OBJECTIVE: To investigate the impact of autophagy on cardiac tissue injury following common bile duct ligation (CBDL) in rats. METHODS: Twenty-four SPF grade healthy adult male Sprague-Dawley (SD) rats were randomly divided into four groups, with 6 rats in each group. The sham-operated (Sham) group underwent only dissection of the common bile duct without ligation. The CBDL group underwent CBDL to simulate jaundice-induced myocardial injury. The autophagy inhibitor 3-methyladenine (3-MA)+CBDL group was intraperitoneally injected with 15 mg/kg 3-MA 2 hours before modeling, and then injected once every other day. The CBDL+autophagy enhancer rapamycin (Rapa) group was intraperitoneally injected with Rapa 1 mg/kg 0.5 hour after modeling, and then injected once every other day. The rats in each group were sacrificed 2 weeks after surgery, and blood was taken from the inferior vena cava. Serum total bilirubin (TBil), alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), and MB isoenzyme of creatine kinase (CK-MB) were detected by using a fully automated animal biochemical analyzer. Serum oxidative stress marker superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were detected by colorimetric assay. The heart tissues of rats were taken and pathological changes were observed under a light microscope after hematoxylin-eosin (HE) staining. Transmission electron microscope was used to observe autophagosomes after double staining with uranyl acetate and lead citrate. The expressions of autophagy-related proteins were detected using Western blotting. RESULTS: Compared with the Sham group, the serum SOD activity of rats in the CBDL group was significantly decreased, while the serum MDA, TBil, ALT, AST, LDH, and CK-MB were significantly increased; the expressions of autophagy-related proteins Beclin-1 and microtubule-associated protein 1 light chain 3-II/I (LC3-II/I) were significantly increased, and p62 protein expression was significantly decreased. Autophagosomes were seen under electron microscopy in the CBDL group, and cardiac histopathological morphology showed focal necrosis in the myocardium as well as infiltration of inflammatory cells, dilatation of small interstitial blood vessels, and myocardial fiber degeneration. Compared with the CBDL group, cardiac tissue injury in rats was attenuated by pretreatment with the autophagy inhibitor 3-MA, with a decrease in inflammatory cell infiltration in myocardial tissue, a reduction in interstitial vasodilatation, and a decrease in the area of myocardial fibrosis; a decrease in the number of autophagosomes by electron microscopy; and a further rise in the viability of serum TBil, ALT, and AST [TBil (μmol/L): 184.40±6.74 vs. 120.70±16.93, ALT (U/L): 501.10±62.18 vs. 178.80±22.30, AST (U/L): 806.50±76.92 vs. 275.50±55.81, all P < 0.01], as well as a decrease in the levels of serum SOD, MDA, LDH, and CK-MB [SOD (kU/L): 85.00±5.29 vs. 107.50±7.86, MDA (μmol/L): 10.72±0.93 vs. 15.06±1.88, LDH (U/L): 387.40±119.50 vs. 831.30±84.35, CK-MB (U/L): 320.10±14.04 vs. 814.70±75.66, all P < 0.05]. The expressions of the autophagy-related proteins Beclin-1 and LC3-II/I in cardiac tissues were significantly decreased [Beclin-1 protein (Beclin-1/GAPDH): 0.67±0.04 vs. 0.89±0.01, LC3-II/I ratio: 0.93±0.03 vs. 1.09±0.01, both P < 0.01], and p62 protein expression was significantly increased (p62/GAPDH: 0.99±0.01 vs. 0.60±0.01, P < 0.01). In contrast, compared with the CBDL group, after administration of the autophagy enhancer Rapa, the rats showed increased cardiac tissue injury, increased inflammatory cell infiltration in myocardial tissues, increased interstitial vasodilatation, and increased area of myocardial fibrosis; an increase in autophagosomes was seen by electron microscopy; the change tendency of serum biochemical indicators and proteins in myocardial tissues were opposite with autophagy inhibition group with a decrease in serum TBil, ALT, and AST [TBil (μmol/L): 22.00±3.21 vs. 120.70±16.93, ALT (U/L): 72.13±5.97 vs. 178.80±22.30, AST (U/L): 135.20±12.95 vs. 275.50±55.81, all P < 0.05], as well as a increase in the levels of serum SOD, MDA, LDH, and CK-MB [SOD (kU/L): 208.00±2.65 vs. 107.50±7.86, MDA (μmol/L): 20.38±0.40 vs. 15.06±1.88, LDH (U/L): 1 268.00±210.90 vs. 831.30±84.35, CK-MB (U/L): 1 150.00±158.70 vs. 814.70±75.66, all P < 0.05]. The protein expressions of Beclin-1 and LC3-II/I in cardiac tissues were significantly increased [Beclin-1 protein (Beclin-1/GAPDH): 0.96±0.01 vs. 0.89±0.01, LC3-II/I ratio: 1.19±0.01 vs. 1.09±0.01, both P < 0.05], and p62 protein expression was significantly decreased (p62/GAPDH: 0.19±0.02 vs. 0.60±0.01, P < 0.01). CONCLUSIONS Activation of autophagy in CBDL rats led to myocardial tissue injury and reduced cardiac function. Inhibition of autophagy improved cardiac tissue injury in CBDL rats, while increasing autophagy exacerbated myocardial tissue injury.
