AIM:To investigate the mechanism of Piezo1 channel activation promoting the increase in intra-cellular Ca2+ concentration([Ca2+]i)of rat coronary artery smooth muscle cells(CASMCs).METHODS:The primary CASMCs of SD rats were cultured,and the expression and subcellular localization of Piezo1 in the cells were observed by immunofluorescence staining.The Piezo1 and stromal interaction molecule 1(STIM1)in CASMCs were knocked down by siRNA transfection,and the expression levels of the proteins were detected by Western blot.Utilizing laser confocal mi-croscopy,the change of[Ca2+]i in CASMCs was detected by Fluo-4 AM fluorescent probes.RESULTS:It was confirmed by immunofluorescence staining that the expression of Piezo1 existed in primary rat CASMCs.Immunofluorescence staining also showed that Piezo1 was co-located with sarco-/endoplasmic reticulum Ca2+-ATPase 2(SERCA2),mitochondrial outer membrane protein TOM20 and nuclear membrane protein lamin B1.Western blot results showed that the protein expres-sion levels of STIM1 and Piezo1 were significantly down-regulated by siRNA transfection(P<0.05).Compared with con-trol group,Yoda1,the agonist of Piezo1,could increase the extracellular Ca2+ influx of CASMCs(P<0.01).However,the Ca2+ influx mediated by Yoda1 was not affected by the inhibition of L-type calcium channels.Treatment with Yoda1 in-creased the intracellular Ca2+ release of CASMCs(P<0.01).However,inhibition of calcium channels on endoplasmic re-ticulum,ryanodine receptor and inositol 1,4,5-triphosphate receptor,did not affect intracellular Ca2+ release mediated by Yoda1.After the Ca2+ in endoplasmic reticulum was emptied using thapsigargin(TG),Yoda1 also mediated the Ca2+ re-lease of other organelles in CASMCs(P<0.01).After inhibition of L-type calcium channels,treatment with store-operated calcium channel(SOCC)inhibitor BTP2 or knockdown of STIM1 led to the decrease in extracellular Ca2+ influx of CASMCs mediated by Yoda1(P<0.01).Treatment with TG increased the release of Ca2+ from the endoplasmic reticulum of CASMCs after knockdown of Piezo1(P<0.05),but the extracellular Ca2+ influx mediated by TG was not affected.After inhibition of L-type calcium channels and SOCC,knockdown of Piezo1 led to the decreases in intracellular Ca2+ release and extracellular Ca2+ influx induced by Yoda1(P<0.01).CONCLUSION:The Piezo1 agonist orchestrates the influx of extracellular Ca2+ by activating Piezo1 channels on the cell membrane and inducing the indirect activation of SOCC.More-over,it facilitates the release of Ca2+ from organelles.Consequently,these pathways synergistically elevate the[Ca2+]i of rat CASMCs.

