Effect of Danggui Buxuetang on Podocyte Pyroptosis in Diabetic Kidney Disease Rats： An Exploration Based on TXNIP/NLRP3/GSDMD Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Danggui Buxuetang on podocyte pyroptosis in diabetic kidney disease （DKD） rats and to explore the possible mechanism of its prevention and treatment of DKD and podocyte pyroptosis. MethodEight of the 50 male SD rats were randomly classified into a normal group， and the remaining 42 were fed a high-glucose and high-fat diet for six weeks and then intraperitoneally injected with 35 mg·kg-1 streptozotocin （STZ） for inducing type 2 diabetes. After successful modeling， they were randomized into the model group， low- （0.72 g·kg-1） and high-dose （1.44 g·kg-1） Danggui Buxuetang group， and irbesartan （0.017 g·kg-1） group and gavaged with the corresponding drugs， while those in the normal group and model group with an equal volume of normal saline， once per day， for 20 weeks. During the medication， the fasting blood glucose （FBG） and 24 h urine protein （24 h-UTP） were measured regularly. After administration， the pathological changes in renal tissues were observed by periodic acid-silver metheramine （PASM） staining， followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope （TEM）. Serum levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） were determined by enzyme-linked immunosorbent assay （ELISA）. The DNA damage in renal tissue cells of rats was detected by in situ nick end-labeling （TUNEL） assay. The protein expression levels of thioredoxin interacting protein （TXNIP）， cysteine-dependent aspartate-directed protease-1 （Caspase-1）， and gasdermin D （GSDMD） in renal tissues of rats were detected by immunohistochemistry （IHC）， the expression levels of nucleotide binding domain like receptor protein 3 （NLRP3） and Wilms tumor protein-1 （WT-1） in podocytes by immunofluorescent （IF） staining， and the expression levels of TXNIP/NLRP3/Caspase-1/GSDMD pathway proteins and Synaptopodin in renal podocytes by Western blot. ResultCompared with the normal group， the model group exhibited increased FBG and 24 h UTP， glomerular hypertrophy， mesangial hyperplasia， increased extracellular matrix， thickened basement membrane， K-W nodules， vacuolar degeneration in renal tubular epithelial cells， foot process fusion or loss， elevated serum IL-1β and IL-18 levels and TUNEL-positive cells in renal tissue， enhanced NLRP3 but diminished WT-1 expression in podocytes， down-regulated Synaptopodin protein expression， and up-regulated TXNIP/NLRP3/Caspase-1/GSDMD protein expression （P<0.01）. Compared with the model group， Danggui Buxuetang high-dose group remarkably lowered FBG， 24-h UTP， and TUNEL-positive cells in renal tissue， improved renal histopathology and podocyte injury and loss， down-regulated NLRP3 expression in podocytes and TXNIP/NLRP3/Caspase-1/GSDMD protein expression levels， and up-regulated WT-1 expression in podocytes and Synaptopodin protein expression （P<0.05， P<0.01）. ConclusionDanggui Buxuetang inhibits podocyte pyroptosis to reduce proteinuria and delays the development of DKD possibly by regulating the TXNIP/NLRP3/GSDMD signaling pathway.

