Zarzio Ameliorates Non-alcoholic Fatty Liver Induced By a High Fat Diet in a Rat Experimental Model: A Histological and Immunohistochemical Study

SUMMARY: The therapeutic effect of a granulocyte-colony stimulating factor (G-CSF) biosimilar drug, zarzio, on non-alcoholic fatty liver disease (NAFLD) in a rat model was investigated in this study. Thirty-two rats were randomly divided into four groups. Groups I and II were fed a standard laboratory diet, whereas groups III and IV were fed a high fat diet (HFD) for 14 weeks. After 12 weeks of feeding, groups I and III were administered normal saline, and groups II and IV were intraperitoneally administered zarzio (200 mg/kg/day) for two consecutive weeks. Hematoxylin-eosin (H&E) staining was used to assess hepatic and pancreatic morphology in all groups, oil red O (ORO) staining for lipid accumulation, Masson's staining for fibrosis, and immunohistochemistry assay for hepatic protein expression of insulin receptor substrate 1 (IRS1), nuclear factor erythroid 2-related factor 2 (Nrf2), tumour necrosis factor alpha (TNF-α) and pancreatic caspase-3. The NAFLD rats (group III) developed hepatic steatosis with increased lipid accumulation, perisinusoidal fibrosis, upregulated IRS1, TNF-α (all P<0.05) without a significant increase in Nrf2 protein expression compared with normal control. In comparison, model rats treated with zarzio (group IV) showed significant rejuvenation of the hepatic architecture, reduction of fat accumulation, and fibrosis. This was accompanied by the upregulation of Nrf2, downregulation of IRS1 and TNF-α protein expression (all P<0.05). No correlation was detected between NAFLD and non-alcoholic fatty pancreas disease (NAFPD). However, the pancreatic β-cells in group III showed increased caspase-3 expression, which was decreased (P<0.05) in group IV. In conclusion, zarzio ameliorates NAFLD by improving the antioxidant capacity of liver cells, reducing hepatic IRS1, TNF-α protein expression and pancreatic β-cells apoptosis, suggesting that zarzio could be used as a potential therapy for NAFLD.

En este estudio se investigó el efecto terapéutico de un fármaco biosimilar del factor estimulante de colonias de granulocitos (G-CSF), zarzio, sobre la enfermedaddel hígado graso no alcohólico (NAFLD) en un modelo de rata. Treinta y dos ratas se dividieron aleatoriamente en cuatro grupos. Los grupos I y II fueron alimentados con una dieta estándar de laboratorio, mientras que los grupos III y IV fueron alimentados con una dieta alta en grasas (HFD) durante 14 semanas. Después de 12 semanas de alimentación, a los grupos I y III se les administró solución salina normal, y a los grupos II y IV se les administró zarzio por vía intraperitoneal (200 mg/kg/ día) durante dos semanas consecutivas. Se utilizó tinción de hematoxilina-eosina (H&E) para evaluar la morfología hepática y pancreática en todos los grupos, tinción con rojo aceite O (ORO) para la acumulación de lípidos, tinción de Masson para la fibrosis y ensayo de inmunohistoquímica para la expresión de la proteína hepática del sustrato 1 del receptor de insulina (IRS1), factor nuclear eritroide 2 relacionado con el factor 2 (Nrf2), factor de necrosis tumoral alfa (TNF-α) y caspasa-3 pancreática. Las ratas NAFLD (grupo III) desarrollaron esteatosis hepática con aumento de la acumulación de lípidos, fibrosis perisinusoidal, IRS1 y TNF-α regulados positivamente (todos P <0,05) sin un aumento significativo en la expresión de la proteína Nrf2 en comparación con el control normal. En comparación, las ratas modelo tratadas con zarzio (grupo IV) mostraron un rejuvenecimiento significativo de la arquitectura hepática, una reducción de la acumulación de grasa y fibrosis. Esto estuvo acompañado por la regulación positiva de Nrf2, la regulación negativa de la expresión de la proteína IRS1 y TNF-α (todas P <0,05). No se detectó correlación entre NAFLD y la enfermedad del páncreas graso no alcohólico (NAFPD). Sin embargo, las células β pancreáticas en el grupo III mostraron una mayor expresión de caspasa-3, que disminuyó (P <0,05) en el grupo IV. En conclusión, zarzio mejora la NAFLD al mejorar la capacidad antioxidante de las células hepáticas, reduciendo el IRS1 hepático, la expresión de la proteína TNF-α y la apoptosis de las células β pancreáticas, lo que sugiere que zarzio podría usarse como una terapia potencial para la NAFLD.

The Effects of White Tea (Camellia Sinensis) and Infliximab Against Cisplatin- Induced Testicular Damage via Oxidative Stress and Apoptosis / Efectos del Té Blanco (Camellia Sinensis) e Infliximab Contra el Daño Testicular Inducido por Cisplatino a Través del Estrés Oxidativo y la Apoptosis

SUMMARY: Cisplatin (Cis) is an important chemotherapeutic agent used in cancer treatment. Males exposed to Cis were reported to exhibit testicular toxicity. Cis-induced testicular toxicity is mediated by oxidative stress, inflammation, testosterone inhibition and apoptosis. Accordingly, this study was conducted to evaluate the potential protective roles of infliximab (IFX), which is an anti- TNF-a agent, and of white tea (Camellia sinensis), which is known to possess antioxidant, anti-apoptotic, and anti-inflammatory effects, against Cis-induced testicular toxicity in rats. Rats were randomly assigned into five groups as follows: control group, Cisplatin (7 mg/kg) treatment group, Cisplatin (7 mg/kg) + infliximab (7 mg/kg) treatment group, cisplatin + white tea (WT) treatment group, and Cisplatin+ WT+IFX combined treatment group. In the present study, Cis exposure reduced the sperm count. It also increased testicular oxidative stress as well as the levels of inflammatory and apoptotic markers. Histopathological assays supported the biochemical findings. Treatment with IFX and/or WT restored testicular histology, preserved spermatogenesis, suppressed oxidative stress and apoptosis, and significantly ameliorated Cis-induced damage. It was concluded that white tea and infliximab could potentially serve as therapeutic options for the protection of testicular tissue against the harmful effects of Cis.