AIM:To investigate the influence of fat mass and obesity-associated(Fto)gene on the aberrant contraction of aortic smooth muscle in diabetes mellitus(DM)mice,and to explore the mechanism of Fto gene underlying the calcium regulation.METHODS:Smooth muscle-specific Fto gene knockout(FtoSMKO)mice were generated using Cre-loxP technology.The experiment involved 3 groups of mice:wild-type(WT)group,DM model group and FtoSMKO-DM group,with 15 mice in each group.In DM group and FtoSMKO-DM group,type 1 DM was induced by intraperitoneal injec-tion of streptozotocin.The mice in WT group were injected with equal volume of citric acid-sodium citrate buffer solution.The influences of different drugs on the contraction responses of aortic smooth muscle in mice were analyzed using a multi-myograph system.The expression level of FTO protein in the aortic tissues was detected by Western blot.RESULTS:(1)Compared with WT mice,the expression levels of FTO protein in the aortic tissues of DM mice were significantly in-creased(P＜0.01).(2)The expression level of FTO protein in smooth muscle was significantly decreased after knockout of Fto gene(P＜0.01).Compared with WT group,the mice in DM group exhibited a significant decrease in body weight and a marked increase in fasting blood glucose level(P＜0.05).There were no noticeable differences in body weight or fasting blood glucose level between FtoSMKO-DM group and DM group(P＞0.05).(3)The contraction responses of aortic smooth muscle in DM group were substantially increased by phenylephrine compared with WT group.Specifically,vaso-constriction responses mediated by non-L-type calcium channels and store-operated calcium channels(SOCC)were signifi-cantly enhanced in DM group.In addition,the responses mediated by inositol 1,4,5-trisphosphate receptors(IP3R),which facilitate calcium release from the sarcoplasmic reticulum,were significantly enhanced.However,the responses mediated by caffeine-activated ryanodine receptors(RyR),which also facilitate calcium release from the sarcoplasmic re-ticulum,were significantly inhibited(P＜0.05).(4)Compared with DM group,the phenylephrine-induced contraction re-sponses of aortic smooth muscle in FtoSMKO-DM group were greatly weakened(P＜0.05).In particular,the vasoconstriction responses mediated by non-L-type calcium channels and SOCC in FtoSMKO-DM group were greatly suppressed(P＜0.05),while those mediated by caffeine-activated RyR were dramatically boosted(P＜0.05).However,IP3R-mediated responses were not affected(P＞0.05).CONCLUSION:Smooth muscle-specific Fto gene knockout suppresses contractile hyperre-sponsiveness in the aortic smooth muscle of DM mice,which may be attributed to involvement of FTO protein in calcium regulation in the vascular smooth muscle.

Objective To evaluate the therapeutic effect of Huangqin Decoction(HQD)on ulcerative colitis(UC)in mice and explore its mechanism.Methods Male Balb/c mice were randomly divided into normal control group,model group,mesalazine group(5-ASA,200 mg/kg),and low-,medium-and high-dose HQD groups(2.275,4.55 and 9.1 g/kg,respectively).With the exception of those in the normal control group,all the mice were exposed to 3%DSS solution in drinking water for 7 days to establish UC models.After treatment with the indicated drugs,the mice were assessed for colon injury and apoptosis using HE,AB-PAS and TUNEL staining,and the expression levels of inflammatory factors were detected with ELISA.Western blotting,immunohistochemistry and qRT-PCR were used to detect the changes in protein expressions associated with the intestinal chemical barrier,mechanical barrier and endoplasmic reticulum stress(ERS).Results HQD treatment significantly reduced DAI score and macro score of UC mice,decreased colonic epithelial cell apoptosis,lowered expressions of IL-6,TNF-α,IL-1β and IL-8,and enhanced the expressions of MUC2 and TFF3.HQD treatment also upregulated the protein expressions of claudin-1,occludin and E-cadherin,reduced the expressions of GRP78,CHOP,caspase-12 and caspase-3,decreased the phosphorylation levels of PERK,eIF2α and IRE1α,and increased the Bcl-2/Bax ratio in the colon tissues of UC mice.Conclusion HQD inhibits colonic epithelial cell apoptosis and improves intestinal barrier function in UC mice possibly by reducing ERS mediated by the PERK and IRE1α signaling pathways.

Objective To evaluate the therapeutic effect of Huangqin Decoction(HQD)on ulcerative colitis(UC)in mice and explore its mechanism.Methods Male Balb/c mice were randomly divided into normal control group,model group,mesalazine group(5-ASA,200 mg/kg),and low-,medium-and high-dose HQD groups(2.275,4.55 and 9.1 g/kg,respectively).With the exception of those in the normal control group,all the mice were exposed to 3%DSS solution in drinking water for 7 days to establish UC models.After treatment with the indicated drugs,the mice were assessed for colon injury and apoptosis using HE,AB-PAS and TUNEL staining,and the expression levels of inflammatory factors were detected with ELISA.Western blotting,immunohistochemistry and qRT-PCR were used to detect the changes in protein expressions associated with the intestinal chemical barrier,mechanical barrier and endoplasmic reticulum stress(ERS).Results HQD treatment significantly reduced DAI score and macro score of UC mice,decreased colonic epithelial cell apoptosis,lowered expressions of IL-6,TNF-α,IL-1β and IL-8,and enhanced the expressions of MUC2 and TFF3.HQD treatment also upregulated the protein expressions of claudin-1,occludin and E-cadherin,reduced the expressions of GRP78,CHOP,caspase-12 and caspase-3,decreased the phosphorylation levels of PERK,eIF2α and IRE1α,and increased the Bcl-2/Bax ratio in the colon tissues of UC mice.Conclusion HQD inhibits colonic epithelial cell apoptosis and improves intestinal barrier function in UC mice possibly by reducing ERS mediated by the PERK and IRE1α signaling pathways.