Effect of Danggui Buxuetang on Podocyte Injury in Diabetic Kidney Disease Rats： An Exploration Based on RIPK1/RIPK3/MLKL Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Danggui Buxuetang on the podocyte injury and receptor-interacting protein kinase 1/receptor-interacting protein kinase3/mixed lineage kinase domain-like protein （RIPK1/RIPK3/MLKL） signaling pathway in diabetic kidney disease （DKD） ratsand to explore its possible mechanism against DKD. MethodEight of the 50 SD rats were randomly classified intoa normal group， and the remaining were fed a high-glucose and high-fat diet for six weeks and then intraperitoneally injected with 0.035 g·kg-1streptozotocin （STZ） for inducing type 2 diabetes. After successful modeling，they were randomized into the model group，high- and low-dose （1.44，0.72 g·kg-1） Danggui Buxuetang groups， and irbesartan （0.017 g·kg-1）group. After 20 weeks of drug intervention， the fasting blood glucose （FBG）， kidney index （KI），and urinary microalbumin-to-urine creatinine ratio （UACR）were detected in each group. The pathological changes in renal tissue were observed by hematoxylin-eosin （HE） staining， followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope. The levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in renal tissue of rats were determined by enzyme-linked immunosorbent assay （ELISA）， and the protein expression levels of RIPK1， RIPK3， and MLKL in rat kidney tissue by immunohistochemistry. The apoptosis rate of podocytes was detected by in situ nick end-labeling （TUNEL） assay. The mRNA expression levels of RIPK1， RIPK3， and MLKL in kidney tissue of rats were measured by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the protein expression levels of RIPK， RIPK3， and MLKL and podocyte marker Wilms tumor protein-1 （WT-1） in rat kidney tissue were assayed by Western blot. ResultCompared with the normal group， the model group exhibited elevated FBG， UACR， and KI （P<0.01）， glomerular hypertrophy， thickened basement membrane， increased extracellular matrix， mesangial hyperplasia， foot process fusion or loss， enhanced apoptosis in renal tissue， up-regulated TNF-α and IL-6 levels （P<0.01） and RIPK1/RIPK3/MLKL mRNA and protein expression （P<0.01）， and down-regulated WT-1 protein expression. Compared with the model group， Danggui Buxuetang high-dose group significantly reduced the levels of FBG， UACR， and KI， improved renal histopathology， podocyte loss， and apoptosis in renal tissue， down-regulated TNF-α and IL-6 levels and RIPK1/RIPK3/MLKL mRNA and protein expression （P<0.05， P<0.01）， and up-regulated WT-1 protein expression. ConclusionDanggui Buxuetang alleviates podocyte injury and delays the development of DKD possibly by regulating the RIPK1/RIPK3/MLKL signaling pathway.

Danggui Buxuetang Alleviates Oxidative Stress and Inflammation in Diabetic Kidney Disease Rats by Improving Mitochondrial Dysfunction of Podocytes / 中国实验方剂学杂志