El cisplatino (Cis) es un importante agente quimioterapéutico utilizado en el tratamiento del cáncer. Se informó que los hombres expuestos a Cis exhibieron toxicidad testicular. La toxicidad testicular inducida por Cis está mediada por el estrés oxidativo, la inflamación, la inhibición de la testosterona y la apoptosis. En consecuencia, este estudio se realizó para evaluar las posibles funciones protectoras de infliximab (IFX), un agente anti-TNF-α, y del té blanco (Camellia sinensis), conocido por sus propiedades antioxidantes, antiapoptóticas y anti-TNF-α -efectos inflamatorios, contra la toxicidad testicular inducida por Cis en ratas. Cinco grupos de ratas se asignaron al azar de la siguiente manera: grupo control, grupo de tratamiento con cisplatino (7 mg/ kg), grupo de tratamiento con cisplatino (7 mg/kg) + infliximab (7 mg/kg), grupo de tratamiento con cisplatino + té blanco (WT), y grupo de tratamiento combinado Cisplatino+ WT+IFX. En el presente estudio, la exposición a Cis redujo el conteo de espermatozoides. También aumentó el estrés oxidativo testicular, así como los niveles de marcadores inflamatorios y apoptóticos. Los ensayos histopatológicos respaldaron los hallazgos bioquímicos. El tratamiento con IFX y/o WT restauró la histología testicular, preservó la espermatogénesis, suprimió el estrés oxidativo y la apoptosis, y mejoró significativamente el daño inducido por Cis. Se concluyó que el té blanco y el infliximab podrían potencialmente servir como opciones terapéuticas para la protección del tejido testicular contra los efectos nocivos de Cis.

Metformin Inhibits ROS/TNF-alpha Axis-Mediated Chronic Kidney Disease Induced by TAA Independent of Leukocyte Infiltration in Association with the Inhibition of Kidney Injury Biomarkers / La Metformina Inhibe la Enfermedad Renal Crónica Mediada por el Eje ROS/TNF-alfa Inducida por TAA Independientemente de la Infiltración de Leucocitos en Asociación con la Inhibición de Biomarcadores de Lesión Renal

SUMMARY: The toxic effects of thioacetamide (TAA) and carbon tetrachloride on the human body are well recognized. In this study, we examined whether TAA intoxication can induce kidney leukocyte infiltration (measured as leukocyte common antigen CD45) associated with the augmentation of the reactive oxygen species (ROS)/tumor necrosis factor-alpha (TNF-α) axis, as well as biomarkers of kidney injury with and without metformin treatment. Rats were either injected with TAA (200 mg/kg; twice a week for 8 weeks) before being sacrificed after 10 weeks (experimental group) or were pre-treated with metformin (200 mg/kg) daily for two weeks prior to TAA injections and continued receiving both agents until the end of the experiment, at week 10 (protective group). Using basic histology staining, immunohistochemistry methods, and blood chemistry analysis, we observed profound kidney tissue injury such as glomerular and tubular damage in the experimental group, which were substantially ameliorated by metformin. Metformin also significantly (p0.05) increase in kidney expression of CD45 positive immunostaining cells. In conclusion, we found that TAA induces kidney injury in association with the augmentation of ROS/TNF-α axis, independent of leukocyte infiltration, which is protected by metformin.

Son bien conocidosos los efectos tóxicos de la tioacetamida (TAA) y el tetracloruro de carbono en el cuerpo humano. En este estudio, examinamos si la intoxicación por TAA puede inducir la infiltración de leucocitos renales (medida como antígeno leucocitario común CD45) asociada con el aumento de las especies reactivas de oxígeno (ROS)/factor de necrosis tumoral-alfa (TNF-α), así como biomarcadores de daño renal con y sin tratamiento con metformina. A las ratas se les inyectó TAA (200 mg/kg; dos veces por semana durante 8 semanas) antes de sacrificarlas a las 10 semanas (grupo experimental) o se les pretrató con metformina (200 mg/kg) diariamente durante dos semanas antes de las inyecciones de TAA y continuaron recibiendo ambos agentes hasta el final del experimento, en la semana 10 (grupo protector). Usando tinción histológica básica, métodos de inmunohistoquímica y análisis químico de la sangre, observamos una lesión profunda del tejido renal, como daño glomerular y tubular en el grupo experimental, que mejoraron sustancialmente con la metformina. La metformina también inhibió significativamente (p0,05) en la expresión renal de células de inmunotinción positivas para CD45. En conclusión, encontramos que el TAA induce la lesión renal en asociación con el aumento del eje ROS/TNF-α, independientemente de la infiltración de leucocitos, que está protegida por metformina.

Comparative Analysis of TNF-alpha, TNF-R1, and TNF-R2 in Patients with Low-impact Fractures Due to Osteoporosis / Análise comparativa de TNF-alfa, TNF-R1 e TNF-R2 em pacientes com fraturas de baixo impacto decorrentes de osteoporose

Abstract Objective To analyze the serum levels of TNF-alpha and its TNF-R1 and TNF-R2 receptors in the blood of patients with low-impact fractures due to osteoporosis, comparing between genders and with healthy patients. Methods The present study was conducted with a blood sample of 62 patients, divided into patients with osteoporosis and healthy patients. The results were obtained using the ELISA method. Cytokine concentrations were determined based on the absorbance values obtained. Results Serum TNF-alpha levels were undetectable in female patients, while in males they were found only in one patient, with no significant difference. Similar results were found in the analyses of TNF-R1 and TNF-R2 levels, a significant increase in levels of TNF-alpha receptors in the groups of patients with osteoporosis compared with the control groupinbothsexes.There wasnosignificant difference between the sexes in the dosage of both receptors within the group with osteoporosis. There was also a positive and significant correlation in the levels of TNF-R1 and TNF-R2 only in women. Conclusion The significant increase in TNF-R1 and TNF-R2 levels in women with osteoporosis suggest that the release and expression of these receptors may be contributing differently to the development of osteoporosis in men and women.

Resumo Objetivo Analisar os níveis séricos de TNF-alfa e de seus receptores TNF-R1 e TNF-R2 no sangue de pacientes com fraturas de baixo impacto, decorrentes de osteoporose, comparando entre os sexos e com pacientes saudáveis. Métodos Oestudofoi realizadocom amostradesanguede 62 pacientes,divididos em pacientes com osteoporose e pacientes saudáveis. Os resultados foram obtidos através do método de ELISA. As concentrações de citocinas foram determinadas com base nos valores de absorbância obtidos. Resultados Os níveis séricos de TNF-alfa foram indetectáveis nos pacientes do sexo feminino, enquanto no masculino encontrou-se somente em um paciente, não havendo diferença significativa. Encontrou-se resultados semelhantes nas análises dos níveis de TNF-R1 e TNF-R2, aumento significativo nos níveis dos receptores de TNF-alfa nos grupos de pacientes com osteoporose em comparação com o grupo controle, em ambos os sexos. Não houve diferença significativa entre os sexos na dosagem de ambos os receptores dentro do grupo com osteoporose. Houve ainda correlação positiva e significativa nos níveis de TNF-R1 e TNF-R2 apenas nas mulheres. Conclusão O aumento significativo nos níveis de TNF-R1 e TNF-R2 em mulheres com osteoporose sugerem que a liberação e expressão destes receptores pode estar contribuindo de maneira distinta no desenvolvimento da osteoporose em homens e mulheres.

