A method to directly collect negatively charged nucleic acids, such as DNA and RNA, in the biosamples simply by applying an electric field in between the sample and collection buffer separated by the nanofilter membrane is proposed. The nanofilter membrane was made of low-stress silicon nitride with a thickness of 100 nm, and multiple pores were perforated in a highly arranged pattern using nanoimprint technology with a pore size of 200 nm and a pore density of 7.22 × 10 8 /cm 2 . The electrophoretic transport of hsa-mir-93-5p across the membrane was confirmed in pure microRNA (miRNA) mimic solution using quantitative reverse transcription-polymerase chain reactions (qRT-PCR). Consistency of the collected miRNA quantity, stability of the system during the experiment, and yield and purity of the prepared sample were discussed in detail to validate the effectiveness of the electrical protocol. Finally, in order to check the applicability of this method to clinical samples, liquid biopsy process was demonstrated by evaluating the miRNA levels in sera of hepatocellular carcinoma patients and healthy controls. This efficient system proposed a simple, physical idea in preparation of nucleic acid from biosamples, and demonstrated its compatibility to biological downstream applications such as qRT-PCR as the conventional nucleic acid extraction protocols.
BACKGROUND CONTEXT: Lumbar intervertebral disk herniation (LDH) is a common disease that causes low back pain, radiating leg pain, and sensory impairment. Preclinical studies rely heavily upon standardized animal models of human diseases to predict clinical treatment efficacy and to identify and investigate potential adverse events in human subjects. The current method for making the LDH model involves harvesting the nucleus pulposus (NP) from autologous coccygeal disks and applying to the lumbar nerve roots just proximal to the corresponding dorsal root ganglion. However, this surgical method generates a model that exhibits very different characteristics of disk herniation than that observed in human. PURPOSE: To produce a rat LDH model that better resembles disk herniation in humans and a standardized and uniform LDH model using Interleukin-1 beta (IL-1b). STUDY DESIGN: Experimental rat LDH model. METHODS: We exposed the L5−6 disk dorsolaterally on the right side through hemi-laminectomy without nerve compression. Herniation was initiated by puncturing the exposed disk with a 30-gauge needle at a depth of 4 mm. Interleukin-1 beta (IL-1b) was injected simultaneously to heighten the pathological processes of disk degeneration, including inflammatory responses, matrix destruction, and herniation of the NP. We performed histological staining to assess morphological changes, immunohistochemistry to analyze inflammation-and pain-related expression within and around the puncture site of the L5−6 disk, and real-time polymerase chain reaction to examine expression of markers for degenerative processes. In addition, we performed locomotor tests on the rats. RESULTS: We found that the IL-1b groups showed that the border between the annulus fibrosis and nucleus pulposus was severely interrupted compared to that of the control (puncture only) group. And, the injection of IL-1b leads to accelerated disk degeneration and inflammation in a more consistent manner in LDH model. Functional deficit was consistently induced by puncturing and injection of IL-1b in the exposed disk. CONCLUSIONS: The method proposed here can be used as an index to control the severity of disk degeneration and inflammation through the injected IL-1b concentration concurrent with surgically induced herniation. CLINICAL SIGNIFICANCE: Our proposed model may facilitate research in drug development to evaluate the efficacy of potential therapeutic agents for disk herniation and neuropathic pain and may also be used for nonclinical studies to more accurately assess the effectiveness of various treatment strategies according to the severity of disk degeneration.
Oxidative stress plays a key role in the pathogenesis of several diseases, including neurodegenerative diseases. Recent studies have reported that mitochondrial dysfunction is a leading cause of the overproduction of reactive oxygen species and oxidative stress. Mitochondrial changes play an important role in preventing oxidative stress. However, there is a lack of experimental evidence supporting this hypothesis. Thymus quinquecostatus CELAK (TQC) extract is a plant from China belonging to the thymus species, which can mediate the inflammatory response and prevent cell damage through its antioxidant activities. This study examines whether TQC can scavenge excess ROS originating from the mitochondria in RAW 264.7 macrophages. We used lipopolysaccharide (LPS) to induce inflammation and oxidative stress in RAW 264.7 macrophages and performed an immunocytochemistry dot blot of 8-hydroxy-2′-deoxyguanosine (8-OHdG) and real-time PCR to analyze the expression levels of genes involved in mitochondrial biogenesis and oxidative metabolism. TQC was found to significantly reduce the intensity of immunostained MitoSOX and 8-OHdG levels in the total genomic DNA within the mitochondria in RAW 264.7 macrophages. The HO-1 and Nrf2 mRNA levels were also significantly increased in the TQC groups. Therefore, we verified that TQC improves mitochondrial function and attenuates oxidative stress induced by LPS. Our results can provide reference for the effect of TQC to develop new therapeutic strategies for various diseases.
Inflammation is an essential biological response that eliminates pathogenic bacteria and repairs tissue after injury. Acute kidney injury (AKI) is associated with systemic and intrarenal inflammation as the inflammatory process decreases renal function and promotes progression to advanced chronic kidney disease. Macrophages are key mediators of the inflammatory response; their activation influences the immune system and may have various effects. Classically activated type I macrophages (M1) produce a variety of pro-inflammatory cytokines at the lesion site. However, anti-inflammatory type II macrophages (M2) are alternatively activated upon exposure to anti-inflammatory cytokines and are associated with wound healing and tissue repair following AKI. Here, we used melittin from bee venom to enhance the polarization of M2 macrophages and promote renal recovery after AKI. Melittin was administered to mice intraperitoneally for 5 days at various concentrations (10, 50, and 100 µg/kg); serum creatinine and blood urea nitrogen (BUN) levels were analyzed 72 h after cisplatin administration to confirm renal dysfunction. Melittin inhibited the cisplatin-induced increase in creatinine and BUN, an indicator of renal dysfunction. The expression of M1 markers (CD16/32) decreased significantly, whereas that of M2 markers (CD206, Arg1nase I) increased after melittin administration. Consistently, tubular necrosis was substantially reduced in melittin-treated mice. Thus, melittin alleviates cisplatin-induced AKI by regulating M2 macrophage expression.
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