Objective A model for studying oral ulcers induced by betel nut-extract was constructed in rats.Changes in the structure and diversity of oral flora were observed to explore the involvement of oral flora and local inflammatory factors in the pathogenesis of oral ulcers induced by betel nut-extract and to provide theoretical support for the prevention and treatment of oral ulcers in the clinic.Methods Thirty SD rats were randomly divided into normal,model and intervention groups(Guilin watermelon cream,8 mg/d for 7 days),with 10 rats/group.The oral mucosa of rats was subcutaneously injected with 10 g/mL of betel nut-extract to generate an oral ulcer model.The histomorphological changes were observed,and ulcer area and ulcer scores were assessed.Local oral tissue tumor necrosis factor-α(TNF-α),interleukin(IL)-2 and IL-8 levels were determined.Oral mucosal tissues were sampled for HE staining and analyzed for the structural distribution of oral flora and the diversity of microbial communities using high-throughput sequencing method.Results Compared with rats in the normal group,those in the model group had an increased ulcer area,significantly increased ulcer scores(P＜0.01),and significantly increased levels of TNF-α,IL-2 and IL-8 in the oral mucosal tissues(P＜0.01).The amount Streptococcus(P＜0.05)and Veillonella(P＜0.001)in the oral saliva of the model group rats was significantly reduced.The model group rats showed oral mucosal epithelial cell hyperplasia or focal necrosis,mucosal lamina propria edema,and hemorrhage accompanied by mass neutrophil and monocyte infiltration.Compared with the model group rats,the intervention group rats had significantly reduced ulcerated area(P＜0.05,P＜0.01)and ulcer scores(P＜0.05).And oral mucosal tissue levels of TNF-α(P＜0.01),IL-2(P＜0.05)and IL-8(P＜0.05),as well as significantly increased Streptococcus(P＜0.001)and Veillonella(P＜0.01)and significantly reduced Staphylococcus(P＜0.01)in the oral saliva.The degree of lesions in the oral mucosal tissues was significantly improved in the intervention group.Conclusions Betel nut-extract can be used to successfully reproduce a rat model of oral ulcer,and it is speculated that the development of oral ulcers after exposure to betel nut-extract may be related to an imbalance in the oral flora and local tissue inflammatory mediators.

Objective:To analyze the gene variants in patients with primary distal renal tubular acidosis (dRTA), and explore the correlation between the genotype and phenotype.Methods:The Sanger direct sequencing or whole-exome sequencing was used to identify causal variants and the variation pathogenicity was evaluated according to 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines in 44 dRTA patients (37 families) diagnosed in the Affiliated Qingdao Municipal Hospital of Qingdao University and the Affiliated Hospital of Qingdao University from April 2010 to September 2020. The clinical features of the patients were summarized, and the correlation between the genotype and phenotype was investigated.Results:Seven variants of SLC4A1 gene, 17 variants of ATP6V0A4 gene, and 15 variants of ATP6V1B1 gene were identified in 44 patients with dRTA, and of which 11 variants were new ones. According to ACMG guidelines, the pathogenic, likely pathogenic, benign variants among the 39 variants were 22, 16 and 1, respectively. Nine patients were autosomal dominant hereditary dRTA caused by SLC4A1 gene mutation, 4 patients with autosomal recessive hereditary dRTA complicated with Southeast Asian ovalocytosis and anemia were caused by SLC4A1 gene mutation, and 14 patients caused by ATP6V0A4 gene mutation and 8 patients caused by ATP6V1B1 gene mutation were autosomal recessive hereditary dRTA; Two children with dRTA were found to carry one monoallelic defect in ATP6V1B1, and no causal gene mutation was identified in 7 patients. One patient showed incomplete dRTA, and the other 43 patients showed complete dRTA. The prevalence of sensory neural hearing loss caused by ATP6V0A4 and ATP6V1B1 mutation were 2/14 and 6/10 respectively. The frequency of chronic kidney disease in adults, children and infants were 4/4, 2/4, and 1/36, separately. After the drug treatment based on potassium citrate and sodium citrate, the growth and development (28/40) and electrolyte disturbance (41/44) of most patients were significantly improved. Conclusions:The present study has identified 39 variants of SLC4A1, ATP6V0A4 and ATP6V1B1 genes in 44 patients with dRTA, including 11 novel ones. There is a close relationship between genotype and phenotype in dRTA patients and most patients' conditions were improved after proper treatment. This study enriches the human gene mutation database and provides valuable references for diagnosis, treatment and genetic counseling in patients with dRTA.