Animals ; Autophagy ; Rats, Sprague-Dawley ; Male ; Ligation ; Rats ; Common Bile Duct/surgery* ; Myocardium/pathology* ; Adenine/pharmacology*

Objective:To summarize the clinical phenotype and genetic features of patients with relapsing encephalopathy with cerebellar ataxia (RECA) caused by ATP1A3 gene R756 variants. Methods:A retrospective analysis was performed on patients carrying the ATP1A3 gene R756 variants, identified by whole-exome sequencing of family members, at Capital Center for Children′s Health, Capital Medical University and Children's Medical Center, Peking University First Hospital from August 2005 to February 2024. Their clinical, laboratory, neuroimaging, electrophysiological and genetic characteristics were summarized. Results:A total of 13 RECA patients were enrolled in this study, including 8 males and 5 females. The age of onset was 8 months to 5 years, with a median age of onset of 18 months. All of 13 patients presented paroxysmal episodes of neurological decompensations triggered by fever and residual symptoms following the acute phase. During acute attack stage, ataxia was observed in all 13 cases, muscle weakness in 12 cases, dysarthria in 12 cases, altered consciousness in 10 cases, dysphagia in 10 cases, dystonic episodes in 4 cases, abnormal eye movement in 2 cases, choreoathetosis in 2 cases, and epileptic seizures in 1 case. All 13 patients had residual symptoms during the nonparoxysmal period, of whom 9 patients had ataxia, 9 patients had dysarthria, 4 patients had dystonia, 3 patients had cognitive disorders, and 1 patient had epileptic seizures. All 13 cases had ATP1A3 missense variants, and variant c.2266C>T/p.R756C was found in 6 cases, c.2267G>A/p.R756H in 5 cases, and c.2267G>T/p.R756L in 2 cases. Nine cases carried de novo variants, 4 with inherited variants. Conclusions:RECA caused by variants of ATP1A3 in residue 756 typically presents with an acute onset during infancy or early childhood, precipitated by febrile episodes and characterized by recurrent episodes of ataxia, with bulbar paralysis, muscle weakness and altered consciousness. Recurrence is common, and the most common persistent symptoms are cerebellar ataxia and dysarthria. A few patients have cognitive impairment. Three types of ATP1A3 gene variants R756C, R756H and R756L are related with RECA, and R756C is the most common variant.

