Objective Multiple carboxylase deficiency(MCD) is an autosomal recessively inherited defect of organic acid metabolism.The underlying mechanism is biotinidase(BT) or holocarboxylase synthetase(HLCS) deficiency.The authors reported 15 cases of MCD(clinical characteristics,diagnosis and treatment) and outcomes of long-term follow-up.Methods The clinical data of 15 patients with MCD admitted to Guangzhou Women and Children's Medical Center between Aug.2001 and Feb.2013 were analyzed.The diagnosis was confirmed by urinary organic acid analysis with gas chromatography/mass spectrometry (GC/MS),blood enzymatic determination and gene analysis.The patients consisted of 12 male and 3 female.The onset age ranged from newborn infants to 16 months.Results 1.Remarkable elevations of urinary lactate,3-oxy-butyric acid,3-OH-isovalerate,methylcitrate,3-methylcrontonylglycine,3-OH-propionate were detected in all of 15 cases.Fourteen cases with HLCS deficiency and 1 case with BT deficiency were confirmed by gene analysis.2.Most of patients with HLCS deficiency presented in the neonatal period or early infancy.The main clinical manifestations were skin rash (14 cases),tachypnea (9 cases),developmental retardation (8 cases),vomiting(5 cases),poor feeding (3 cases),developmental regradation (1 case),convulsion (1 case).Laboratory evaluation showed persistent metabolic acidosis and varied degree of ketosis,lactic acidosis,hyperuricacidemia,ammoniemia and hypoglycemia.Biotin was given to 13 patients in 10 mg/d and their metabolic disorders were corrected within 48 h,except one who gave up treatment and died.Treatment with Biotin in 5 mg/d provided clinical stability and normal neurodevelopmental outcome on follow-up for 3-11 (6.47 ± 2.70) years.3.One patient with BT deficiency presented with severe neurological symptoms(impaired consciousness,limbs trembling,tachypnea with irregular respiratory rhythm) at 16 months old.Demyelination of corpus callosum was evident on magnetic resonance imaging.Biotin treatment was given to him on the second of onset,and 1-year follow-up indicated significant improvement of his clinical symptoms,but the right limb weakness did not completely recover.Conclusions The main clinical features of HLCS deficiency include frequent skin rash,tachypnea,and metabolic disorders.BT deficiency may produce variable neurologic manifestations including demyelination of corpus callosum.Urinary organic acid analysis with GC/MS is critical to the early diagnosis of MCD.Prompt biotin treatment is recommended to correct metabolic derangements and continued therapy is essential to the improvement of long-term prognosis.Delayed commencement of therapy in BT deficiency can result in irreversible neurological damage.

ObjectiveTo explore the protective mechanism of paeoniflorin on mice with ulcerative colitis （UC） through the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） autophagy pathway. MethodUC mouse model was established by allowing mice freely drink 4% DSS， and 56 BALB/c male mice were randomly divided into model group， AMPK inhibitor group （20 mg·kg^-1）， paeoniflorin （50 mg·kg^-1） + inhibitor （20 mg·kg^-1） group， and high dose （50 mg·kg^-1）， medium dose （25 mg·kg^-1）， and low dose （12.5 mg·kg^-1） paeoniflorin groups. After seven days of drug intervention， the protective effect of paeoniflorin on mice with UC was determined by comparing the body weight， disease activity index （DAI） changes， and Hematoxylin-eosin （HE） staining results. Enzyme linked immunosorbent assay （ELISA） was used to detect the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the serum of mice in each group， and immunofluorescence was utilized to detect microtubule-associated protein 1 light chain 3 （LC3） content in the colon， AMPK， mTOR proteins， and their phosphorylated proteins including p-AMPK and p-mTOR in the colon tissue were detected by Western blot， and the mRNA expression levels of AMPK， mTOR， Beclin1， LC3， and p62 were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the model group showed a decrease in body mass， an increase in DAI score， and severe pathological damage to the colon. The levels of inflammatory factors including TNF-α and IL-6 increased in serum （P<0.01）， while the protein levels of LC3 and p-AMPK/AMPK were down-regulated in colon tissue， and those of p-mTOR/mTOR were up-regulated （P<0.01）. The mRNA expression levels of AMPK and LC3 were down-regulated， while the mRNA expression levels of mTOR and p62 were up-regulated （P<0.01）. Compared with the model group and the paeoniflorin + inhibitor group， the mice treated with paeoniflorin showed an increase in body mass， a decrease in DAI score， a reduction in pathological damage to colon tissue， and a reduction in the levels of inflammatory factors of TNF-α and IL-6 in serum （P<0.05）. The protein levels of LC3 and p-AMPK/AMPK in colon tissue were up-regulated， while the protein levels of p-mTOR/mTOR were down-regulated （P<0.01）. The mRNA expression levels of AMPK， Beclin1， and LC3 were up-regulated， while the mRNA expression of mTOR and p62 were down-regulated （P<0.01）. The colon tissue of the inhibitor group was severely damaged， and the trend of various indicators was completely opposite to that of the high dose paeoniflorin group. ConclusionPaeoniflorin can enhance autophagy and reduce inflammatory damage in mice with UC by activating the AMPK/mTOR signaling pathway and thus play a protective role.