Objective: To identify and analyze the variants of the KCNJ1 gene in five Chinese patients with Bartter syndrome type 2 (BS2), and to describe their clinical features as well as treatment results. Methods: Data and blood samples of five BS2 patients and their relatives confirmed by Qingdao Municipal Hospital from June 2012 to January 2019 were collected. Whole-exome-sequencing (WES) based on the second generation high throughput sequencing was performed to detect variants. The 2015 American College of Medical Genetics and Genomics Standards and Guidelines were applied to analyze the pathogenicity of the variants. The clinical features and laboratory results were retrospectively studied. The response to treatment and follow-up data were reviewed. Results: Ten variants including six novel ones of KCNJ1 gene were identified through WES and verified by Sanger dideoxy sequencing. Missense variants accounted for the highest proportion. The common symptoms and signs of five BS2 patients from high to low incidence were polydipsia and polyuria (5/5), one of them (1/5) presented with diabetes insipidus; maternal polyhydramnios and premature delivery (4/5); growth retardation (3/5). Initially, two patients presented with hypochloremic metabolic alkalosis and hypokalemia, whereas the acid-base disturbance was absent in the others. One patient experienced hyperkalemia. In terms of calcium-phosphorus metabolism, one patient had evident parathyroid hormone (PTH) resistance (hypocalcemia, hyperphosphatemia and markedly elevated serum intact PTH levels), three presented with PTH overacting (hypercalcemia, hypophosphatemia and mild elevated serum intact PTH levels), and one showed normal blood calcium and phosphorus concentrations with high-normal serum intact PTH levels. All patients had nephrocalcinosis or hypercalciuria, and one of them complicated with nephrolithiasis. Indomethacin helped to correct the growth retardation, halt polydipsia polyuria, decrease the elevated urinary calcium excretion, and normalize electrolyte disturbance as well as PTH parameters in some patients. Conclusions This investigation identifies ten variants of KCNJ1 gene, including six ones that have not been previously reported, which will enrich the human gene mutation database (HGMD). These patients in our study have atypical BS phenotype, so that careful differentiation from other parathyroid diseases will be required for clinicians.

To study the protective effect of Xingnaojing Injection on early global brain ischemia-induced deep coma in rats.
 Methods: The deep coma model was induced by global brain ischemia by using four-vessel occlusion method in male SD rats. According to the body weight, the rats were randomly divided into 8 groups: a model control group, three different dose of Xingnaojing Injection (1.8, 3.6 and 5.4 mL.kg-1) groups, a Xingnaojing Injection (3.6 mL.kg-1) plus PI3K inhibitor group, a naloxone injection (0.04 mL.kg-1) group and a naloxone injection (0.04 mL.kg-1) plus Xingnaojing Injection (3.6 mL.kg-1) group (n=8 per group). In addition, eight animals served as the sham group were performed same operation with the model group excepting no blockage of the blood vessels. After the operation, three different doses of Xingnaojing Injection and/or naloxone injection were given intravenously once a day for three days. Ten μL PI3K inhibitor (LY294002, 10 mmol/L) was injected via anterior cerebral ventricle at once after global brain ischemia. The awakening time after the first drug treatment, the grasping power and the autonomous activity within 10 min after the last drug treatment were recorded. The levels of both dopamine (DA) and glutamate (Glu) in cerebrospinal fluid were detected by ELISA. The pathological changes were observed in brain tissue slices with HE staining and the protein levels of Akt/p-Akt and cAMP-response element binding protein (CREB)/p-CREB in hippocampus were detected by Western blotting.
 Results: Comparing with the model group, single administration of Xingnaojing Injection could significantly shorten the waking time (P<0.05) and continuous administration of Xingnaojing Injection for 3 d could increase grasping power, distance, frequency and duration of autonomous activities (P<0.05 or P<0.01) in the deep coma rat. Also, Xingnaojing Injection could inhibit these increases in neurotransmitters DA and Glu contents (P<0.05 or P<0.01), and improve pathological changes of hippocampal tissue. Xingnaojing Injection significantly induced protein phosphorylation of both Akt and CREB (P<0.05 or P<0.01); this effect was inhibited by PI3K inhibitor (P<0.05 or P<0.01). Moreover, the protective effects of naloxone on awakening time, grasping power, the autonomous activity and hippocampus damage in global brain ischemia-induced deep coma could be enhanced by joint use of Xingnaojing Injection (P<0.05 or P<0.01).
 Conclusion: Xingnaojing Injection could significantly improve deep coma induced by global brain ischemia in rat, which is related to inducing PI3K/Akt-dependent protein phosphorylation of CREB, and reducing hippocampal damage. The protective effect of Xingnaojing Injection is synergistically enhanced by naloxone.
Animals ; Brain ; Brain Ischemia ; Coma ; Drugs, Chinese Herbal ; Male ; Phosphatidylinositol 3-Kinases ; Rats ; Rats, Sprague-Dawley