Objective:To summarize the clinical phenotype and genetic features of patients with relapsing encephalopathy with cerebellar ataxia (RECA) caused by ATP1A3 gene R756 variants. Methods:A retrospective analysis was performed on patients carrying the ATP1A3 gene R756 variants, identified by whole-exome sequencing of family members, at Capital Center for Children′s Health, Capital Medical University and Children's Medical Center, Peking University First Hospital from August 2005 to February 2024. Their clinical, laboratory, neuroimaging, electrophysiological and genetic characteristics were summarized. Results:A total of 13 RECA patients were enrolled in this study, including 8 males and 5 females. The age of onset was 8 months to 5 years, with a median age of onset of 18 months. All of 13 patients presented paroxysmal episodes of neurological decompensations triggered by fever and residual symptoms following the acute phase. During acute attack stage, ataxia was observed in all 13 cases, muscle weakness in 12 cases, dysarthria in 12 cases, altered consciousness in 10 cases, dysphagia in 10 cases, dystonic episodes in 4 cases, abnormal eye movement in 2 cases, choreoathetosis in 2 cases, and epileptic seizures in 1 case. All 13 patients had residual symptoms during the nonparoxysmal period, of whom 9 patients had ataxia, 9 patients had dysarthria, 4 patients had dystonia, 3 patients had cognitive disorders, and 1 patient had epileptic seizures. All 13 cases had ATP1A3 missense variants, and variant c.2266C>T/p.R756C was found in 6 cases, c.2267G>A/p.R756H in 5 cases, and c.2267G>T/p.R756L in 2 cases. Nine cases carried de novo variants, 4 with inherited variants. Conclusions:RECA caused by variants of ATP1A3 in residue 756 typically presents with an acute onset during infancy or early childhood, precipitated by febrile episodes and characterized by recurrent episodes of ataxia, with bulbar paralysis, muscle weakness and altered consciousness. Recurrence is common, and the most common persistent symptoms are cerebellar ataxia and dysarthria. A few patients have cognitive impairment. Three types of ATP1A3 gene variants R756C, R756H and R756L are related with RECA, and R756C is the most common variant.

Objective:To observe the effect of hyperbilirubinemia on glomerulus of rats, and to explore its dose-response and mechanism.Methods:Twenty-four adult male Sprague-Dawley (SD) rats were divided into four groups according to the random number table method, with 6 rats in each group. Hyperbilirubinemia rat model was reproduced by intraperitoneal injection of bilirubin once every 12 hours for 4 times, at doses of 50, 100, and 200 mg/kg in low, medium, and high dose bilirubin group (LB group, MB group, HB group), respectively. The rats in negative control group (NC group) were given the same solvent without bilirubin powder. Urine was collected 24 hours after administration, and total protein (TP) level was detected. Then the rats were sacrificed, the blood was collected by cardiac puncture, and the total bilirubin (TBil) and direct bilirubin (DBil) levels were measured by automatic biochemical analyzer. The renal tissue was collected and stained with periodic acid-Schiff (PAS) staine, the glomerular morphology was observed under light microscope, and the glomerular injury score was performed. Podocyte morphology was observed by transmission electron microscopy after uranium acetate and lead citrate double staining. The activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) were determined by colorimetric method. The expression level of podocyte specific marker Wilms tumor protein-1 (WT-1) was determined by Western blotting.Results:With the increase of bilirubin dose, the contents of 24-hour urine TP, blood TBil, blood DBil and MDA content in kidney tissue were gradually increased, and the SOD activity and WT-1 expression in kidney tissue were gradually decreased. The differences between LB group, MB group, HB group and NC group were statistically significant [24-hour urine TP (mg): 24.85±2.22, 52.57±3.66, 56.84±3.49 vs. 7.50±1.33; blood TBil (μmol/L): 37.75±2.19, 81.37±2.13, 125.13±9.96 vs. 5.53±0.41; blood DBil (μmol/L): 15.50±1.96, 37.88±1.05, 64.53±2.89 vs. 2.38±0.35; kidney MDA (μmol/g): 3.14±0.65, 5.01±0.28, 7.50±1.08 vs. 2.30±0.20; kidney SOD (kU/g): 95.91±10.43, 57.06±15.90, 37.12±11.72 vs. 113.91±12.16; kidney WT-1 protein (WT-1/GAPDH): 0.280±0.006, 0.239±0.006, 0.198±0.001 vs. 0.361±0.005; all P < 0.05]. It was shown under light microscope that uneven thickness of mesangial membrane and basement membrane of the glomerulus, and some of them were accompanied by hyperplasia and widening. The glomerular injury score increased with the increase in bilirubin dose. The differences between LB group, MB group, HB group and NC group were statistically significant (17.50±1.05, 25.00±1.41, 34.00±1.41 vs. 11.67±0.82, all P < 0.05). Transmission electron microscopy showed that with the increase of bilirubin dose, the damage of glomerular podocytes was aggravated. Conclusions:Hyperbilirubinemia induced damage to glomerulus in a dose-dependent manner. In the lethal dose range, the higher the dose, the stronger the damage, which might be related to the oxidative stress promoted by bilirubin and the damage of glomerular podocytes.