ObjectiveTo explore the mechanism of Huanglian Jiedutang in inhibiting the pyroptosis mediated by NOD-like receptor protein 3 （NLRP3） inflammasomes and alleviating the acute liver injury （ALI） induced by lipopolysaccharide （LPS） in the mouse model of sepsis. MethodFifty-four male C57BL/6 mice were randomized into blank， model， low- （3.08 g·kg^-1）， medium- （6.15 g·kg^-1）， and high-dose （12.30 g·kg^-1） Huanglian Jiedutang， and positive control （dexamethasone） groups （n=9）. The mice were administrated with Huanglian Jiedutang at different doses by gavage for 7 days， and then LPS （15 mg·kg^-1） was injected intraperitoneally for the modeling of sepsis. In the positive control group， dexamethasone （0.05 g·kg^-1） was injected intraperitoneally 1.5 h after modeling， and the mouse sepsis score （MSS） was recorded 12 h after modeling. The mice were sacrificed for the collection of blood and liver tissue samples. The levels of alanine transaminase （ALT） and aspartate transaminase （AST） were measured by a biochemical analyzer. The levels of tumor necrosis factor （TNF）-α， interleukin （IL）-6， IL-1β， and IL-18 in the serum were measured by enzyme-linked immunosorbent assay kits. Hematoxylin-eosin staining was used to observe the pathological changes in the liver tissue. The content of NLRP3 was observed by the immunofluorescence assay. The expression of apoptosis-associated speck-like protein containing CARD （ASC） was detected by immunohistochemistry. The protein levels of NLRP3， ASC， Caspase-1， and gasdermin D （GSDMD） in the liver tissue were determined by Western blot. Real-time quantitative polymerase chain reaction（Real-time PCR） was employed to determine the mRNA levels of GSDMD， Caspase-1， IL-1β， and IL-18. ResultCompared with the blank group， the model group showed elevated levels of ALT and AST （P<0.01） and risen levels of inflammatory cytokines in the serum （P<0.01）. In addition， the modeling resulted in edema and necrosis in the liver， and up-regulated the protein levels of GSDMD， NLRP3， ASC， and Caspase-1 （P<0.01） and the mRNA levels of GSDMD， Caspase-1， IL-1β， and IL-18 （P<0.01）. Compared with the model group， the drug intervention groups showed reduced content of inflammatory cytokines （P<0.01）， alleviated pathological damage in the liver tissue， and down-regulated protein levels of GSDMD， NLRP3， ASC， and Caspase-1 （P<0.05，P<0.01） and mRNA levels of GSDMD， Caspase-1， IL-1β， and IL-18 （P<0.05，P<0.01） in the liver tissue. ConclusionHuanglian Jiedutang can inhibit pyroptosis and reduce inflammation by inhibiting the activation of NLRP3 inflammasomes， thus demonstrating a therapeutic effect on acute liver injury in the mouse model of sepsis induced by LPS.