OBJECTIVE: To detect variants of IVD gene among 4 neonates with suspected isovalerate acidemia in order to provide a guidance for clinical treatment. METHODS: 111 986 newborns and 7461 hospitalized children with suspected metabolic disorders were screened for acyl carnitine by tandem mass spectrometry. Those showing a significant increase in serum isovaleryl carnitine (C5) were analyzed for urinary organic acid and variants of the IVD gene. RESULTS: Four cases of isovalerate acidemia were detected, which included 2 asymptomatic newborns (0.018‰, 2/111 986) and 2 children suspected for metabolic genetic diseases (0.268‰, 2/7461). The formers had no obvious clinical symptoms. Analysis of acyl carnitine has suggested a significant increase in C5, and urinary organic acid analysis has shown an increase in isovaleryl glycine and 3-hydroxyisovalerate. Laboratory tests of the two hospitalized children revealed high blood ammonia, hyperglycemia, decreased red blood cells, white blood cells, platelets and metabolic acidosis. The main clinical manifestations have included sweaty foot-like odor, feeding difficulty, confusion, drowsiness, and coma. Eight variants (5 types) were detected, which included c.158G>A (p.Arg53His), c.214G>A (p.Asp72Asn), c.548C>T (p.Ala183Val), c.757A>G (p.Thr253Ala) and 1208A>G (p.Tyr403Cys). Among these, c.548C>T and c.757A>G were unreported previously. None of the variants was detected by next generation sequencing of 2095 healthy newborns, and all variants were predicted to be likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics. CONCLUSION The incidence of isovalerate acidemia in Liuzhou area is quite high. Screening of metabolic genetic diseases is therefore recommended for newborns with abnormal metabolism. The discovery of novel variants has enriched the mutational spectrum of the IVD gene.
Infant, Newborn ; Child ; Humans ; Acidosis ; Carnitine ; Erythrocytes ; High-Throughput Nucleotide Sequencing

There are two source plants for the traditional Chinese medicine Murrayae Folium et Cacumen (MFC) in Chinese Pharmacopoeia, i.e. Murraya exotica L. and M. paniculata (L.) Jack. Herein, a chemical comparison of M. exotica and M. paniculata by high performance liquid chromatography (HPLC) fingerprint analysis coupled with chemometrics and network pharmacology was performed. The main peaks in the fingerprints were identified by liquid chromatography coupled with ion trap/time-of-flight mass spectrometry (LC-IT-TOF-MS) and authenticated by references. The chemometrics results showed that the HPLC fingerprints of these two species were clearly divided into two categories using hierarchical cluster analysis (HCA) and principal component analysis (PCA), and a total of 13 significantly differentiated markers were screened out by orthogonal partial least squares-discriminant analysis (OPLS-DA). However, the following network pharmacology analysis showed that these discriminated markers were found to act via many common targets and metabolic pathways, indicating the possibly similar pharmacological effects and mechanisms for M. exotica and M. paniculata. The above results provide valuable evidence for the equivalent use of these two plants in clinical settings. Moreover, the chromatographic fingerprint analysis coupled with chemometrics and network pharmacology supplies an efficient approach for the comparative analysis of multi-source TCMs like MFC.