Captopril inhibits thioacetamide-induced chronic liver injury associated with the suppression of inflammation / hypoxia- inducible factor 1-alpha / profibrogenic axis-mediated hepatotoxicity in rats / Captopril inhibe la lesión hepática crónica inducida por tioacetamida asociada con la supresión de la inflamación/factor 1-alfa inducible por hipoxia/hepatotoxicidad mediada por el eje profibrogénico en rata

SUMMARY: Liver transplantation is the only available method to treat liver failure induced by chronic liver injury. We sought to determine whether the angiotensin-converting enzyme inhibitor, captopril, can inhibit the development of chronic liver injury induced by the hepatotoxic agent thioacetamide (TAA) in association with the suppression of inflammation (hsCRP, TNF-α, and IL-6) / hypoxia- inducible factor 1-alpha (HIF-1α) / profibrosis (TIMP-1, MMP-9, and α-SMA) axis that mediates liver injury. Therefore, the model group of rats was injected for eight weeks with 200 mg/kg TAA starting at week two. The protective group was pretreated with 150 mg/ kg captopril daily for two weeks prior to TAA injections and continued receiving both capropril and TAA agents until being humanely scrificed at week 10. We observed a substantial damage to liver tissue in the model group as demonstrated by a significant (p<0.0001) increase in blood and hepatic tissue levels of high sensitivity C-reactive protein (hsCRP), tumor necrosis factor-a (TNF-α), interleukin- 6 (L-6), HIF-1α, tissue inhibitor of metalloproteinases-1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), alpha-smooth muscle actin (α-SMA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). All these parameters were significantly (p<0.0244) protected by captopril. Also, a significant (p<0.0001) positive correlation was observed between a-SMA (profibrosis) and the serum and tissue levels of hsCRP, TNF-α, HIF-1α, TIMP-1, MMP-9, and ALT. Thus, these findings suggest that the induction of chronic liver injury by the hepatotoxic compound, TAA is associated with the upregulation of inflammation/HIF-1α/profibrosis, with captopril exhibiting beneficial hepatic pleotropic effects.

El trasplante de hígado es el único método disponible para tratar la insuficiencia hepática inducida por una lesión hepática crónica. Buscamos determinar si el inhibidor de la enzima convertidora de angiotensina, captopril, puede inhibir el desarrollo de lesión hepática crónica inducida por el agente hepatotóxico tioacetamida (TAA) en asociación con la supresión de la inflamación (hsCRP, TNF-α e IL-6) / factor inducible por hipoxia 1-alfa (HIF-1α) / profibrosis (TIMP-1, MMP-9 y α- SMA) eje que media la lesión hepática. Por lo tanto, al grupo modelo de ratas se le inyectó durante ocho semanas 200 mg/kg de TAA a partir de la semana dos. El grupo protector fue pretratado con 150 mg/kg de captopril al día durante dos semanas antes de las inyecciones de TAA y continuó recibiendo capropril y agentes TAA hasta que fue sacrificado en la semana 10. Observamos un daño sustancial en el tejido hepático en el grupo modelo, como lo demuestra un aumento significativo (p<0,0001) de los niveles en sangre y tejido hepático de proteína C reactiva de alta sensibilidad (hsCRP), factor de necrosis tumoral-α (TNF-a), interleucina-6 (L-6), HIF-1α, inhibidor tisular de metaloproteinasas-1 (TIMP-1), metaloproteinasa de matriz-9 (MMP-9), actina de músculo liso alfa (α-SMA), alanina aminotransferasa (ALT) y aspartato aminotransferasa (AST). Todos estos parámetros estaban significativamente (p<0,0244) protegidos por captopril. Además, se observó una correlación positiva significativa (p<0,0001) entre α-SMA (profibrosis) y los niveles séricos y tisulares de hsCRP, TNF-α, HIF-1α, TIMP- 1, MMP-9 y ALT. Por lo tanto, estos hallazgos sugieren que la inducción de daño hepático crónico por el compuesto hepatotóxico, TAA, está asociada con la regulación al alza de la inflamación/HIF-1α/profibrosis, con captopril exhibiendo efectos pleotrópicos hepáticos beneficiosos.

Quercetin and resveratrol are associated with the downregulation of TNFalpha/NF-kB/inos axis-mediated acute liver injury in rats induced by paracetamol poisoning / La quercetina y el resveratrol están asociados con la regulación decreciente de la lesión hepática aguda mediada por el eje TNF-alfa/NF-kB/inos en ratas inducida por envenenamiento con paracetamol

SUMMARY: Paracetamol (known as acetaminophen, or APAP) poisoning causes acute liver damage that can lead to organ failure and death. We sought to determine that APAP overdose can augment tumor necrosis factor-alpha (TNF-α)/ nuclear factor kappa B (NF-kB)/induced nitic oxide synthase (iNOS) axis-mediated hepatotoxicity in rats, and the anti-inflammatory polyphenolic compounds, quercetin (QUR) plus resveratrol (RES) can ameliorate these parameters. Therefore, we induced acute hepatotoxicity in rats using APAP overdose (2 g/kg, orally) and the protective group of rats were treated with 50 mg/kg QUR plus 30 mg/kg RES for one week before APAP ingestion. Animals were killed at day 8. APAP poisoning caused the induction of hepatic tissue levels of TNF-α, NF-kB, and iNOS, which were significantly (p<0.05) decreased by QUR+RES. QUR+RES, also inhibited liver injury biomarkers, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Additionally, a link between liver injury and TNF-α /NF-kB / iNOS axis mediated hepatotoxicity was observed. Thus, the presented data backing the conclusion that intoxication by paracetamol increases TNF-α / NF-kB / iNOS axis -mediated hepatotoxicity, and is protected by a combination of quercetin and resveratrol.

