Pharmacological Inhibition of Macrophage Toll-like Receptor 4/Nuclear Factor-kappa B Alleviates Rhabdomyolysis-induced Acute Kidney Injury.

BACKGROUND:: Acute kidney injury (AKI) is the most common and life-threatening systemic complication of rhabdomyolysis. Inflammation plays an important role in the development of rhabdomyolysis-induced AKI. This study aimed to investigate the kidney model of AKI caused by rhabdomyolysis to verify the role of macrophage Toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signaling pathway. METHODS:: C57BL/6 mice were injected with a 50% glycerin solution at bilateral back limbs to induce rhabdomyolysis, and CLI-095 or pyrrolidine dithiocarbamate (PDTC) was intraperitoneally injected at 0.5 h before molding. Serum creatinine levels, creatine kinase, the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6, and hematoxylin and eosin stainings of kidney tissues were tested. The infiltration of macrophage, mRNA levels, and protein expression of TLR4 and NF-κB were investigated by immunofluorescence double-staining techniques, reverse transcriptase-quantitative polymerase chain reaction, and Western blotting, respectively. In vitro, macrophage RAW264.7 was stimulated by ferrous myoglobin; the cytokines, TLR4 and NF-κB expressions were also detected. RESULTS:: In an in vivo study, using CLI-095 or PDTC to block TLR4/NF-κB, functional and histologic results showed that the inhibition of TLR4 or NF-κB alleviated glycerol-induced renal damages (P < 0.01). CLI-095 or PDTC administration suppressed proinflammatory cytokine (TNF-α, IL-6, and IL-1ß) production and macrophage infiltration into the kidney (P < 0.01). Moreover, in an in vitro study, CLI-095 or PDTC suppressed myoglobin-induced expression of TLR4, NF-κB, and proinflammatory cytokine levels in macrophage RAW264.7 cells (P < 0.01). CONCLUSION:: The pharmacological inhibition of TLR4/NF-κB exhibited protective effects on rhabdomyolysis-induced AKI by the regulation of proinflammatory cytokine production and macrophage infiltration.

Enzymatic hydrolysis of Grass Carp fish skin hydrolysates able to promote the proliferation of Streptococcus thermophilus.

BACKGROUND: The promotion effect on proliferation of Streptococcus thermophilus by enzymatic hydrolysates of aquatic products was firstly studied. The effect of influencing factors of the hydrolysis on the growth of S. thermophilus was investigated. RESULT: Grass Carp fish skin was hydrolysed to peptides by enzymatic hydrolysis using protease ProteAX, and for the S. thermophilus growth, the optimal enzymatic hydrolysis conditions were temperature of 60 °C, initial pH of 9.0, enzyme concentration of 10 g kg-1 , hydrolysis time of 80 min, and ratio of material to liquid of 1:2. The Grass Carp fish skin hydrolysate (GCFSH) prepared under the optimum conditions was fractionated to five fragments (GCFSH 1, GCFSH 2, GCFSH 3, GCFSH 4, GCFSH 5) according to molecular weight sizes, in which the fragments GCFSH 4 and GCFSH 5, with molecular weights of less than 1000 Da, significantly promoted the growth of S. thermophilus. CONCLUSION: The hydrolysis process of Grass Carp fish skin can be simplified, and the peptides with molecular weights below 1000 Da in the hydrolysates are the best nitrogen source for proliferation of S. thermophilus. This work can provide a fundamental theoretical basis for the production of multi-component functional foods, especially in milk drinks or yogurt. © 2017 Society of Chemical Industry.

Resolvin D1 Protects Lipopolysaccharide-induced Acute Kidney Injury by Down-regulating Nuclear Factor-kappa B Signal and Inhibiting Apoptosis.