ObjectiveTo investigate the intervention effect of Danggui Buxuetang on oxidative stress and inflammatory response in diabetic kidney disease （DKD） rats from its improvement of podocyte mitochondrial dysfunction. MethodSD rats were randomly divided into the control group and modeling group， and the ones in the latter group rats were fed a high-glucose and high-fat diet and then intraperitoneally injected with a small dose of streptozotocin （STZ） for inducing type 2 diabetes. The successfully modeled rats were randomized into the model group， high- and low-dose （1.44 and 0.72 g·kg-1） Danggui Buxuetang groups， and irbesartan （0.017 g·kg-1）group and gavaged with the corresponding drugs， while those in the normal and model groups with an equal volume of normal saline. After 20 weeks of drug intervention， the urinary microalbumin-to-urine creatinine ratio （UACR） and serum malondialdehyde （MDA） content and manganese superoxide dismutase （MnSOD） activity in each group were measured. The pathological changes in renal tissue were observed by Masson trichrome staining， and periodic acid-silver metheramine （PASM） staining， followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope （TEM）. The expression level of reactive oxygen species （ROS） in rat kidney tissue was detected using a fluorescent probe dihydroethidium （DHE）. The protein expression levels of peroxisome proliferator-activated receptor γ -coactivator -1α （PGC-1α）， nucleotide-binding domain like receptor protein 3 （NLRP3）， and Wilms tumor protein-1 （WT-1） were measured by immunohistochemistry （IHC）， and the expression levels of NLRP3， interleukin-1β （IL-1β），and WT-1 in podocytes by immunofluorescence （IF） assay. The mRNA expression levels of PGC-1α and NLRP3 in the renal tissues were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the protein expression levels of PGC-1α， MnSOD， NLRP3， and IL-1β were assayed by Western blot. ResultCompared with the normal group， the model group exhibited elevated UACR and MDA content， weakened MnSOD activity （P<0.01）， glomerular hypertrophy， thickened basement membrane， mesangial hyperplasia， increased extracellular matrix， K-W nodules， podocyte mitochondrial swelling， disordered mitochondrial cristae， foot process fusion or loss， vacuolization， increased ROS （P<0.01）， enhanced NLRP3 and IL-1β but diminished WT-1 expression in podocytes， down-regulated PGC-1α mRNA expression （P<0.01） and PGC-1α and MnSOD protein expression （P<0.01）， and up-regulated NLRP3 mRNA expression and NLRP3 and IL-1β protein expression （P<0.01）. Compared with the model group， Danggui Buxuetang high-dose group significantly decreased UACR and MDA， enhanced MnSOD activity （P<0.05， P<0.01）， improved renal histopathology and podocyte mitochondrial ultrastructure， decreased ROS （P<0.05， P<0.01） and NLRP3 and IL-1β expression in podocytes， enhanced WT-1 expression in podocytes， up-regulated the mRNA and protein levels of PGC-1α and MnSOD， and down-regulated the mRNA and protein levels of NLRP3 and IL-1β （P<0.05， P<0.01）. ConclusionDanggui Buxuetang alleviates oxidative stress， reduces inflammatory response， protects kidney， and delays the progression of DKD possibly by improving the mitochondrial dysfunction in podocytes of DKD rats.

Deficiency of TRIM32 Impairs Motor Function and Purkinje Cells in Mid-Aged Mice.

Proper functioning of the cerebellum is crucial to motor balance and coordination in adult mammals. Purkinje cells (PCs), the sole output neurons of the cerebellar cortex, play essential roles in cerebellar motor function. Tripartite motif-containing protein 32 (TRIM32) is an E3 ubiquitin ligase that is involved in balance activities of neurogenesis in the subventricular zone of the mammalian brain and in the development of many nervous system diseases, such as Alzheimer's disease, autism spectrum disorder, attention deficit hyperactivity disorder. However, the role of TRIM32 in cerebellar motor function has never been examined. In this study we found that motor balance and coordination of mid-aged TRIM32 deficient mice were poorer than those of wild-type littermates. Immunohistochemical staining was performed to assess cerebella morphology and TRIM32 expression in PCs. Golgi staining showed that the extent of dendritic arborization and dendritic spine density of PCs were decreased in the absence of TRIM32. The loss of TRIM32 was also associated with a decrease in the number of synapses between parallel fibers and PCs, and in synapses between climbing fibers and PCs. In addition, deficiency of TRIM32 decreased Type I inositol 1,4,5-trisphosphate 5-phosphatase (INPP5A) levels in cerebellum. Overall, this study is the first to elucidate a role of TRIM32 in cerebellar motor function and a possible mechanism, thereby highlighting the importance of TRIM32 in the cerebellum.

A clinical case report of brain abscess caused by Nocardia brasiliensis in a non-immunocompromised patient and a relevant literature review.

BACKGROUND: Brain abscess due to the Nocardia genus is rare and usually found in immunocompromised patients. The most common subtype implicated is Nocardia farcinica while brain abscess due to Nocardia brasiliensis is comparatively rare. Diagnosis of brain abscess is based mainly on bacteriological culture from pus collected at the site of infection, and brain imaging. Stereotaxic aspiration or surgical resection combined with adequate duration of treatment with antibiotics to which the bacteria are sensitive represent effective treatment strategies. CASE PRESENTATION: We report a rare case of brain abscess caused by Nocardia brasiliensis in a non-immunocompromised patient. He admitted to our hospital twice with a headache. Stereotaxic aspiration was performed at the patient's first appointment at the hospital, and a craniotomy was used to excise the lesion during subsequent abscess recurrence. CONCLUSION: Early diagnosis, reasonable surgical intervention, and adequate duration of treatment with effective antibiotics are critical for treating brain abscess.

