OBJECTIVE: To investigate the effects of astragaloside IV (AS-IV) on podocyte injury of diabetic nephropathy (DN) and reveal its potential mechanism. METHODS: In in vitro experiment, podocytes were divided into 4 groups, normal, high glucose (HG), inositol-requiring enzyme 1 (IRE-1) α activator (HG+thapsigargin 1 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups. Additionally, podocytes were divided into 4 groups, including normal, HG, AS-IV (HG+AS-IV 20 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups, respectively. After 24 h treatment, the morphology of podocytes and endoplasmic reticulum (ER) was observed by electron microscopy. The expressions of glucose-regulated protein 78 (GRP78) and IRE-1α were detected by cellular immunofluorescence. In in vivo experiment, DN rat model was established via a consecutive 3-day intraperitoneal streptozotocin (STZ) injections. A total of 40 rats were assigned into the normal, DN, AS-IV [AS-IV 40 mg/(kg·d)], and IRE-1α inhibitor [STF-083010, 10 mg/(kg·d)] groups (n=10), respectively. The general condition, 24-h urine volume, random blood glucose, urinary protein excretion rate (UAER), urea nitrogen (BUN), and serum creatinine (SCr) levels of rats were measured after 8 weeks of intervention. Pathological changes in the renal tissue were observed by hematoxylin and eosin (HE) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expressions of GRP78, IRE-1α, nuclear factor kappa Bp65 (NF-κBp65), interleukin (IL)-1β, NLR family pyrin domain containing 3 (NLRP3), caspase-1, gasdermin D-N (GSDMD-N), and nephrin at the mRNA and protein levels in vivo and in vitro, respectively. RESULTS: Cytoplasmic vacuolation and ER swelling were observed in the HG and IRE-1α activator groups. Podocyte morphology and ER expansion were improved in AS-IV and IRE-1α inhibitor groups compared with HG group. Cellular immunofluorescence showed that compared with the normal group, the fluorescence intensity of GRP78 and IRE-1α in the HG and IRE-1α activator groups were significantly increased whereas decreased in AS-IV and IRE-1α inhibitor groups (P<0.05). Compared with the normal group, the mRNA and protein expressions of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N in the HG group was increased (P<0.05). Compared with HG group, the expression of above indices was decreased in the AS-IV and IRE-1α inhibitor groups, and the expression in the IRE-1α activator group was increased (P<0.05). The expression of nephrin was decreased in the HG group, and increased in AS-IV and IRE-1α inhibitor groups (P<0.05). The in vivo experiment results revealed that compared to the normal group, the levels of blood glucose, triglyceride, total cholesterol, BUN, blood creatinine and urinary protein in the DN group were higher (P<0.05). Compared with DN group, the above indices in AS-IV and IRE-1α inhibitor groups were decreased (P<0.05). HE staining revealed glomerular hypertrophy, mesangial widening and mesangial cell proliferation in the renal tissue of the DN group. Compared with the DN group, the above pathological changes in renal tissue of AS-IV and IRE-1α inhibitor groups were alleviated. Quantitative RT-PCR and Western blot results of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N were consistent with immunofluorescence analysis. CONCLUSION AS-IV could reduce ERS and inflammation, improve podocyte pyroptosis, thus exerting a podocyte-protective effect in DN, through regulating IRE-1α/NF-κ B/NLRP3 signaling pathway.
Podocytes/metabolism* ; Animals ; Diabetic Nephropathies/metabolism* ; Saponins/therapeutic use* ; Triterpenes/therapeutic use* ; Signal Transduction/drug effects* ; NF-kappa B/metabolism* ; Protein Serine-Threonine Kinases/metabolism* ; Male ; Rats, Sprague-Dawley ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Endoribonucleases/metabolism* ; Endoplasmic Reticulum Chaperone BiP ; Rats ; Diabetes Mellitus, Experimental/complications* ; Endoplasmic Reticulum/metabolism* ; Multienzyme Complexes