BACKGROUND: Resolvin D1 (RvD1) is a newly found anti-inflammatory bioactive compound derived from polyunsaturated fatty acids. The current study aimed to explore the protective effect of RvD1 on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) and its possible mechanism. METHODS: Both in vivo and in vitro studies were conducted. Male BALB/c mice were randomly divided into control group (saline), LPS group (LPS 5 mg/kg), RvD1 group (RvD1 5 µg/kg + LPS 5 mg/kg), and blockage group (Boc-MLP 5 µg/kg + RvD1 5 µg/kg + LPS 5 mg/kg). Boc-MLP is a RvD1 receptor blocker. The mice were intraperitoneally injected with these drugs and recorded for general condition for 48 h, while the blood and kidneys were harvested at 2, 6, 12, 24, and 48 h time points, respectively (n = 6 in each group at each time point). Human proximal tubule epithelial cells (HK-2) were randomly divided into control group (medium only), LPS group (LPS 5 µg/ml), RvD1 group (RvD1 10 ng/ml + LPS 5 µg/ml), and blockage group (Boc-MLP 10 ng/ml + RvD1 10 ng/ml + LPS 5 µg/ml). The cells were harvested for RNA at 2, 4, 6, 12, and 24 h time points, respectively (n = 6 in each group at each time point). Blood creatinine was tested by using an Abbott i-STAT portable blood gas analyzer. Tumor necrosis factor-α (TNF-α) level was detected by ELISA. Kidney pathology was observed under hematoxylin and eosin (HE) staining and transmission electron microscope (TEM). We hired immune-histological staining, Western blotting, and fluorescence quantitative polymerase chain reaction to detect the expression of RvD1 receptor ALX, nuclear factor-kappa B (NF-κB) signaling pathway as well as caspase-3. Kidney apoptosis was evaluated by TUNEL staining. RESULTS: RvD1 receptor ALX was detected on renal tubular epithelials. Kaplan-Meier analysis indicated that RvD1 improved 48 h animal survival (80%) compared with LPS group (40%) and RvD1 blockage group (60%), while RvD1 also ameliorated kidney pathological injury in HE staining and TEM scan. After LPS stimulation, the mRNA expression of toll-like receptor 4, myeloid differentiation factor 88, and TNF-α in both mice kidneys and HK-2 cells were all up-regulated, while RvD1 substantially inhibited the up-regulation of these genes. Western blotting showed that the phosphorylated-IκB/IκB ratio in LPS group was significantly higher than that in the control group, which was inhibited in the RvD1 group. RvD1 could inhibit the up-regulation of cleaved-caspase-3 protein stimulated by LPS, which was prohibited in RvD1 blockage group. RvD1 group also had a lower proportion of apoptotic nuclei in mice kidney by TUNEL staining compared with LPS group. CONCLUSION: In LPS-induced AKI, RvD1 could decrease TNF-α level, ameliorate kidney pathological injury, protect kidney function, and improve animal survival by down-regulating NF-κB inflammatory signal as well as inhibiting renal cell apoptosis.

Factors related to fatigue in Chinese patients with end-stage renal disease receiving maintenance hemodialysis: a multi-center cross-sectional study.

BACKGROUD: Fatigue is considered as a common symptom in patients with end-stage renal disease (ESRD) and can significantly decrease patients' quality of life. This study aimed to assess fatigue in hemodialysis patients and to investigate risk factors of fatigue in Chinese patients receiving maintenance hemodialysis (MHD) in China. METHODS: Eligible patients completed questionnaires including demographic information, a Chinese version of the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue), the Family APGAR Index (APGAR), the medical outcomes study health status-Social Functioning subscale (SF-36,SocF), and the Pittsburgh sleep quality index (PSQI). Laboratory parameters were abstracted by medical records review. The multiple linear regression model was used to relate parameters with the FACIT-Fatigue score. RESULTS: A total of 345 MHD patients (216 men and 129 women, age 55.6 ± 12.8) were recruited in this study. The score of FACIT-Fatigue was 39 (Interquartile Range, 31-44). Fatigue was correlated with PSQI scores (p < 0.001), SocF scores (p < 0.001), comorbidity (p = 0.006), exercise time <1 hour per day (p = 0.003), adequacy of dialysis (Kt/V) < 1.2 (p = 0.016), APGAR scores (p = 0.014), and high Scr (p = 0.043). CONCLUSIONS: Fatigue is related to sleep disturbance, social and family functioning, taking physical exercise time, comorbidity condition, Kt/V and serum creatinine level in Chinese MHD patients. Future studies and interventions should focus on developing strategies and improving the quality of life in patients by addressing these significant contributing factors.

[Preliminary study on the pathogenic mechanism of myoglobin-induced endoplasmic reticulum stress and apoptosis in crush syndrome].