El envenenamiento por paracetamol (conocido como acetaminofeno o APAP) causa daño hepático agudo que puede provocar una insuficiencia orgánica y la muerte. El objetivo de este trabajo fue determinar si la sobredosis de APAP puede aumentar la hepatotoxicidad mediada por el eje del factor de necrosis tumoral alfa (TNF-α)/factor nuclear kappa B (NF-kB)/óxido nítico sintasa inducida (iNOS) en ratas, y si el polifenólico antiinflamatorio compuesto por quercetina (QUR) más resveratrol (RES) pueden mejorar estos parámetros. Por lo tanto, inducimos hepatotoxicidad aguda en ratas usando una sobredosis de APAP (2 g/kg, por vía oral). El grupo protector de ratas se trató con 50 mg/ kg de QUR más 30 mg/kg de RES durante una semana antes de la ingestión de APAP. Los animales se sacrificaron el día 8. El envenenamiento con APAP en el tejido hepático provocó la inducción de niveles de TNF-α, NF-kB e iNOS, que se redujeron significativamente (p<0,05) con QUR+RES. QUR+RES, también inhibió los biomarcadores de daño hepático, la alanina aminotransferasa (ALT) y el aspartato aminotransferasa (AST). Además, se observó una relación entre la lesión hepática y la hepatotoxicidad mediada por el eje TNF-α /NF-kB/iNOS. Por lo tanto, los datos presentados respaldan la conclusión de que la intoxicación por paracetamol aumenta la hepatotoxicidad mediada por el eje TNF-α /NF-kB / iNOS, y está protegida por una combinación de quercetina y resveratrol.

Protective mechanisms of Leontopodium leontopodioides extracts on lipopolysaccharide-induced acute kidney injury viathe NF-κB/NLRP3 pathway / 中国天然药物

Sepsis-induced uncontrolled systemic inflammatory response syndrome (SIRS) is a critical cause of multiple organ failure. Acute kidney injury (AKI) is one of the most serious complications associated with an extremely high mortality rate in SIRS, and it lacked simple, safe, and effective treatment strategies. Leontopodium leontopodioides (Willd.) Beauv (LLB) is commonly used in traditional Chinese medicine for the treatment of acute and chronic nephritis. However, it remains unclear whether lipopolysaccharide (LPS) affects LPS-induced AKI. To identify the molecular mechanisms of LLB in LPS-induced HK-2 cells and mice, LLB was prepared by extraction with 70% methanol, while a lipopolysaccharide (LPS)-induced HK-2 cell model and an AKI model were established in this study. Renal histopathology staining was performed to observe the morphology changes. The cell supernatant and kidney tissues were collected for determining the levels of inflammatory factors and protein expression by ELISA, immunofluorescence, and Western blot. The results indicated that LLB significantly reduced the expression of IL-6 and TNF-α in LPS-induced HK-2 cells, as well as the secretion of IL-6, TNF-α, and IL-1β in the supernatant. The same results were observed in LPS-induced AKI serum. Further studies revealed that LLB remarkably improved oxidative stress and apoptosis based on the content of MDA, SOD, and CAT in serum and TUNEL staining results. Notably, LLB significantly reduced the mortality due to LPS infection. Renal histopathology staining results supported these results. Furthermore, immunofluorescence and Western blot results confirmed that LLB significantly reduced the expression of the protein related to the NF-κB signaling pathway and NLRP3, ASC, and Caspase-1 which were significantly increased through LPS stimulation. These findings clearly demonstrated the potential use of LLB in the treatment of AKI and the crucial role of the NF-κB/NLRP3 pathway in the process through which LLB attenuates AKI induced by LPS.

MAGED4B Promotes Glioma Progression via Inactivation of the TNF-α-induced Apoptotic Pathway by Down-regulating TRIM27 Expression / 神经科学通报·英文版

MAGED4B belongs to the melanoma-associated antigen family; originally found in melanoma, it is expressed in various types of cancer, and is especially enriched in glioblastoma. However, the functional role and molecular mechanisms of MAGED4B in glioma are still unclear. In this study, we found that the MAGED4B level was higher in glioma tissue than that in non-cancer tissue, and the level was positively correlated with glioma grade, tumor diameter, Ki-67 level, and patient age. The patients with higher levels had a worse prognosis than those with lower MAGED4B levels. In glioma cells, MAGED4B overexpression promoted proliferation, invasion, and migration, as well as decreasing apoptosis and the chemosensitivity to cisplatin and temozolomide. On the contrary, MAGED4B knockdown in glioma cells inhibited proliferation, invasion, and migration, as well as increasing apoptosis and the chemosensitivity to cisplatin and temozolomide. MAGED4B knockdown also inhibited the growth of gliomas implanted into the rat brain. The interaction between MAGED4B and tripartite motif-containing 27 (TRIM27) in glioma cells was detected by co-immunoprecipitation assay, which showed that MAGED4B was co-localized with TRIM27. In addition, MAGED4B overexpression down-regulated the TRIM27 protein level, and this was blocked by carbobenzoxyl-L-leucyl-L-leucyl-L-leucine (MG132), an inhibitor of the proteasome. On the contrary, MAGED4B knockdown up-regulated the TRIM27 level. Furthermore, MAGED4B overexpression increased TRIM27 ubiquitination in the presence of MG132. Accordingly, MAGED4B down-regulated the protein levels of genes downstream of ubiquitin-specific protease 7 (USP7) involved in the tumor necrosis factor-alpha (TNF-α)-induced apoptotic pathway. These findings indicate that MAGED4B promotes glioma growth via a TRIM27/USP7/receptor-interacting serine/threonine-protein kinase 1 (RIP1)-dependent TNF-α-induced apoptotic pathway, which suggests that MAGED4B is a potential target for glioma diagnosis and treatment.

Soybean isoflavones alleviate cerebral ischemia/reperfusion injury in rats by inhibiting ferroptosis and inflammatory cascade reaction / 南方医科大学学报

OBJECTIVE@#To explore the mechanism that mediates the effect of soybean isoflavones (SI) against cerebral ischemia/reperfusion (I/R) injury in light of the regulation of regional cerebral blood flow (rCBF), ferroptosis, inflammatory response and blood-brain barrier (BBB) permeability.@*METHODS@#A total of 120 male SD rats were equally randomized into sham-operated group (Sham group), cerebral I/R injury group and SI pretreatment group (SI group). Focal cerebral I/R injury was induced in the latter two groups using a modified monofilament occlusion technique, and the intraoperative changes of real-time cerebral cortex blood flow were monitored using a laser Doppler flowmeter (LDF). The postoperative changes of cerebral pathological morphology and the ultrastructure of the neurons and the BBB were observed with optical and transmission electron microscopy. The neurological deficits of the rats was assessed, and the severities of cerebral infarction, brain edema and BBB disruption were quantified. The contents of Fe2+, GSH, MDA and MPO in the ischemic penumbra were determined with spectrophotometric tests. Serum levels of TNF-α and IL-1βwere analyzed using ELISA, and the expressions of GPX4, MMP-9 and occludin around the lesion were detected with Western blotting and immunohistochemistry.@*RESULTS@#The rCBF was sharply reduced in the rats in I/R group and SI group after successful insertion of the monofilament. Compared with those in Sham group, the rats in I/R group showed significantly increased neurological deficit scores, cerebral infarction volume, brain water content and Evans blue permeability (P < 0.01), decreased Fe2+ level, increased MDA level, decreased GSH content and GPX4 expression (P < 0.01), increased MPO content and serum levels of TNF-α and IL-1β (P < 0.01), increased MMP-9 expression and lowered occludin expression (P < 0.01). All these changes were significantly ameliorated in rats pretreated with IS prior to I/R injury (P < 0.05 or 0.01).@*CONCLUSION@#SI preconditioning reduces cerebral I/R injury in rats possibly by improving rCBF, inhibiting ferroptosis and inflammatory response and protecting the BBB.