Verbascoside inhibits progression of glioblastoma cells by promoting Let-7g-5p and down-regulating HMGA2 via Wnt/beta-catenin signalling blockade.

Glioblastoma (GBM) continues to show a poor prognosis despite advances in diagnostic and therapeutic approaches. The discovery of reliable prognostic indicators may significantly improve treatment outcome of GBM. In this study, we aimed to explore the function of verbascoside (VB) in GBM and its effects on GBM cell biological processes via let-7g-5p and HMGA2. Differentially expressed GBM-related microRNAs (miRNAs) were initially screened. Different concentrations of VB were applied to U87 and U251 GBM cells, and 50 µmol/L of VB was selected for subsequent experiments. Cells were transfected with let-7g-5p inhibitor or mimic, and overexpression of HMGA2 or siRNA against HMGA2 was induced, followed by treatment with VB. The regulatory relationships between VB, let-7g-5p, HMGA2 and Wnt/ß-catenin signalling pathway were determined. The results showed that HMGA2 was a direct target gene of let-7g-5p. VB treatment or let-7g-5p overexpression inhibited HMGA2 expression and the activation of Wnt/ß-catenin signalling pathway, which further inhibited cell viability, invasion, migration, tumour growth and promoted GBM cell apoptosis and autophagy. On the contrary, HMGA2 overexpression promoted cell viability, invasion, migration, tumour growth while inhibiting GBM cell apoptosis and autophagy. We demonstrated that VB inhibits cell viability and promotes cell autophagy in GBM cells by up-regulating let-7g-5p and down-regulating HMGA2 via Wnt/ß-catenin signalling blockade.

Absence of TRIM32 Leads to Reduced GABAergic Interneuron Generation and Autism-like Behaviors in Mice via Suppressing mTOR Signaling.

Mammalian target of rapamycin (mTOR) signaling plays essential roles in brain development. Hyperactive mTOR is an essential pathological mechanism in autism spectrum disorder (ASD). Here, we show that tripartite motif protein 32 (TRIM32), as a maintainer of mTOR activity through promoting the proteasomal degradation of G protein signaling protein 10 (RGS10), regulates the proliferation of medial/lateral ganglionic eminence (M/LGE) progenitors. Deficiency of TRIM32 results in an impaired generation of GABAergic interneurons and autism-like behaviors in mice, concomitant with an elevated autophagy, which can be rescued by treatment embryonically with 3BDO, an mTOR activator. Transplantation of M/LGE progenitors or treatment postnatally with clonazepam, an agonist of the GABAA receptor, rescues the hyperexcitability and the autistic behaviors of TRIM32-/- mice, indicating a causal contribution of GABAergic disinhibition. Thus, the present study suggests a novel mechanism for ASD etiology in that TRIM32 deficiency-caused hypoactive mTOR, which is linked to an elevated autophagy, leads to autism-like behaviors via impairing generation of GABAergic interneurons. TRIM32-/- mouse is a novel autism model mouse.

Verbascoside Inhibits Glioblastoma Cell Proliferation, Migration and Invasion While Promoting Apoptosis Through Upregulation of Protein Tyrosine Phosphatase SHP-1 and Inhibition of STAT3 Phosphorylation.