OBJECTIVE: To explore the pathogenic mechanism of myoglobin-induced endoplasmic reticulum stress (ERS) and apoptosis in tubular epithelial cells in acute kidney injury (AKI) mouse model of crush syndrome. METHODS: Eighteen C56BL/6 mice were randomly divided into control group, modeling 8 h group and modeling 24 h group. The AKI model of crush syndrome was established by intramuscular injection of 50% glycerol saline solution into thigh (8 microL/g), while equivalent volume of physiological saline was injected in control group. AKI was diagnosed when serum creatinine (sCr) level increased to double value of control group. The mice in the experimental groups were sacrificed at the time points of 8 h and 24 h after injection respectively. Serum Cr was detected and renal tissues was observed under electron microscopy. Apoptosis was detected by TUNEL technique. Marker proteins and mRNA of apoptosis and ERS were detected by immunohistochemistry and real-time PCR. Human kidney proximal tubular cell (HK-2) cells cultured in vitro were randomly divided into control, intervention 6 h and intervention 12 h groups. Control group were incubated in standard cell culture (DMEM/F12) and the two intervention groups were incubated in special DMEM/F12 in which ferrohemoglobin was added. After 6 h and 12 h incubation, the cells were collected and apoptosis was detected by flow cytometry. RESULTS: AKI model of crush syndrome was successfully established, which was proved with sCr doubling at the 8 h after the intramuscular injection of glycerol saline. Swelling of endoplasmic reticulum and mitochondria in proximal tubular epithelial cells was more obvious in the two model groups than that in control group. TUNEL staining showed the percentage of positive cells in AKI groups was higher than that in control group (P<0.05). Immunohistochemistry and real-time PCR showed the expressions of caspase3, caspase12 and CHOP in AKI groups were higher than those in control group (P<0.05). Flow cytometry showed cell apoptosis ratio was higher in model groups than control groiap (P<0.05). CONCLUSION: Myoglobin induced ERS and apoptosis may be involved in the pathogenesis of AKI in crush syndrome.

Monitoring oxidative stress in acute-on-chronic liver failure by advanced oxidation protein products.

AIM: Increased oxidative stress is important in the pathogenesis of acute-on-chronic liver failure (ACLF). This study aimed to investigate whether advanced oxidation protein products (AOPP) levels can monitor oxidative stress of ACLF patients. Furthermore, we aimed to study plasma exchange (PE) treatment and determine whether it can eliminate AOPP. METHODS: We measured AOPP levels in 50 ACLF patients, 30 patients with compensated liver cirrhosis (CR), 30 patients with chronic hepatitis B (CHB) and 50 healthy controls by spectrophotometric assay. AOPP concentrations were also measured before and after PE treatment in ACLF patients. As an apoptosis marker, serum cytokeratin 18 (CK18 M 30) levels were detected to investigate the relationship between AOPP and apoptosis in ACLF patients. RESULTS: Significantly higher AOPP levels at admission were found in patients with ACLF compared with CR, CHB and healthy controls (69.45 ± 29.04 µmol/L vs. 19.67 ± 7.02 µmol/L, 26.75 ± 5.21 µmol/L and 21.35 ± 6.15 µmol/L, respectively; P < 0.001). There was a positive relationship with total bilirubin, Child-Pugh, model for end-stage liver disease scores and CK18 M 30. In ACLF patients, AOPP levels were higher in non-survivors than survivors. An AOPP cut-off of 74.21 µmol/L was used for predicting poor prognosis. Multivariate Cox regression analysis demonstrated that AOPP were independent risk factors for prognosis. Dynamic change of AOPP levels associated with prognosis appeared earlier than total bilirubin. Following PE treatment, AOPP levels reduced to 34.65 ± 18.14 µmol/L (P < 0.001). CONCLUSIONS: Advanced oxidation protein products were suitable for monitoring the levels of oxidative stress in ACLF patients. Increased AOPP may serve as an important biological marker of worse outcome. In addition, PE therapy was effective in reducing AOPP.

[Spectroscopic studies on the interaction of Elsinochrome A with myoglobin].

The interaction between Elsinochrome A (EA) and myoglobin (Mb) was investigated using UV-Vis and fluorescence spectroscopy. The results suggested that there was a strong interaction between EA and Mb. In the dark, the interaction occurred on the surfaces amidic acid of Mb, but when illuminated, the interactions happened both on amidic acid and the interior structure of hemachrome of Mb. According to the values of the quenching constant, the thermodynamics parameters, the binding constants and the binding sites, it was showed that the binding interaction of EA and Mb was mainly hydrophobic in nature and the quenching mechanism was static quenching procedure. The change in the micro-circumstance of aminos of myoglobin was studied by synchronous fluorescence spectroscopy.

A nanoencapsulated hypocrellin A prepared by an improved microemulsion method for photodynamic treatment.