Objective To study the protective effect of shenfu injection on cerebral ischemia reperfusion injury in rats.Methods 120 male Sprague Dawley (SD) rats(320-350 g) were randomly divided into sham operation group,model control group,Nimodipine injection group,low,medium and high dose group of shenfu injection according to gender weight.20 males in each group were given medicine once a day for 7 days before operation.The cerebral ischemia model was established by thread embolization after 5 days of administration.In the sham operation group,the other operations were the same as those in the model group except for carotid artery ligation and thread insertion.After 24 hours of perfusion,the neurological score,abdominal aorta blood flow,malondialdehyde (MDA),superoxide dismutase (SOD) and glutathione (GHS) levels in brain tissues were measured.Triphenyltetrazolium chloride (TTC) staining was used to calculate the area of cerebral infarction and pathological examination of brain tissues.Results Compared with the model control group,the middle and high dosage of shenfu injection could obviously improve the nerve function and increase the percentage of cerebral infarction area (P ＜ 0.05);the high dosage group of shenfu injection could obviously decrease the whole blood viscosity (P ＜ 0.01);the middle and high dosage of shenfu injection could obviously reduce the level of MDA in rat brain tissue (P ＜ 0.01) while increasing the levels of SOD and GSH (P ＜0.01),finally could significantly improve the pathological changes of brain tissues such as mild swelling of nerve fibers,mild neuronal degeneration,inflammatory interstitial edema and inflammation.Conclusions Shenfu injection has obvious protective effect on cerebral ischemia reperfusion model in rats.

Objective To analyze the mutations of SLC12A1 gene in nine Chinese families with Bartter syndrome type I (BS1),and analyze the relationship between genotype and phenotype.Methods The next generation sequencing was used to detect mutations in nine BS1 patients including eight with antenatal BS (aBS) and one with classical BS (cBS).Clinical characteristics and biochemical findings at the first admission as well as follow-up were reviewed.Results 15 different mutations of SLC12A1 gene were identified,including 11 novel ones.Among nine probands,seven were compound heterozygotes,two were homozygotes.All patients presented with polydipsia and polyuria,and eight with growth retardation.All patients had lower than-normal serum chloride concentration,metabolic alkalosis,and elevated basal renin activity and aldosterone,and seven had hypokalemia.Through treatment of indomethacin and/or potassium chloride,biochemical indicators could roughly restored normal.Conclusion These findings will enrich the human gene mutation database (HGMD) and provide valuable references to the genetic counseling and diagnosis for Chinese population.

Objective To analyze and identify the mutations in SGLT2 gene of nine Chinese families with FRG, and determine the renal threshold for glucose excretion (RTG), so as to explore the association of genotype and RTG. Methods All coding regions of SGLT2 gene, including intron exon boundaries, were analyzed using PCR followed by direct sequence analysis. Quantitative test for 24?hour urine glucose and RTG were measured among 9 probands (21 patients) and their family members from 9 pedigrees (total 25 subjects). The differences in renal glucose thresholds between patients with different genotypes (heterozygotes and compound heterozygotes; c.886(-10_-31) del heterozygotes and other heterozygotes) were compared. Results Twelve mutations were identified by SGLT2 gene analysis, including 10 novel ones that were not included in HGMD:c.331T>C, p.W111R;c.374T>C, p.M125T; c.394C>T, p.R132C; c.612G>C, p.Q204H; c.829C>T, p.P277S; c.880G>A, p.D294N;c.1129G>A, p.G377S; c.1194C>A, p.F398L; c.1540C>T, p.P514S; c.1573C>T, p.H525Y. In thisstudy, the mutation c.886(-10_-31)del that is specific to Chinese population accounted for about 28％of the total alleles (5/18). The RTG values of patients with compound heterozygous mutations were much lower than those with simple heterozygous mutations [(1.28 ±0.10) vs (5.14±0.77) mmol/L; P<0.001];and c.886(-10_-31)del heterozygotes had significant lower RTG values than others [(4.43 ± 0.37) vs (5.70 ± 0.51) mmol/L, P<0.001]. Conclusions Ten novel mutations which may be related to FRG are found in this study, and c.886(-10-31)del may be a hot?spot mutation in Chinese patients. Compound heterozygotes had much lower RTG values than simple heterozygotes.