OBJECTIVE: To explore the mechanism of colon dialysis with Yishen Decoction (YS) in improving the autophagy disorder of intestinal epithelial cells in chronic renal failure (CRF) in vivo and in vitro. METHODS: Thirty male SD rats were randomly divided into normal, CRF, and colonic dialysis with YS groups by a random number table method (n=10). The CRF model was established by orally gavage of adenine 200 mg/(kg•d) for 4 weeks. CRF rats in the YS group were treated with colonic dialysis using YS 20 g/(kg•d) for 14 consecutive days. The serum creatinine (SCr) and urea nitrogen (BUN) levels were detected by enzyme-linked immunosorbent assay. Pathological changes of kidney and colon tissues were observed by hematoxylin and eosin staining. Autophagosome changes in colonic epithelial cells was observed with electron microscopy. In vitro experiments, human colon cancer epithelial cells (T84) were cultured and divided into normal, urea model (74U), YS colon dialysis, autophagy activator rapamycin (Ra), autophagy inhibitor 3-methyladenine (3-MA), and SIRT1 activator resveratrol (Re) groups. RT-PCR and Western blot were used to detect the mRNA and protein expressions of zonula occludens-1 (ZO-1), Claudin-1, silent information regulator sirtuin 1 (SIRT1), LC3, and Beclin-1 both in vitro and in vivo. RESULTS: Colonic dialysis with YS decreased SCr and BUN levels in CRF rats (P<0.05), and alleviated the pathological changes of renal and colon tissues. Expressions of SIRT1, ZO-1, Claudin-1, Beclin-1, and LC3II/I were increased in the YS group compared with the CRF group in vivo (P<0.05). In in vitro study, compared with normal group, the expressions of SIRT1, ZO-1, and Claudin-1 were decreased, and expressions of Beclin-1, and LC3II/I were increased in the 74U group (P<0.05). Compared with the 74U group, expressions of SIRT1, ZO-1, and Claudin-1 were increased, whereas Beclin-1, and LC3II/I were decreased in the YS group (P<0.05). The treatment of 3-MA and rapamycin regulated autophagy and the expression of SIRT1. SIRT1 activator intervention up-regulated autophagy as well as the expressions of ZO-1 and Claudin-1 compared with the 74U group (P<0.05). CONCLUSION Colonic dialysis with YS could improve autophagy disorder and repair CRF intestinal mucosal barrier injury by regulating SIRT1 expression in intestinal epithelial cells.
Animals ; Sirtuin 1/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Autophagy/drug effects* ; Male ; Intestinal Mucosa/drug effects* ; Rats, Sprague-Dawley ; Epithelial Cells/metabolism* ; Colon/drug effects* ; Humans ; Kidney Failure, Chronic/drug therapy* ; Signal Transduction/drug effects* ; Renal Dialysis ; Rats ; Kidney/drug effects*

Insulin is an important protein hormone that participates in multiple metabolic pathways.Biosynthetic insulin has been widely used in the treatment of type 1 and type 2 diabetes.Currently,the number of reported cases of insulin overdose both at home and abroad is gradually increasing,and insulin homicide is no longer a means of"committing murder without leaving a trace".At present,there are no systematic protocols for the identification of insulin overdose in the field of forensic medi-cine in China.This article introduces the causes,toxicological characteristics,forensic examination,labo-ratory testing methods and indicator reference of insulin overdose.Based on the identification practice and research results and referring to relevant studies on insulin overdose at home and abroad,this pa-per aims to provide recommendations and references for the formulation of forensic identification guide-lines for insulin overdose cases.

Objective To study the bioequivalence of test and reference olmesartan tablet in Chinese healthy subjects after single dose under fasting and fed conditions.Methods A single-center,random,open,single-dose,two-preparations,double-period,crossover study was adopted.A total of 48 healthy adult male and female subjects(24 cases of fasting test and 24 cases of fed test)were included in the random crossover administration.Single oral dose 20 mg of test and reference were taken under fasting and postprandial conditions,respectively.Plasma concentration of olmesartan in plasma were determined by liquid chromatography tandem mass spectrometry.The main pharmacokinetic parameters were calculated by Phoenix WinNonlin 8.0 software.Results The main pharmacokinetic parameters of the test and reference preparations of olmesartan tablets in the fasting group were as follows:Cmax were(653.06±133.53)and(617.37±151.16)ng·mL-1,AUC0-t were(4 201.18±1 035.21)and(4 087.38±889.99)ng·mL-1·h,AUC0-∞ were(4 254.30±1 058.90)and(4 135.69±905.29)ng·mL-1·h.The main pharmacokinetic parameters of the test and reference preparations of olmesartan tablets in the postprandial group were as follows:Cmax were(574.78±177.05)and(579.98±107.74)ng·mL-1,AUC0-t were(3 288.37±866.06)and(3 181.51±801.06)ng·mL-1·h,AUC0-∞ were(3 326.11±874.26)and(3 242.01±823.09)ng·mL-1·h.Under fasting and postprandial conditions,the 90％confidence intervals of the main pharmacokinetic parameters of the test and reference preparations are both 80.00％-125.00％.Conclusion Under fasting and postprandial conditions,a single oral dose of test and reference preparations olmesartan tablets in Chinese healthy adult volunteers showed bioequivalence.