A new hypocrellin A (HA) encapsulated silica nanoparticles was prepared by an improved microemulsion method based on the unique character of cetyl trimethyl ammonium bromide (CTAB). Stable aqueous dispersions of the HA-loaded nanoparticles, with the diameter about 50 nm, owned superior photo-stability and singlet oxygen generation ability to free HA. In vitro studies demonstrated the active uptake of HA-doped nanoparticles into the cytosol of HeLa (human cervix epithelioid carcinoma) cells. Significant morphology change and phototoxicity to such impregnated tumor cells was observed upon irradiation with light. Thus, the potential of using this method to prepare silica nanoparticles as drug carriers for photodynamic therapy has been demonstrated.

Mitochondria-targeting photosensitizer-encapsulated amorphous nanocage as a bimodal reagent for drug delivery and biodiagnose in vitro.

The use of ceramic nano-carriers containing anti-cancer drugs for targeted delivery that span both fundamental and applied research has attracted the interest of the scientific community. In this paper, a hydrophobic photodynamic therapy drug, hypocrellin A (HA), was successfully encapsulated in water-soluble amorphous silica nanocage (HANC) by an improved sol-gel method. These nanocages are of ultrasmall size, highly monodispersed, stable in aqueous suspension, and retain the optical properties of HA. Moreover, these nanocages can be effectively delivered, subsequently taken up by cancer cells and finally targeted to mitochondria. In addition, incubation time dependent photodynamic efficacy difference between HANC and HA was investigated for the first time. Especially, the nanocages, owning extremely high stable fluorescence comparing with free HA, also have potentials as efficient probes for optical biodiagnose in vitro. All these properties of HANC could possibly make it especially promising to be used as a bimodal reagent for photodynamic therapy and biodiagnose.

A new sol-gel silica nanovehicle preparation for photodynamic therapy in vitro.

We report a facile silica nanovehicle preparation procedure for hydrophobic drug delivery, which is carried out in water without adding any surfactant and additional catalyst. This strategy includes hydrophobic drug nanopaticle preparation by reprecipitation method and in situ hydrolyzation and polymerization to encapsulate this naoparticle using only N-(beta-amimoethyl)-gamma-aminopropyltriethoxysilane (AETPS). To demonstrate this technique hypocrellin A (HA), a hydrophobic photosensitizing anticancer drug, is embedded into silica nanovehicle using this simple method. The resulting HA encapsulated nanovehicles (HANV) are monodisperse and stable in aqueous solution. Comparative studies with free HA and entrapped HA have demonstrated that the encapsulation effect on the embedded photosensitizer nanoparticle significantly enhances the efficacy of singlet oxygen generation and, thereby, the in vitro photodynamic efficacy.

[Study on interaction of caffeine with myoglobin by fluorescence spectroscopy].

The interaction of caffein and myoglobin was investigated by fluorescence spectroscopy and synchronous fluorescence spectroscopy. The intrinsic fluorescence of myoglobin was significantly quenched by caffein under the physiological condition (pH 7.4). The results indicated that caffeine was capable of binding with myoglobin to form a 1:1 complex and the quenching mechanism of myoglobin affected by caffeine was shown to be a static quenching procedure by calculating quenching constant, binding sites and binding constant. According to the thermodynamic parameters, the main binding force of the interaction is electrostatic force and hydrophobic force. The change in the micro-circumstance of aminos of myoglobin was analyzed by synchronous fluorescence spectrometry. The result indicated that caffeine can change the conformation of the protein, leading to the change in the micro-environment of tryptophane and tyrosine residues from hydrophobic environment to hydrophilic environment to different extent.

[Study on interaction between hypocrellin A and hemoglobin or myoglobin using synchronous fluorescence spectra].

The effects of hypocrellin A (HA) on the conformational changes of hemoglobin and myoglobin were studied using synchronous fluorescence spectroscopy. The results indicated that HA can change the conformation of these two proteins, leading to the change in the micro-environment of tryptophane and tyrosine residues from hydrophobic environment to hydrophilic environment to different extent.

[Effect of aggregation extent of microperoxidase-8 on its interaction with La3+].

Oxygen of two propionic acid groups of the heme group in an MP-8 molecule, leading to an increase in the non-planarity of the porphyrin ring of the heme group. The increasing extent in the non-planarity of the porphyrin ring of the heme group due to the interaction of La3+ and MP-8 molecule would be significantly decreased because NaCl could increase the aggregation extent of MP-8 molecules. Although methanol can reduce the aggregation extent of MP-8 molecules, it has little influence on the increasing extent in the non-planarity of the porphyrin ring of the heme group due to the interaction of La3+ and MP-8 molecule.

