberine using real time RT-PCR and found it changes expressions of adipokines. We hypothesized that anti-adipogenicity of berberine mediates anti-inflammtory effect and explored leptin as a candidate mediator of this signaling. We studied this hypothesis by western blot analysis, but our results showed that berberine has no effect on the phosphorylations of STAT-3 and ERK which have important roles on leptin signaling. These results led us to conclude that the anti-inflammatory effect of berberine is not mediated by the inhibition of leptin signal transduction. Moreover, we have found that berberine down-regulates NF-kappaB signaling, one of the inflammation-related signaling pathway, through western blot analysis. Taken together, the anti-inflammatory effect of berberine is not mediated by leptin, and berberine induces anti-inflammatory effect independent of leptin signaling.

Inhibition of tyrosine phosphorylation of vascular endothelial growth factor receptors in human umbilical vein endothelial cells: a potent anti-angiogenic lipid-rich extract from shark.

We have previously reported that an ethanolic extract of dried shark muscle mixed with olive oil (shark muscle-olive oil [SMO]) has potent anti-angiogenic activity and that this extract appears to inhibit the binding of vascular endothelial growth factor (VEGF) to its receptor(s). In this study, we investigated the effects of SMO on the phosphorylation of VEGF receptor(s) in human umbilical vein endothelial cells (HUVECs). In vitro cell proliferation assays showed that SMO significantly reversed the VEGF-promoted increase in HUVEC proliferation. Western blot analysis revealed that SMO treatment markedly inhibited the VEGF-promoted tyrosine phosphorylation of VEGF receptor-2 (KDR) and VEGF receptor-1 (Flt-1) in a dose-dependent manner. These results demonstrated that SMO might interfere with or block the binding of VEGF with its receptors, and thereby inhibit the VEGF receptor(s) signal transduction pathway and so inhibit angiogenesis.

Anti-obesity and hypolipidemic effects of black soybean anthocyanins.

The effect of anthocyanins extracted from black soybean (Glycine max L.) seed coats on body weight, adipose tissue weight, and serum lipids was evaluated in rats fed a high fat diet (HFD). Rats were raised on a normal diet (ND) (based on the AIN-93M diet), HFD (ND supplemented with 16% lard oil), HFD containing 10% black soybean, and HFD containing 0.037% black soybean anthocyanins (equivalent to that in the 10% black soybean diet). Weight gain was significantly lowered in the rats fed HFD plus black soybean anthocyanins compared with the rats fed HFD alone (P < .05) and reversed to the level of the rats fed ND. The black soybean diet also decreased body weight gain compared with the HFD (P < .05). The black soybean anthocyanins-added diet suppressed the HFD-induced weight gain in liver intermediately and tended to decrease the weights of epididymal and perirenal fat pads. The black soybean anthocyanins were also effective in improving the lipid profile. They significantly reduced the levels of serum triglyceride and cholesterol (P < .05), while they markedly increased the high-density lipoprotein-cholesterol concentration, which was decreased in the rats fed HFD (P < .05). These results indicate that the anthocyanins in black soybean seed coats have an anti-obesity effect, which can reverse the effects of HFD on body weight, adipose tissue weight, and serum lipid contents.

Isomer-specific effect of conjugated linoleic acid on inflammatory adipokines associated with fat accumulation in 3T3-L1 adipocytes.

The purpose of this study was to examine the isomer-specific effect of conjugated linoleic acid (CLA) on inflammatory markers associated with fat accumulation in cultures of differentiating 3T3-L1 adipocytes. trans-10,cis-12 CLA (t10c12 CLA) reduced leptin secretion and fat accumulation. Linoleic acid (LA) and cis-9,trans-11 CLA (c9t11 CLA) increased them, but not significantly. t10c12 CLA and LA showed similar effects on mRNA expression of inflammatory markers. t10c12 CLA and LA tended to up-regulate the mRNA levels of inflammatory cytokines such as interleukin (IL)-6 (not significantly), tumor necrosis factor (TNF)-alpha, and C-reactive protein (CRP) with no significant change in the secretion of adiponectin, an anti-inflammatory adipokine. However, c9t11 CLA induced no significant change in the mRNA expression of IL-6, TNF-alpha, or CRP, but significantly increased adiponectin secretion. In conclusion, CLA exerted isomer-specific effects on fat accumulation and mRNA expression of inflammatory markers in 3T3-L1 adipocytes. t10c12 CLA up-regulated inflammatory markers in spite of the decreased fat accumulation, and TNF-alpha might be one of the causal factors.