BACKGROUND/AIMS: As a natural antioxidant, verbascoside (VB) is proved to be a promising method for the treatment of oxidative-stress-related neurodegenerative diseases. Thus, this study aimed to investigate the effects of VB on glioblastoma cell proliferation, apoptosis, migration, and invasion as well as the mechanism involving signal transducer and activator of transcription 3 (STAT3) and Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1). METHODS: U87 cells were assigned to different treatments. The MTT assay was used to test cell proliferation, flow cytometry was used to detect cell apoptosis, and a Transwell assay was used for cell migration and invasion. We analyzed the glioblastoma tumor growth in a xenograft mouse model. Western blot analysis was employed to determine the protein expression of related genes. RESULTS: Glioblastoma cells exhibited decreased cell proliferation, migration, invasion, and increased apoptosis when treated with VB or TMZ. Western blot analysis revealed elevated SHP-1 expression and reduced phosphorylated (p)-STAT3 expression in glioblastoma cells treated with VB compared with controls. Correspondingly, in a xenograft mouse model treated with VB, glioblastoma tumor volume and growth were decreased. Glioblastoma xenograft tumors treated with VB showed elevated SHP-1, Bax, cleaved caspase-3, and cleaved PARP expression and reduced p-STAT3, Bcl-2, survivin, MMP-2, and MMP-9 expression. siRNA-SHP-1 inhibited the VB effects on glioblastoma. CONCLUSION: This study demonstrates that VB inhibits glioblastoma cell proliferation, migration, and invasion while promoting apoptosis via SHP-1 activation and inhibition of STAT3 phosphorylation.

Toll-Like Receptor 4 Deficiency Impairs Motor Coordination.

The cerebellum plays an essential role in balance and motor coordination. Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex and are critical for the execution of its functions, including motor coordination. Toll-like receptor (TLR) 4 is involved in the innate immune response and is abundantly expressed in the central nervous system; however, little is known about its role in cerebellum-related motor functions. To address this question, we evaluated motor behavior in TLR4 deficient mice. We found that TLR4(-∕-) mice showed impaired motor coordination. Morphological analyses revealed that TLR4 deficiency was associated with a reduction in the thickness of the molecular layer of the cerebellum. TLR4 was highly expressed in PCs but not in Bergmann glia or cerebellar granule cells; however, loss of TLR4 decreased the number of PCs. These findings suggest a novel role for TLR4 in cerebellum-related motor coordination through maintenance of the PC population.

Disrupted-in-Schizophrenia-1 Attenuates Amyloid-ß Generation and Cognitive Deficits in APP/PS1 Transgenic Mice by Reduction of ß-Site APP-Cleaving Enzyme 1 Levels.

Disrupted-in-Schizophrenia-1 (DISC1) is a genetic risk factor for a wide range of major mental disorders, including schizophrenia, major depression, and bipolar disorders. Recent reports suggest a potential role of DISC1 in the pathogenesis of Alzheimer's disease (AD), by referring to an interaction between DISC1 and amyloid precursor protein (APP), and to an association of a single-nucleotide polymorphism in a DISC1 intron and late onset of AD. However, the function of DISC1 in AD remains unknown. In this study, decreased levels of DISC1 were observed in the cortex and hippocampus of 8-month-old APP/PS1 transgenic mice, an animal model of AD. Overexpression of DISC1 reduced, whereas knockdown of DISC1 increased protein levels, but not mRNA levels of ß-site APP-Cleaving Enzyme 1 (BACE1), a key enzyme in amyloid-ß (Aß) generation. Reduction of BACE1 protein levels by overexpression of DISC1 was accompanied by an accelerating decline rate of BACE1, and was blocked by the lysosomal inhibitor chloroquine, rather than proteasome inhibitor MG-132. Moreover, overexpression of DISC1 in the hippocampus with an adeno-associated virus reduced the levels of BACE1, soluble Aß40/42, amyloid plaque density, and rescued cognitive deficits of APP/PS1 transgenic mice. These results indicate that DISC1 attenuates Aß generation and cognitive deficits of APP/PS1 transgenic mice through promoting lysosomal degradation of BACE1. Our findings provide new insights into the role of DISC1 in AD pathogenesis and link a potential function of DISC1 to the psychiatric symptoms of AD.