Circular RNA circRSF1 binds to HuR to promote radiation-induced inflammatory phenotype in hepatic stellate cells / 南方医科大学学报

OBJECTIVE@#To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function.@*METHODS@#Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR.@*RESULTS@#Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05).@*CONCLUSION@#circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.

TRPV1 participates in the protective effect of propolis on colonic tissue of ulcerative colitis / 中南大学学报(医学版)

OBJECTIVES@#Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) mainly characterized by inflammation, ulceration and erosion of colonic mucosa and submucosa. Transient receptor potential vanilloid 1 (TRPV1) is an important mediator of visceral pain and inflammatory bowel disease. This study aims to investigate the protective effect of water soluble propolis (WSP) on UC colon inflammatory tissue and the role of TRPV1.@*METHODS@#Male SD rats were randomly divided into 6 groups (n=8): a normal control (NC) group, an ulcerative colitis model (UC) group, a low-WSP (L-WSP) group, a medium-WSP (M-WSP) group, a high-WSP (H-WSP) group, and a salazosulfapyridine (SASP) group. The rats in the NC group drank water freely, and the other groups drank 4% dextran sulfate sodium (DSS) solution freely for 7 d to replicate the ulcerative colitis model. Based on the successful replication of the UC, the L-WSP, M-WSP, and H-WSP groups were given 50, 100, and 200 mg/kg of water-soluble propolis by gavage for 7 d, and the SASP group was given 100 mg/kg of sulfasalazine by gavage for 7 d. The body weight of rats in each group was measured at the same time every day, the fecal traits and occult blood were observed to record the disease activity index (DAI). After intragastric administration, the animals were sacrificed after fasted 24 h. Serum and colonic tissue were collected, and the changes of MDA, IL-6 and TNF-α were detected. The pathological changes of colon tissues were observed by HE staining, and the expression of TRPV1 in colon tissues was observed by Western blotting, immunohistochemistry, and immunofluorescence.@*RESULTS@#The animals in each group that drank DSS freely showed symptoms such as weight loss, decreased appetite, depressed state, and hematochezia, indicating that the model was successfully established. Compared with the NC group, DAI scores of other groups were increased (all P<0.05). MDA, IL-6, TNF-α in serum and colon tissues of the UC group were increased compared with the NC group (all P<0.01), and they were decreased after WSP and SASP treatment (all P<0.01). The results of showed that the colon tissue structure was obviously broken and inflammatory infiltration in the UC group, while the H-WSP group and the SASP group significantly improved the colon tissue and alleviated inflammatory infiltration. The expression of TRPV1 in colon tissues in the UC group was increased compared with the NC group (all P<0.01), and it was decreased after WSP and SASP treatment.@*CONCLUSIONS@#WSP can alleviate the inflammatory state of ulcerative colitis induced by DSS, which might be related to the inhibition of inflammatory factors release, and down-regulation or desensitization of TRPV1.

Ozonated oil alleviates dinitrochlorobenzene-induced allergic contact dermatitis via inhibiting the FcεRI/Syk signaling pathway / 中南大学学报(医学版)

OBJECTIVES@#Ozone is widely applied to treat allergic skin diseases such as eczema, atopic dermatitis, and contact dermatitis. However, the specific mechanism remains unclear. This study aims to investigate the effects of ozonated oil on treating 2,4-dinitrochlorobenzene (DNCB)-induced allergic contact dermatitis (ACD) and the underling mechanisms.@*METHODS@#Besides the blank control (Ctrl) group, all other mice were treated with DNCB to establish an ACD-like mouse model and were randomized into following groups: a model group, a basal oil group, an ozonated oil group, a FcεRI-overexpressed plasmid (FcεRI-OE) group, and a FcεRI empty plasmid (FcεRI-NC) group. The basal oil group and the ozonated oil group were treated with basal oil and ozonated oil, respectively. The FcεRI-OE group and the FcεRI-NC group were intradermally injected 25 µg FcεRI overexpression plasmid and 25 µg FcεRI empty plasmid when treating with ozonated oil, respectively. We recorded skin lesions daily and used reflectance confocal microscope (RCM) to evaluate thickness and inflammatory changes of skin lesions. Hematoxylin-eosin (HE) staining, real-time PCR, RNA-sequencing (RNA-seq), and immunohistochemistry were performed to detct and analyze the skin lesions.@*RESULTS@#Ozonated oil significantly alleviated DNCB-induced ACD-like dermatitis and reduced the expressions of IFN-γ, IL-17A, IL-1β, TNF-α, and other related inflammatory factors (all P<0.05). RNA-seq analysis revealed that ozonated oil significantly inhibited the activation of the DNCB-induced FcεRI/Syk signaling pathway, confirmed by real-time PCR and immunohistochemistry (all P<0.05). Compared with the ozonated oil group and the FcεRI-NC group, the mRNA expression levels of IFN-γ, IL-17A, IL-1β, IL-6, TNF-α, and other inflammatory genes in the FcεRI-OE group were significantly increased (all P<0.05), and the mRNA and protein expression levels of FcεRI and Syk were significantly elevated in the FcεRI-OE group as well (all P<0.05).@*CONCLUSIONS@#Ozonated oil significantly improves ACD-like dermatitis and alleviated DNCB-induced ACD-like dermatitis via inhibiting the FcεRI/Syk signaling pathway.