Mycobacterium intracellular CHPC 1.5701 strain was isolated from the sputum specimen of the patient.This study attempted to establish a guinea pig infection model to evaluate its virulence,so as to provide basic scientific basis for the prevention and treatment of Mycobacterium intracellular infection.Mycobacterium intracellular CHPC 1.5701 was cultured in 7H10 medium.The morphology of the colony changed from colorless to light yellow smooth colony,and the acid-fast staining was positive.Mycobacterium intracellular suspension was diluted with 0.9％sodium chloride solution to 1 × 1010 CFU/mL,1×109 CFU/mL,1 × 108 CFU/mL,1 × 107 CFU/mL and 1 × 106 CFU/mL,respectively.Healthy Hartly guinea pigs with negative skin test of pure protein derivatives of tuberculin were selected and randomly divided into 6 groups with 10 guinea pigs in each group,half male and half female.Guinea pigs in the ex-perimental group were divided into 5 groups,which were intra-peritoneally injected with 5 different concentrations of 1.0 mL suspension/guinea pig,and those in the control group were in-traperitoneally injected with 0.9％sodium chloride solution 1.0 mL suspension/guinea pig.The guinea pigs was then observed,weighed every week,dissected 5 weeks later,lung,spleen and liver tissues were taken,and the tissue lesions were analyzed by hematoxylin-eosin(HE)staining,and the bacteria load was detected by tissue homogenate culture in 7H10 medium.The results showed that Mycobacterium intracellular challenge test had no significant effect on body weight of male guinea pigs(P＞0.05),but could reduce body weight of female guinea pigs(P＜0.01).No pathological changes of organ tissues were ob-served in guinea pigs in the control group,while HE staining of lung,spleen and liver tissues of guinea pigs in challenge experi-mental groups showed alveolar wall alveolar epithelial hyperplasia,monocyte infiltration in liver and spleen,granulomatous in-flammation and other pathological changes,and the degree of pathological changes was related to the injection dose of Mycobac-terium intracellular.Mycobacteria intracellular could be isolated from tissue culture of some organs,among which the spleen had a large amount of bacteria.Intrabitoneal injection of Mycobacteria intracellular CHPC 1.5701 can affect the body weight of female guinea pigs and cause lung,liver and spleen infection of guinea pigs,but no caseous necrosis of guinea pigs was ob-served.Compared with the reported data of Mycobacterium tuberculosis virulence test,it is concluded that Mycobacterium in-tracellular has certain virulence to guinea pigs and belongs to low virulence and low pathogenicity bacteria...

Mycobacterium intracellular CHPC 1.5701 strain was isolated from the sputum specimen of the patient.This study attempted to establish a guinea pig infection model to evaluate its virulence,so as to provide basic scientific basis for the prevention and treatment of Mycobacterium intracellular infection.Mycobacterium intracellular CHPC 1.5701 was cultured in 7H10 medium.The morphology of the colony changed from colorless to light yellow smooth colony,and the acid-fast staining was positive.Mycobacterium intracellular suspension was diluted with 0.9％sodium chloride solution to 1 × 1010 CFU/mL,1×109 CFU/mL,1 × 108 CFU/mL,1 × 107 CFU/mL and 1 × 106 CFU/mL,respectively.Healthy Hartly guinea pigs with negative skin test of pure protein derivatives of tuberculin were selected and randomly divided into 6 groups with 10 guinea pigs in each group,half male and half female.Guinea pigs in the ex-perimental group were divided into 5 groups,which were intra-peritoneally injected with 5 different concentrations of 1.0 mL suspension/guinea pig,and those in the control group were in-traperitoneally injected with 0.9％sodium chloride solution 1.0 mL suspension/guinea pig.The guinea pigs was then observed,weighed every week,dissected 5 weeks later,lung,spleen and liver tissues were taken,and the tissue lesions were analyzed by hematoxylin-eosin(HE)staining,and the bacteria load was detected by tissue homogenate culture in 7H10 medium.The results showed that Mycobacterium intracellular challenge test had no significant effect on body weight of male guinea pigs(P＞0.05),but could reduce body weight of female guinea pigs(P＜0.01).No pathological changes of organ tissues were ob-served in guinea pigs in the control group,while HE staining of lung,spleen and liver tissues of guinea pigs in challenge experi-mental groups showed alveolar wall alveolar epithelial hyperplasia,monocyte infiltration in liver and spleen,granulomatous in-flammation and other pathological changes,and the degree of pathological changes was related to the injection dose of Mycobac-terium intracellular.Mycobacteria intracellular could be isolated from tissue culture of some organs,among which the spleen had a large amount of bacteria.Intrabitoneal injection of Mycobacteria intracellular CHPC 1.5701 can affect the body weight of female guinea pigs and cause lung,liver and spleen infection of guinea pigs,but no caseous necrosis of guinea pigs was ob-served.Compared with the reported data of Mycobacterium tuberculosis virulence test,it is concluded that Mycobacterium in-tracellular has certain virulence to guinea pigs and belongs to low virulence and low pathogenicity bacteria...