Objective:To summarize the clinical phenotype and gene mutation characteristics of male patients with epilepsy caused by mosaic PCDH19 mutation. Methods:The clinical data of 3 male patients with epilepsy caused by mosaic PCDH19 mutation were analyzed.Microdroplet digital polymerase chain reaction (mDDPCR) was used for the detection of mosaicism in the three probands and their family members.Relevant literatures were reviewed. Results:The seizure onset age were 5 months, 9 months and 6 months of life respectively.Focal seizures occurred in 2 cases and multiple seizure types occurred in 1 case.Three patients presented with clusters of seizures.Fever sensitivity was observed in 2 cases out of the 3 cases.Two patients had intellectual disability and 1 patient had autistic manifestation.The clinical phenotype in 2 patient fulfilled the diagnosis of Dravet syndrome. PCDH19 mosaic mutations c. 317T>A(p.M106K), c.158dupT(p.D54Gfs*35) and c. 1639G>C(p.A547P) were detected respectively, and were identified as de novo after parental validation.Mutant allele fractions (MAF) in the blood samples were identified as 81.18%, 37.08%, 77.64%, respectively.The MAF of multiple tissues in 1 patient varied from 78.67% to 98.46%.Review of literature revealed that a total of 11 cases with mosaic PCDH19 mutation were reported.Among them, seizure onset occurred between 5 and 31 months of age.Focal seizures occurred in 9 cases, 3 cases of the 9 cases had only focal seizures.Generalized tonic clonic seizures occurred in 4 cases.Two or more seizures were observed in 6 cases.Clustering of seizures was found in all patient and sensitivity to fever was observed in 9 patients.Seven patients had mild to severe intellectual disability and 5 patients had autistic features. Conclusions:The clinical phenotypes of male patients with epilepsy caused by PCDH19 mosaic mutation are characterized by clustering of seizures, sensitivity to fever, focal seizures in most cases, varied degree of intellectual disability and autistic features in partial.

Objective:To follow up and clarify the prognosis of 670 pediatric patients with Dravet syndrome (DS).Methods:The clinical data of DS pediatric patients treated in the Department of Pediatrics, Peking University First Hospital from February 2005 to August 2016 were recorded, and genetic testing was carried out.DS pediatric patients were followed up via subsequent visits at the outpatient and telephone interview.Results:Among 670 cases with DS, 556 cases (556/670 cases, 83.0%) carried SCN1A mutations.In the follow-up, 608 cases were contacted (608/670 cases, 90.7%) and 62 cases (62/670 cases, 9.3%) were lost.The last follow-up median age was 8 years 5 months.Eighty-two cases (82/608 cases, 13.5%) were seizure-free for more than 1 year, with a median age of 9 years and 2 months.Thirty-eight cases relapsed (38/82 cases, 46.3%), mainly induced by fever (34 cases) or mi-ssing antiepileptic drugs (2 cases). Analysis of the relative factors of patients that were seizure-free for more than 1 year showed that children with missense SCN1A mutations, inherited mutations and an older age had a relatively good outcome for seizure control.Twenty-five cases (25/608 cases, 4.1%) were deceased, with a median age of 4 years.The mortality factors included multiple organ dysfunction syndromes after prolonged status epilepticus (12 cases), possible sudden unexpected death in epilepsy (7 cases), asphyxiation after vomiting with or without a seizure (2 cases), and an accidental injury (1 case). The fatal causes in the remaining 3 cases were unknown. Conclusions:DS is an intractable epileptic syndrome, but few patients may have a seizure remission (seizure free for more than 1 year). Patients with mi-ssense SCN1A mutations, inherited mutations and an older age have a relatively good outcome for seizure control.The mortality rate is high in DS patients.The causes of mortality include multiple organ dysfunction syndromes after prolonged status epilepticus, possible sudden unexpected death in epilepsy, and so on.