Antiobesity effect of Kochujang (Korean fermented red pepper paste) extract in 3T3-L1 adipocytes.

Kochujang (Korean fermented red pepper paste) is a mixture of fermented soybeans, wheat, and red pepper powder. Kochujang has been reported to reduce body fat gain and lipid levels of adipose tissues and serum in rats. We studied the inhibitory effect of Kochujang on lipid accumulation and investigated the molecular mechanism of the action in 3T3-L1 adipocytes by measuring the expression levels of adipocyte-specific genes by real-time reverse transcription-polymerase chain reaction. When 3T3-L1 adipocytes were treated with Kochujang extract (KE), the sizes of adipocytes and leptin secretion were decreased. Hormone-sensitive lipase (HSL) was transcriptionally up-regulated at 4 hours, and glycerol secretion was increased at both 4 hours and 24 hours. Moreover, mRNA expression levels of both sterol regulatory element-binding protein 1-c (SREBP-1c) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma), which are critical transcription factors for adipogenesis, were markedly down-regulated. Tumor necrosis factor-alpha (TNF-alpha) is reported to impair pre-adipocyte differentiation and induce lipolysis and apoptosis. KE treatment of 3T3-L1 adipocytes decreased TNF-alpha mRNA levels, but had no apparent affect on apoptosis. Taken together, our study shows that Kochujang decreased lipid accumulation in 3T3-L1 adipocytes by inhibiting adipogenesis through down-regulation of SREBP-1c and PPAR-gamma and by stimulation of lipolysis due to increased HSL activity. TNF-alpha might not be involved in the reduction of lipid accumulation by KE.

Berberine reduces the expression of adipogenic enzymes and inflammatory molecules of 3T3-L1 adipocyte.

Berberine (BBR), an isoquinoline alkaloid, has a wide range of pharmacological effects, yet its exact mechanism is unknown. In order to understand the anti-adipogenic effect of BBR, we studied the change of expression of several adipogenic enzymes of 3T3-L1 cells by BBR treatment. First, we measured the change of leptin and glycerol in the medium of 3T3-L1 cells treated with 1 micrometer, 5 micrometer and 10 micrometer concentrations of BBR. We also measured the changes of adipogenic and lipolytic factors of 3T3-L1. In 3T3-L1 cells, both leptin and adipogenic factors (SREBP-1c, C/EBP-alpha, PPAR-gamma, fatty acid synthase, acetyl-CoA carboxylase, acyl-CoA synthase and lipoprotein lipase) were reduced by BBR treatment. Glycerol secretion was increased, whereas expression of lipolytic enzymes (hormone-sensitive lipase and perilipin) mRNA was slightly decreased. Next, we measured the change of inflammation markers of 3T3-L1 cells by BBR treatment. This resulted in the down-regulation of mRNA level of inflammation markers such as TNF-alpha, IL-6, C- reactive protein and haptoglobin. Taken together, our data shows that BBR has both anti-adipogenic and anti-inflammatory effects on 3T3-L1 adipocytes, and the anti-adipogenic effect seems to be due to the down-regulation of adipogenic enzymes and transcription factors.

Effect of retinoic acid on leptin, glycerol, and glucose levels in mature rat adipocytes in vitro.

To elucidate the effects of retinoic acids (RAs) on adipogenesis and insulin sensitivity, we treated mature adipocytes with two different kinds of RA, 9-cis-RA and all-trans-RA. Both 9-cis- and all-trans-RA inhibited the secretion of leptin. However, the inhibition was significantly decreased at a higher dose of each RA. The inhibitory effect of 9-cis-RA was synergistically enhanced by the addition of rosiglitazone, a synthetic ligand for peroxisome proliferator-activated receptor (PPAR) gamma. 9-cis-RA also leads to adipogenesis in a dose-dependent manner. On the contrary, all-trans-RA does not increase adipogenesis in a dose-dependent manner. To clarify the antidiabetic effects of RA, glucose uptake was assessed by estimating glucose concentrations in the medium. 9-cis-RA reduced glucose levels in the culture media, but all-trans-RA did not. In conclusion, all-trans-RA does not alter adipogenesis and glucose uptake but does inhibit leptin secretion. 9-cis-RA, however, seems to increase both adipogenesis and glucose uptake through activation of the retinoid X receptor/PPARgamma heterodimer.