PURPOSE: Child head injury under impact scenarios (e.g. falls, vehicle crashes, etc.) is an important topic in the field of injury biomechanics. The head of piglet was commonly used as the surrogate to investigate the biomechanical response and mechanisms of pediatric head injuries because of the similar cellular structures and material properties. However, up to date, piglet head models with accurate geometry and material properties, which have been validated by impact experiments, are seldom. We aim to develop such a model for future research. METHODS: In this study, first, the detailed anatomical structures of the piglet head, including the skull, suture, brain, pia mater, dura mater, cerebrospinal fluid, scalp and soft tissue, were constructed based on CT scans. Then, a structured butterfly method was adopted to mesh the complex geometries of the piglet head to generate high-quality elements and each component was assigned corresponding constitutive material models. Finally, the guided drop tower tests were conducted and the force-time histories were ectracted to validate the piglet head finite element model. RESULTS: Simulations were conducted on the developed finite element model under impact conditions and the simulation results were compared with the experimental data from the guided drop tower tests and the published literature. The average peak force and duration of the guide drop tower test were similar to that of the simulation, with an error below 10%. The inaccuracy was below 20%. The average peak force and duration reported in the literature were comparable to those of the simulation, with the exception of the duration for an impact energy of 11 J. The results showed that the model was capable to capture the response of the pig head. CONCLUSION This study can provide an effective tool for investigating child head injury mechanisms and protection strategies under impact loading conditions.
Animals ; Swine ; Finite Element Analysis ; Skull/injuries* ; Craniocerebral Trauma/diagnostic imaging* ; Brain ; Biomechanical Phenomena ; Scalp

AIM:To evaluate the underlying aetiology and clinical characteristics of retinal detachment(RD)in school-age pediatric monocular RD.METHODS:Patients with RD and contralateral blind(monocular RD)aged 7-14 years, from November 2015 to May 2021 in our hospital were retrospectively reviewed. Demographic characteristics and etiology of RD, clinical type, surgical modality, type of intraocular tamponade, pre-and postoperative visual and anatomical outcomes were recorded and evaluated.RESULTS: There were 27 children(27 eyes)with monocular RD at least 6mo follow-up. The average age at presentation was 10.63±2.30 years. Familial exudative vitreoretinopathy(FEVR)(11/27, 41%), postoperative congenital glaucoma(6/27, 22%)and Stickler syndrome(3/27, 11%)were main underlying etiologies. Among them, rhegmatogenous retinal detachment(RRD)comprised 78%(21/27)of the patients, of which 81% patients(17/21)had proliferative vitreoretinopathy(PVR)C3 or worse. Pars plana vitrectomy(PPV)was done in 85%(23/27)of the patients, of which 83%(19/23)received silicone oil tamponade. Best corrected visual acuity(BCVA, LogMAR)worse than 1.7 was seen in 78%(21/27)of the patients at final visit, and 82%(22/27)had reattached retina, but 41%(11/27)of the patients remained status of silicone oil tamponade at last visit.CONCLUSION:School-age pediatric monocular RD is often associated with underlying congenital or hereditary conditions, and often presented with severe RD and severe PVR reaction which needed vitrectomy combined with silicon oil tamponade, and with poor visual and anatomical short-term prognosis.

The mitochondrial enzyme glutaminase C (GAC) is highly expressed in a variety of cancer cells, resulting in increased glutamine metabolism and cancer development. Therefore, GAC has become a potential target for anti-tumor drug development. However, current GAC inhibitors shared similar structural characteristics, few new scaffolds were reported. By conducting a prokaryotic Escherichia coli expression system, human GAC protein of high-purity was obtained through lysozyme digestion combined with ultrasound dissociation, and cobalt magnetic beads purification, Moreover, we performed studies to validate interaction between small molecules and GAC protein through thermal shift assay, drug affinity responsive target stability assay, protein crosslinking and GAC enzyme activity detection. Meanwhile, a comprehensive small molecule-protein interaction confirmation and systematic pharmacodynamic study in vitro were carried out on compound C19, which was a reported GAC inhibitor screened from the Enamine database. Results showed that C19 directly bind to GAC protein, disturbed GAC tetramers formation, and inhibited its enzyme catalytic activity. By interfering GAC function, C19 dose-dependently suppressed GAC-mediated glutamine metabolism, reduced glutamate in cancer cells, and thus alleviated A549 and NCI-H1299 non-small cell lung cancer cell growth. Together, C19 was identified as a lead compound, providing a new strategy for the structural design of drugs targeting GAC.

APACHE ; Humans ; Microtubule-Associated Proteins ; Multiple Organ Failure ; Sepsis ; Shock, Septic