Effect and mechanism of Bovis Calculus on ulcerative colitis by inhibiting IL-17/IL-17RA/Act1 signaling pathway / 中国中药杂志

This study aimed to elucidate the effect and underlying mechanism of Bovis Calculus in the treatment of ulcerative colitis(UC) through network pharmacological prediction and animal experimental verification. Databases such as BATMAN-TCM were used to mine the potential targets of Bovis Calculus against UC, and the pathway enrichment analysis was conducted. Seventy healthy C57BL/6J mice were randomly divided into a blank group, a model group, a solvent model(2% polysorbate 80) group, a salazosulfapyridine(SASP, 0.40 g·kg~(-1)) group, and high-, medium-, and low-dose Bovis Calculus Sativus(BCS, 0.20, 0.10, and 0.05 g·kg~(-1)) groups according to the body weight. The UC model was established in mice by drinking 3% dextran sulfate sodium(DSS) solution for 7 days. The mice in the groups with drug intervention received corresponding drugs for 3 days before modeling by gavage, and continued to take drugs for 7 days while modeling(continuous administration for 10 days). During the experiment, the body weight of mice was observed, and the disease activity index(DAI) score was recorded. After 7 days of modeling, the colon length was mea-sured, and the pathological changes in colon tissues were observed by hematoxylin-eosin(HE) staining. The levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6), and interleukin-17(IL-17) in colon tissues of mice were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA expression of IL-17, IL-17RA, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1β, CXCL1, CXCL2, and CXCL10 was evaluated by real-time polymerase chain reaction(RT-PCR). The protein expression of IL-17, IL-17RA, Act1, p-p38 MAPK, and p-ERK1/2 was investigated by Western blot. The results of network pharmacological prediction showed that Bovis Calculus might play a therapeutic role through the IL-17 signaling pathway and the TNF signaling pathway. As revealed by the results of animal experiments, on the 10th day of drug administration, compared with the solvent model group, all the BCS groups showed significantly increased body weight, decreased DAI score, increased colon length, improved pathological damage of colon mucosa, and significantly inhibited expression of TNF-α,IL-6,IL-1β, and IL-17 in colon tissues. The high-dose BCS(0.20 g·kg~(-1)) could significantly reduce the mRNA expression levels of IL-17, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1β, CXCL1, and CXCL2 in colon tissues of UC model mice, tend to down-regulate mRNA expression levels of IL-17RA and CXCL10, significantly inhibit the protein expression of IL-17RA,Act1,and p-ERK1/2, and tend to decrease the protein expression of IL-17 and p-p38 MAPK. This study, for the first time from the whole-organ-tissue-molecular level, reveals that BCS may reduce the expression of pro-inflammatory cytokines and chemokines by inhibiting the IL-17/IL-17RA/Act1 signaling pathway, thereby improving the inflammatory injury of colon tissues in DSS-induced UC mice and exerting the effect of clearing heat and removing toxins.

Effects of intranasal administration of tripterygium glycoside-bearing liposomes on behavioral cognitive impairment of mice induced by central nervous system inflammation / 中国中药杂志

Tripterygium glycosides liposome(TPGL) were prepared by thin film-dispersion method, which were optimized accor-ding to their morphological structures, average particle size and encapsulation rate. The measured particle size was(137.39±2.28) nm, and the encapsulation rate was 88.33%±1.82%. The mouse model of central nervous system inflammation was established by stereotaxic injection of lipopolysaccharide(LPS). TPGL and tripterygium glycosides(TPG) were administered intranasally for 21 days. The effects of intranasal administration of TPG and TPGL on behavioral cognitive impairment of mice due to LPS-induced central ner-vous system inflammation were estimated by animal behavioral tests, hematoxylin-eosin(HE) staining of hippocampus, real-time quantitative polymerase chain reaction(RT-qPCR) and immunofluorescence. Compared with TPG, TPGL caused less damage to the nasal mucosa, olfactory bulb, liver and kidney of mice administered intranasally. The behavioral performance of treated mice was significantly improved in water maze, Y maze and nesting experiment. Neuronal cell damage was reduced, and the expression levels of inflammation and apoptosis related genes [tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), BCL2-associated X(Bax), etc.] and glial activation markers [ionized calcium binding adaptor molecule 1(IBA1) and glial fibrillary acidic protein(GFAP)] were decreased. These results indicated that liposome technique combined with nasal delivery alleviated the toxic side effects of TPG, and also significantly ameliorated the cognitive impairment of mice induced by central nervous system inflammation.

Study on construction of c-Met specific CAR-T cells and its killing effect on non-small cell lung carcinoma / 中华肿瘤杂志

Objective: To produce chimeric antigen receptor T cells (CAR-T) targeting human hepatocyte growth factor/c-Met (HGF/c-Met) protein and detect its cytotoxicity against non-small cell lung cancer (NSCLC) cells H1975 in vitro. Methods: The whole gene sequence of c-Met CAR containing c-Met single-chain fragment variable was synthesized and linked to lentiviral vector plasmid, plasmid electrophoresis was used to detect the correctness of target gene. HEK293 cells were transfected with plasmid and the concentrated solution of the virus particles was collected. c-Met CAR lentivirus was transfected into T cells to obtain second-generation c-Met CAR-T and the expression of CAR sequences was verified by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, and the positive rate and cell subtypes of c-Met CAR-T cells were detected by flow cytometry. The positive expression of c-Met protein in NSCLC cell line H1975 was verified by flow cytometry, and the negative expression of c-Met protein in ovarian cancer cell line A2780 was selected as the control. The cytotoxicity of c-Met CAR-T to H1975 was detected by lactate dehydrogenase (LDH) cytotoxicity assay at 1∶1, 5∶1, 10∶1 and 20∶1 of effector: target cell ratio (E∶T). Enzyme-linked immunosorbent assay (ELISA) was used to detect the release of cytokines such as TNF-α, IL-2 and IFN-γ from c-Met CAR-T co-cultured with H1975. Results: The size of band was consistent with that of designed c-Met CAR, suggesting that the c-Met CAR plasmid was successfully constructed. The results of gene sequencing were consistent with the original design sequence and lentivirus was successfully constructed. CAR molecules expression in T cells infected with lentivirus was detected by western blot and RT-qPCR, which showed c-Met CAR-T were successfully constructed. Flow cytometry results showed that the infection efficiency of c-Met CAR in T cells was over 38.4%, and the proportion of CD8(+) T cells was increased after lentivirus infection. The NSCLC cell line H1975 highly expressed c-Met while ovarian cancer cell line A2780 negatively expressed c-Met. LDH cytotoxicity assay indicated that the killing efficiency was positively correlated with the E∶T, and higher than that of control group, and the killing rate reached 51.12% when the E∶T was 20∶1. ELISA results showed that c-Met CAR-T cells released more IL-2, TNF-α and IFN-γ in target cell stimulation, but there was no statistical difference between c-Met CAR-T and T cells in the non-target group. Conclusions: Human NSCLC cell H1975 expresses high level of c-Met which can be used as a target for immunotherapy. CAR-T cells targeting c-Met have been successfully produced and have high killing effect on c-Met positive NSCLC cells in vitro.