Objective: To analyze the etiology and clinical characteristics of hypertension in children; to analyze the risk factors for primary hypertension with target organ damage, and to provide evidence for clinical diagnosis of hypertension in children. Methods: Two hundred and thirty-two hospitalized children with hypertension at Children′s Hospital Institute of Pediatrics from April 2013 to December 2017 were retrospectively analyzed, including general situation, clinical manifestation, laboratory examination, auxiliary examination and damage assessment of target organs. Results: (1)There were totally 232 eligible cases, consisting of 184 males(79.3%) and 48 females(20.7%). Among these children: 38 cases(16.4%), 126 cases(54.3%), and 68 cases(29.3%) were diagnosed as prehypertension, hypertension in stage Ⅰ, and hypertension in stage Ⅱ, respectively.(2)The etiological composition of hypertension: there were 181 cases(78%) diagnosed as primary hypertension and 51 cases(22%) diagnosed as secondary hypertension, respectively; the proportion of the primary hypertension children during the period of preschool, school-age, and adolescent was 1.6%(3 cases), 21.0%(38 cases) and 77.4%(140 cases), respectively.The etiology of secondary hypertension was mostly of the renal parenchymal hypertension and renal vascular hypertension(43.1%). (3) The clinical manifestations of primary hypertension were mostly of asymptomatic or mild symptoms, totally 148 cases(64%), and then dizziness, headache, chest pain, chest tightness, palpitation, blurred vision, and other clinical symptoms.(4) The high risk factors for primary hypertension with target-organ damage included cesarean section, glucose metabolism disturbance and body mass index >24 kg/m². Conclusions In recent years, the proportion of primary hypertension among hospitalized children has increased.The changes in the etiology of secondary hypertension are not found.Primary hypertension children are often accompanied with target organ damage.It is of great significance to make blood pressure monitoring as a routine physical examination for primary and middle school students, so as to detect hypertension in children earlier, and make active intervention and improve the prognosis of hypertension in children.

Objective: To summarize the clinical and genetic features of β-propeller protein-associated neurodegeneration (BPAN). Methods: The clinical data of 17 patients with BPAN with WDR45 gene variants were retrospectively collected at Children’s Hospital of Fudan University, Peking University First Hospital, Capital Institute of Pediatrics, Shengjing Hospital of China Medical University and Shanghai Children's Hospital from June 2016 to December 2018, and their clinical manifestations, electroencephalogram, neuroimaging and genetics were analyzed. Results: Seventeen cases (13 females, 4 males), aged 1.1-8.8 years, were included. The median age of seizure onset was 14.5 months, from 3 months to 24 months of age, manifested with epileptic spasm in 6 cases and focal seizures in 5 cases. Eight patients had only one seizure type and 8 patients had two or more seizure types. Nine patients had complete remission of seizures. All 16 patients with seizures had developmental delay before the seizure onset, of whom 13 patients had moderate to severe seizures. The brain magnetic resonance imaging (MRI) was abnormal in 13 patients, including cerebral atrophy (10 cases) and thinning of the corpus callosum (9 cases). The brain magnetic susceptibility weighted imaging (SWI) in preschool stage showed prominent T2 hypointense signals in bilateral globus pallidus and brainstem ventral in two cases. Five seizure types (spasm, focal, absence, myodonic and generalized tonic clonic seizures)were found on ictal electroencephalogram(EEG) recordings. Compared to female patients(17(6-24) months of ege), male cases had earlier seizure onset (3, 4, 5, 18 months of age) . All patients had de novo variations in WDR45(6 nonsense, 4 frameshift, 3 missense and 4 splicing variations), with hemizygous variants in 3 males, mosaic variants in a male and heterozygous variants in 13 females, within which 5 variations had not been reported (c.977-1C>T,c.976+1G>C,c.10C>T,c.806del and c.110T>C). Conclusions The patients with BPAN have profound developmental delay and are vulnerable to seizures. The male patients with BPAN tend to have more severer clinical phenotype than females. Early brain SWI could facilitate the timely diagnosis of this disease.