Objective To investigate the clinical immune status of patients with severe sepsis shock.Methods A total of 45 patients with sepsis in general ward and 45 severe sepsis shock patients treated in our hospital were selected for the study,and were as control group and observation group,respectively.The T lymphocyte,B lymphocyte and CD3+ CD4+,CD3+ CD8+,NK cells,immunoglobulin IgG,IgA,IgM levels,complement C3,C4 were detected in the two groups.The cellular immune function,humoral immune function and inflammatory reactions were compared between the two groups.Results The T lymphocyte count in the observation group and the control group were (60.0 ±16.2)％ and (77.2 ± 18.5)％,respectively,and The B lymphocyte count were (25.1 ± 10.2)％,and (14.3 ±6.9)％,respectively.The CD3+ CD8+ in the observation group was significantly lower,and CD4+/CD8+ was significantly higher than that in the control group (P ＜ 0.05).The percentage of NK cells and CD3 + CD8 + in the two groups showed no significant difference (P ＞ 0.05).The concentration of C4 and IgM in the observation group were significantly lower than that in the control group (P ＜ 0.05),and there was no significant difference between the two groups in C3,IgG and IgA (P ＞ 0.05).The WBC count,procalcitonin and C reactive protein in the observation group were significantly higher than that in the control group (P ＜ 0.05).Conclusion Severe septic shock patients have severe immune disorder.

Objective To investigate the clinical immune status of patients with severe sepsis shock.Methods A total of 45 patients with sepsis in general ward and 45 severe sepsis shock patients treated in our hospital were selected for the study,and were as control group and observation group,respectively.The T lymphocyte,B lymphocyte and CD3+ CD4+,CD3+ CD8+,NK cells,immunoglobulin IgG,IgA,IgM levels,complement C3,C4 were detected in the two groups.The cellular immune function,humoral immune function and inflammatory reactions were compared between the two groups.Results The T lymphocyte count in the observation group and the control group were (60.0 ±16.2)％ and (77.2 ± 18.5)％,respectively,and The B lymphocyte count were (25.1 ± 10.2)％,and (14.3 ±6.9)％,respectively.The CD3+ CD8+ in the observation group was significantly lower,and CD4+/CD8+ was significantly higher than that in the control group (P ＜ 0.05).The percentage of NK cells and CD3 + CD8 + in the two groups showed no significant difference (P ＞ 0.05).The concentration of C4 and IgM in the observation group were significantly lower than that in the control group (P ＜ 0.05),and there was no significant difference between the two groups in C3,IgG and IgA (P ＞ 0.05).The WBC count,procalcitonin and C reactive protein in the observation group were significantly higher than that in the control group (P ＜ 0.05).Conclusion Severe septic shock patients have severe immune disorder.

AIM:To investigate the effects of celecoxib on viability , apoptosis and autophagy in acute myeloid leukemia (AML) cell lines HL-60 and HL-60A.METHODS:The HL-60 cells and HL-60A cells were cultured with vari-ous concentrations (0, 20, 40, 60, 80 and 100μmol/L) of celecoxib.The inhibitory effect of celecoxib on the cell viabil-ity was evaluated by MTT assay .Apoptosis was analyzed by Annexin-V/PI staining.Apoptosis-related and autophagy-relat-ed proteins were determined by Western blot .RESULTS:IC50 of celecoxib were 49.4 μmol/L, 32.0 μmol/L and 25.1μmol/L for HL-60 cells treated with celecoxib for 24 h, 48 h and 72 h, respectively.For HL-60A cells, the corresponding IC50 were 69.1 μmol/L, 42.5 μmol/L and 29.6 μmol/L, respectively.The results of flow cytometry analysis showed the proportions of Annexin-Ⅴ+PI-, Annexin-Ⅴ+PI+and Annexin-Ⅴ-PI+cells were increased in the HL-60 cells, and those of Annexin-Ⅴ+PI-and Annexin-Ⅴ+PI+cells were increased in the HL-60A cells treated with celecoxib for 24 h. After treated with celecoxib , the induction of apoptosis was observed , the apoptosis-related proteins cleaved caspase-3 and cleaved PARP were upregulated , the autophagy-related proteins LC3 II and P62 were both increased , and mTOR, p-mTOR, 4-EBP and p-4-EBP were not changed , indicating that celecoxib inhibited autophagy in the AML cells without the mTOR pathway involvement .CONCLUSION:Celecoxib inhibits the viability of HL-60 cells and HL-60A cells in a time-and dose-dependent manner by its effects of inducing apoptosis and necrosis .Celecoxib inhibits mTOR-independent autoph-agy in AML cells, indicating a possible way of using celecoxib for enhancing the antitumor activity of therapeutic agents to induce cytoprotective autophagy in the AML cells .