[Study on interaction between CT-DNA and cytochrome C using UV-vis spectroscopy and electrochemistry].

The interaction between CT-DNA immobilized on the positively charged nylon membrane and cytochrome C was studied using UV-Vis reflectance absorption spectroscopy and electrochemistry. It was found that cytchrome C can be adsorbed on the negative charged CT-DNA surface with the active positive charge area. Therefore, CT-DNA can promote the direct electrochemical reaction of cytochrome C.

[Spectral study on the interaction mechanism between thionine and calf thymus DNA].

The interaction mechanism of thionine (TH) and calf thymus DNA (CT-DNA) was studied with UV-Vis absorption spectoscopy, fluorescence spectroscopy, circular dichroism (CD), and X-ray photoelectron spectroscopy (XPS). It was demonstrated that in the pH 7.2 phosphate buffer solution, the interaction of TH and CT-DNA is in the intercalative mode. The absorbance of the absorption peak of TH decreases and the peak position shifts to the red direction. The binding constant (K) of TH with CT-DNA is 1.45x 10(4) mol x L(-1). The fluorescence intensities of TH decrease drastically with increasing the concentration of CT-DNA. The Stem-Volmer quenching constant (Ksv) was calculated to be 1.01 x 10(4) mol x L(-1). The intercalative sites are mainly located at the G-C sequences of the CT-DNA molecule through the S atom in the TH molecule according to the experimental data obtained from the fluorescence spectra and XPS analysis. The secondary structure conformation of CT-DNA is changed due to its interaction with TH.

[Preparation of Ge/SiO2 nanometer-crystal doped glass and study of its photoluminescence property].

Ge/SiO2 nanometer-crystal doped glass was prepared through sol-gel method. The gel was synthesized through the hydrolysis of a complex solution of Si (OC2H5)4 and Ge(OC2H5)4. The product gel was then heated at 700 degrees C in H2. Ge crystals appeared after the heating-reducing process. X ray diffraction analysis showed that there were Ge cubic crystals formed in the gel glass. The photoluminescence spectroscopic analysis showed that the Ge/SiO2 fluorescence peaked at 576 nm with excitation wavelength of 494 nm. By using the Brus quantum confinement model, the fluorescence peak energy suggested that the mean size of Ge crystal was about 3 nm.

[Fluorescence spectroscopic study of interaction between Fe-protoporphyrin in myoglobin and Cu(II) ions].

In this paper, the interaction between Cu(II) ions and Fe-protoporphyrin in horse-heart myoglobin (FePP-Mb) was studied. As a result, some of the Fe(II) ions in FePP-Mb were found to be replaced by Cu(II) ions forming CuPP-Mb, by adding Cu(II) ions into the myoglobin solution. The interaction became stronger when adding more Cu(II) ions into the myoglobin solution. By studying the metal ions' interaction with myoglobin proteins as macromolecules and discussing the interaction mechanism, this work provides a theoretical basis for the further study of hazardous metal ions' interaction with the human body and its mechanism. The fluorescence spectroscopic method used in this study has higher sensitivity than the ordinary UV and CD methods.

[FTIR studies on the chemical composition of wheat straw in different layers].

The differences of chemical composition in various layers of wheat straw were studied by infrared spectroscopy with OMNI sampler. The dependence of surface chemical composition of wheat straw on the processing methods was also studied. The results show that there is large amount of SiO2(-1,070 cm-1, -800 cm-1) in the surface of wheat straw. The out layer contains large amount of non-aromatic esters(nu c-o = 1,733 cm-1). There is few OH-containing (-3,300 cm-1) on the surface. The lignin(1,731, 1,650, 1,595, 1,505, 1,455, 1,425, 1,234, 1,039 and 835 cm-1) and cellulose(3,350, 2,935, 2,850, 1,650, 1,455, 1,430, 1,367, 1,317, 1,160, 1,034 and 891 cm-1) are the main species in the middle and inner layers of wheat straw. The large amount of surface SiO2 is mainly responsible for the poor adhesive property in the manufacture of the man-made board. Only a small amount of hydrocarbon and non-aromatic esters is dissolved out when treating the wheat straw with hot water, however, the aqueous NaOH treatment may make esters and SiO2 dissolved out, and then the processing of the man-made board becomes praticable.