Objective: To analyze the clinical characteristics of patients with PCDH19-female limited epilepsy (PCDH19-FE). Methods: The clinical data of 60 female epilepsy patients with PCDH19 gene heterozygous variations at the Department of Pediatrics, Peking University First Hospital from October 2007 to December 2018 were collected and analyzed retrospectively, their clinical manifestations, accessory examination and follow-up treatment were summarized. Results: Data of a total of 60 cases of PCDH19-FE were collected. The seizure onset occurred between 4 and 42 months of age (median: 11 months of age). Focal seizures occurred in 47 patients (78%), generalized tonic-clonic seizures (GTCS) occurred in 30 patients (50%), and other rare types of seizures included atypical absence, myoclonic, clonic, tonic, and atonic seizures. Two or more seizures types existed in 24 patients (40%), and seven patients (12%) had attacks of status epilepticus. Sensitivity to fever was observed in 47 out of them (78%) and clustering of seizures as found in all patients. During the interictal phase, focal discharges were monitored in 22 cases (22/45, 49%), multifocal discharges in 12 cases (12/45, 27%), widely discharging in 2 cases (4%), and both focal and widely discharging in 9 cases (20%). Clinical seizures were detected in 30 patients during the electroencephalogram (EEG) recording, including focal seizures in 22 cases, GTCS seizures in 8 cases, tonic seizure in three cases, myoclonic seizure followed by GTCS in one case, and two types of seizures in four cases. Before seizure onset, 57 patients had normal development and three patients had delayed language development. After seizure onset, varied degrees of intelligence disability were present in 38 cases (63%), language delay in 36 cases (60%), and gait instability in 10 cases (17%). Autistic features occurred in 17 cases (28%); and other behavioral problems like learning difficulties, personality, or emotional disorders existed in 33 cases (55%). Age at last follow-up ranged from one year and 3 months to 22 years and 3 months of age, 17 patients (28%) were seizure-free for more than 2 years (5 to 22 years at the last follow-up). The efficiency of antiepileptic drugs were 65% (33/51) in sodium valproate, 63% (27/43) in levetiracetam and 59% (20/34) in topiramate. Conclusions The clinical features of PCDH19-FE are characterized by clustering of seizures, focal seizures in most cases, sensitivity to fever mostly, focal discharges principally in EEG, varied degrees of intellectual disability or movement disorder, combined with autism spectrum disorders in partial and high efficiency in sodium valproate or levetiracetam treatment.

To summarize the clinical and genetic features of β?propeller protein?associated neurodegeneration (BPAN). Methods The clinical data of 17 patients with BPAN with WDR45 gene variants were retrospectively collected at Children's Hospital of Fudan University, Peking University First Hospital, Capital Institute of Pediatrics, Shengjing Hospital of China Medical University and Shanghai Children's Hospital from June 2016 to December 2018, and their clinical manifestations, electroencephalogram, neuroimaging and genetics were analyzed. Results Seventeen cases (13 females, 4 males), aged 1.1-8.8 years, were included. The median age of seizure onset was 14.5 months, from 3 months to 24 months of age, manifested with epileptic spasm in 6 cases and focal seizures in 5 cases. Eight patients had only one seizure type and 8 patients had two or more seizure types. Nine patients had complete remission of seizures. All 16 patients with seizures had developmental delay before the seizure onset, of whom 13 patients had moderate to severe seizures. The brain magnetic resonance imaging (MRI) was abnormal in 13 patients, including cerebral atrophy (10 cases) and thinning of the corpus callosum (9 cases). The brain magnetic susceptibility weighted imaging (SWI) in preschool stage showed prominent T2 hypointense signals in bilateral globus pallidus and brainstem ventral in two cases. Five seizure types (spasm, focal, absence, myodonic and generalized tonic clonic seizures) were found on ictal electroencephalogram(EEG)recordings. Compared to female patients(17(6-24) months of ege), male cases had earlier seizure onset (3, 4, 5, 18 months of age). All patients had de novo variations in WDR45 (6 nonsense, 4 frameshift, 3 missense and 4 splicing variations), with hemizygous variants in 3 males, mosaic variants in a male and heterozygous variants in 13 females, within which 5 variations had not been reported (c.977?1C>T,c.976+1G>C,c.10C>T,c.806del and c.110T>C). Conclusions The patients with BPAN have profound developmental delay and are vulnerable to seizures. The male patients with BPAN tend to have more severer clinical phenotype than females. Early brain SWI could facilitate the timely diagnosis of this disease.