Cervical spondylosis of nerve root type with qi stagnation and blood stasis treated with warming needle with different lengths of moxa stick: a randomized controlled trial / 中国针灸

OBJECTIVE@#To compare the clinical efficacy on cervical spondylosis of nerve root type with qi stagnation and blood stasis treated with warming needle with different lengths of moxa stick.@*METHODS@#Six hundred patients with cervical spondylosis of nerve root type with qi stagnation and blood stasis were randomly divided into 4 groups: a 4 cm length group (150 cases, 5 cases dropped off, 2 cases suspended), a 3 cm length group (150 cases, 6 cases dropped off, 2 cases suspended), a 2 cm length group (150 cases, 6 cases dropped off), and a routine acupuncture group (150 cases, 6 cases dropped off). Warming needle with moxa stick in the length of 4 cm, 3 cm and 2 cm was delivered in the 4 cm length group, the 3 cm length group and the 2 cm length group, respectively. In the routine acupuncture group, simple acupuncture was applied. The acupoints selected in the above groups included Dazhui (GV 14) and bilateral Jiaji (EX-B 2) of C5 and C7, Fengchi (GB 20), Jianzhen (SI 9), Quchi (LI 11), Zhongzhu (TE 3), etc. In each group, the intervention was delivered once daily and 5 times a week. One course of intervention was composed of 2 weeks and 2 courses were required. The TCM syndrome score, the score of clinical assessment scale for cervical spondylosis (CASCS), the score of the brachial plexus traction test of the affected upper limb, F wave occurrence rate and conduction velocity of the ulnar nerve, the median nerve and the radial nerve of the affected upper limb were compared before and after treatment in the patients of each group. The levels of serum inflammatory factors, i.e. interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and hypersensitive C-reactive protein (hs-CRP), were measured before and after treatment in the patients of each group. The clinical cfficacy was evaluated in the 4 groups.@*RESULTS@#After treatment, the results of TCM syndrome evaluation, i.e. the scores of neck pain, activity limitation and upper limb numbness and pain, as well as the total scores; and the scores of brachial plexus traction test were reduced when compared with those before treatment in each group (P<0.01, P<0.05). The scores of subjective symptoms and adaptability, and the total scores of CASCS were elevated in comparison with those before treatment in each group (P<0.01, P<0.05). In the 4 cm length group, compared with the other 3 groups, the scores of neck pain and activity limitation for TCM syndrome evaluation, and its total score were lower (P<0.05, P<0.01); and the scores of subjective symptoms and adaptability, and the total score of CASCS were higher (P<0.05, P<0.01). The score of the brachial plexus traction test in the 4 cm length group was lower than that of the routine acupuncture group (P<0.05). After treatment, F wave occurrence rates and conduction velocity of median nerve and radial nerve were increased when compared with those before treatment in each group (P<0.05, P<0.01). F wave occurrence rate and conduction velocity of the radial nerve in the 4 cm length group were higher than those of the other 3 groups (P<0.05), and those of the median nerve were higher when compared with the routine acupuncture group (P<0.05). After treatment, the levels of serum IL-1β, IL-6 and TNF-α were all reduced when compared with those before treatment in each group (P<0.01, P<0.05); the level of serum IL-6 in the 4 cm length group was lower than those of the other 3 groups and serum level of TNF-α was lower compared with that in the routine acupuncture group (P<0.05). The total effective rate of the 4 cm length group was 78.3% (112/143), which was higher when compared with the 3 cm length group (67.6%, 96/142), the 2 cm length group (65.3%, 94/144) and the routine acupuncture group (53.5%, 77/144), respectively (P<0.05).@*CONCLUSION@#Warming needle with moxa stick of 4 cm in length effectively relieves the clinical symptoms of cervical spondylosis of nerve root type with qi stagnation and blood stasis, improves the nerve function of the upper limbs, and reduces the inflammatory responses caused by nerve compression. The clinical efficacy of this therapy with moxa stick of 4 cm in length is superior to the warming needle with moxa sticks of 3 cm and 2 cm, as well as the routine acupuncture.

Puerarin alleviates lipopolysaccharide-induced acute kidney injury in mice by modulating the SIRT1/NF-κB pathway / 南方医科大学学报

OBJECTIVE@#To investigate the role of the SIRT1/NF-κB pathway in mediating the effect of puerarin against lipopolysaccharide (LPS)-induced acute kidney injury (AKI).@*METHODS@#Fifteen BALB/C mice were randomized into control group, LPS group and puerarin treatment group, and in the latter two groups, the mice were given an intraperitoneal injection of LPS (5 mg/kg), followed by daily injection of normal saline for 3 days or injection of puerarin (25 mg/kg) given 1 h later and then on a daily basis for 3 days. On day 5 after modeling, the kidney tissues were taken for histological observation and detection of cell apoptosis. The renal function indexes including urea nitrogen (BUN), serum creatinine (Scr) and kidney injury molecule 1 (KIM-1) and the levels of tumor necrosis factor (TNF-α) and interleukin 1β (IL-1β) were measured, and the expressions of SIRT1 and NF-κB-p65(acetyl K310) in the renal tissues were detected.@*RESULTS@#Intraperitoneal injection of LPS caused obvious glomerular capillary dilatation, hyperemia, renal interstitial edema, and renal tubular epithelial cell swelling and deformation in the mice. The mouse models of LPS-induced AKI also showed significantly increased renal tubular injury score and renal cell apoptosis (P < 0.01) with increased serum levels of BUN, Scr, KIM-1, TNF-α and IL-1β (P < 0.01), enhanced renal expressions of TNF-α, IL-1β and NF-κB p65(acetyl K310) (P < 0.01) and lowered renal expression of SIRT1 (P < 0.05). Treatment with puerarin effectively alleviated LPS-induced renal interstitial edema and renal tubular epithelial cell shedding, lowered renal tubular injury score (P < 0.01) and renal cell apoptosis rate (P < 0.01), and decreased serum levels of BUN, Scr, KIM, TNF-α and IL-1β (P < 0.01). Puerarin treatment significantly reduced TNF-α, IL-1β and NF-κB p65 (acetyl K310) expression in the renal tissue (P < 0.05) and increased SIRT1 expression by 17% (P < 0.05) in the mouse models.@*CONCLUSION@#Puerarin can effectively alleviate LPS-induced AKI in mice possibly by modulating the SIRT1/NF-κB signaling pathway.

Role of gut microbiota in perioperative neurocognitive disorders after cardiopulmonary bypass surgery in rats with humanized gut flora / 南方医科大学学报

OBJECTIVE@#To investigate whether gut microbiota disturbance after cardiopulmonary bypass (CPB) contributes to the development of perioperative neurocognitive disorders (PND).@*METHODS@#Fecal samples were collected from healthy individuals and patients with PND after CPB to prepare suspensions of fecal bacteria, which were transplanted into the colorectum of two groups of pseudo-germ-free adult male SD rats (group NP and group P, respectively), with the rats without transplantation as the control group (n=10). The feces of the rats were collected for macrogenomic sequencing analysis, and serum levels of IL-1β, IL-6 and TNF-α were measured with ELISA. The expression levels of GFAP and p-Tau protein in the hippocampus of the rats were detected using Western blotting, and the cognitive function changes of the rats were assessed with Morris water maze test.@*RESULTS@#In all the 3 groups, macrogenomic sequencing analysis showed clustering and clear partitions of the gut microbiota after the transplantation. The relative abundances of Klebsiella in the control group (P < 0.005), Akkermansia in group P (P < 0.005) and Bacteroides in group NP (P < 0.005) were significantly increased after the transplantation. Compared with those in the control group, the rats in group NP and group P showed significantly decreased serum levels of IL-1β, IL-6 and TNF-α and lowered expression levels of GFAP and p-Tau proteins (all P < 0.05). Escape platform crossings and swimming duration in the interest quadrant increased significantly in group NP (P < 0.05), but the increase was not statistically significant in group N. Compared with those in group P, the rats in group NP had significantly lower serum levels of IL-1β, IL-6 and TNF-α and protein expressions of GFAP and p-Tau (all P < 0.05) with better performance in water maze test (P < 0.05).@*CONCLUSION@#In patients receiving CPB, disturbances in gut mirobiota contributes to the development of PND possibly in relation with inflammatory response.

Pachymic acid protects against Crohn's disease-like intestinal barrier injury and colitis in miceby suppressingintestinal epithelial cell apoptosis via inhibiting PI3K/AKT signaling / 南方医科大学学报

OBJECTIVE@#To investigate the effect of pachymic acid (PA) against TNBS-induced Crohn's disease (CD)-like colitis in mice and explore the possible mechanism.@*METHODS@#Twenty-four C57BL/6J mice were randomized equally into control group, TNBS-induced colitis model group and PA treatment group. PA treatment was administered via intraperitoneal injection at the daily dose of 5 mg/kg for 7 days, and the mice in the control and model groups were treated with saline. After the treatments, the mice were euthanized for examination of the disease activity index (DAI) of colitis, body weight changes, colon length, intestinal inflammation, intestinal barrier function and apoptosis of intestinal epithelial cells, and the expressions of TNF-α, IL-6 and IL-1β in the colonic mucosa were detected using ELISA. The possible treatment targets of PA in CD were predicted by network pharmacology. String platform and Cytoscape 3.7.2 software were used to construct the protein-protein interaction (PPI) network. David database was used to analyze the GO function and KEGG pathway; The phosphorylation of PI3K/AKT in the colonic mucosal was detected with Western blotting.@*RESULTS@#PA significantly alleviated colitis in TNBS-treated mice as shown by improvements in the DAI, body weight loss, colon length, and histological inflammation score and lowered levels of TNF-α, IL-6 and IL-1β. PA treatment also significantly improved FITC-dextran permeability, serum I-FABP level and colonic transepithelial electrical resistance, and inhibited apoptosis of the intestinal epithelial cells in TNBS-treated mice. A total of 248 intersection targets were identified between PA and CD, and the core targets included EGFR, HRAS, SRC, MMP9, STAT3, AKT1, CASP3, ALB, HSP90AA1 and HIF1A. GO and KEGG analysis showed that PA negatively regulated apoptosis in close relation with PI3K/AKT signaling. Molecular docking showed that PA had a strong binding ability with AKT1, ALB, EGFR, HSP90AA1, SRC and STAT3. In TNBS-treated mice, PA significantly decreased p-PI3K and p-AKT expressions in the colonic mucosa.@*CONCLUSION@#PA ameliorates TNBS-induced intestinal barrier injury in mice by antagonizing apoptosis of intestinal epithelial cells possibly by inhibiting PI3K/AKT signaling.

Berberine inhibits autophagy and promotes apoptosis of fibroblast-like synovial cells from rheumatoid arthritis patients through the ROS/mTOR signaling pathway / 南方医科大学学报

OBJECTIVE@#To evaluate the regulatory effect of berberine on autophagy and apoptosis balance of fibroblast-like synoviocytes (FLSs) from patients with in rheumatoid arthritis (RA) and explore the mechanism.@*METHODS@#The inhibitory effect of 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L berberine on RA-FLS proliferation was assessed using CCK-8 method. Annexin V/PI and JC-1 immunofluorescence staining was used to analyze the effect of berberine (30 μmol/L) on apoptosis of 25 ng/mL TNF-α- induced RA-FLSs, and Western blotting was performed to detect the changes in the expression levels of autophagy- and apoptosis-related proteins. The cells were further treated with the autophagy inducer RAPA and the autophagy inhibitor chloroquine to observe the changes in autophagic flow by laser confocal detection of mCherry-EGFP-LC3B. RA-FLSs were treated with the reactive oxygen species (ROS) mimic H2O2 or the ROS inhibitor NAC, and the effects of berberine on ROS, mTOR and p-mTOR levels were observed.@*RESULTS@#The results of CCK-8 assay showed that berberine significantly inhibited the proliferation of RA-FLSs in a time- and concentration-dependent manner. Flow cytometry and JC-1 staining showed that berberine (30 μmol/L) significantly increased apoptosis rate (P < 0.01) and reduced the mitochondrial membrane potential of RA-FLSs (P < 0.05). Berberine treatment obviously decreased the ratios of Bcl-2/Bax (P < 0.05) and LC3B-II/I (P < 0.01) and increased the expression of p62 protein in the cells (P < 0.05). Detection of mCherry-EGFP-LC3B autophagy flow revealed obvious autophagy flow block in berberine-treated RA-FLSs. Berberine significantly reduced the level of ROS in TNF-α-induced RA-FLSs and upregulated the expression level of autophagy-related protein p-mTOR (P < 0.01); this effect was regulated by ROS level, and the combined use of RAPA significantly reduced the pro-apoptotic effect of berberine in RA-FLSs (P < 0.01).@*CONCLUSION@#Berberine can inhibit autophagy and promote apoptosis of RA-FLSs by regulating the ROS-mTOR pathway.