Objective:To explore the value of 3D/4D translabial ultrasound in the diagnosis and differential diagnosis of female urethral diverticulum.Methods:The paired chi-square test was used to compare the diagnostic accuracy of 2D translabial ultrasound and 3D/4D translabial ultrasound in 21 suspected urethral diverticulum patients who visited the Third Affiliated Hospital of Sun Yat-Sen University from November 2013 to January 2020. And Kappa consistency test was used to evaluate the consistency between these two methods and the pathological results.Results:All 21 patients with suspected urethral diverticulum were diagnosed by postoperative pathology, of which 16 cases were urethral diverticula and 5 cases were paraurethral cysts. The typical appearance of urethral diverticulum on 3D/4D translabial ultrasound was a cystic structure traversing the urethral rhabdosphincter. Additionally, septa, calcification or solid neoplasm can also be seen in some cases. The sensitivity/specificity of 2D translabial ultrasound and 3D/4D translabial ultrasound were 75.00%/100% and 93.75%/100%, respectively, no statistical differences were found both in sensitivity and specificity between these two methods(all P>0.05). The Kappa consistency test showed that the consistency between 2D translabial ultrasound and pathological diagnosis was general (Kappa=0.588, P=0.003), while the consistency between 3D/4D translabial ultrasound and pathological diagnosis was good (Kappa=0.877, P<0.001). Conclusions:3D/4D translabial ultrasound has a high value in the diagnosis of female urethral diverticulum, and it is worthy of clinical application.

Objective To detect the long term effect of pure and multiple concussions on spatial cognitive of rats.Methods One hundred and eighty 7-week-old Spragne-Dawley male rats with weight of 280 ± 30g were chosen and randomly divided into a control group and a concussion group.The cerebral concussion was induced in the rats using a metallic pendulum striker concussive device.After the first strike,the brain injury group was randomly divided into a pure cerebral concussion(PCC)group and a multiple cerebral concussion(MCC) group.After the second strike,the MCC group was randomly divided into two-fold cerebral concussion(2MCC) group and three-fold cerebral concussion(3MCC) group.The striking interval was 24h.One,3 and 6 months after trauma,their cognitive function was tested using Morris water maze.Results One month later after injury,there was no significant difference in the escape latency between the control group and PCC group.Significant differences in the measurement were observed between the control/PCC group and 2MCC group on the 7th day after the injury,also between the control/PCC and 3MCC groups on the 6th and 7th day.And there were significant differences between the 2MCC and 3MCC groups on the 6th and 7th days.The non-platform test did not observe any significant differences among the four groups.Three months after injury,there was still no significant difference between the control group and PCC group,PCC and 2MCC groups,as well as 2MCC and 3MCC groups in the escape latency.However,there was significant difference between the control group and 2MCC group on the 5th,6th and 7th days,between the control group and 3MCC group on the 4th,5th,6th and 7th days,as well as between PCC group and 3MCC group on the 6th and 7th days.In the non-platform test,there was no significant difference between the control group and PCC group,between PCC group and 2MCC group,as well as between 2MCC group and 3MCC group.However,2MCC and 3MCC groups spent significantly less time in the former platform quadrant,when compared with the control group and 3MCC group spent significantly less time than PCC group.Six months after injury,significant differences in the escape latency were observed between the control group and PCC group on the 6th and 7th days,and 2MCC group on the 5th,6th and 7th days,also and 3MCC groups on the 2nd,3rd,4th,5th,6th and 7th days,still between PCC group and 2MCC group on the 6th and 7th days,as well as between PCC group and 3MCC group on the 4th,5th,6th and 7th days.Moreover,there was significant difference between 2MCC and 3MCC groups only on the 7th day.In the non-platform test,PCC group,2MCC group and 3MCC group spent significantly less time in the former platform quadrant compared with the control group.Moreover,in this test significant differences were found between PCC group and 2MCC/3MCC group,but not between 2MCC group and 3MCC group.Conclusion With the increase of cerebral concussion times,earlier and more serious damage of spatial cognition will appear,with a significant cumulative effect in rats.Such rat model can be used to study the pathological changes of cognitive impairment in chronic traumatic encephalopathy.

Objective To investigate the association between the RoSCo scoring system and the postopera tive complications of laparoscopic partial nephrectomy.Methods The clinical data of 127 patients with laparoscopic partial nephrectomy in our hospital from 2010 to 2015 were retrospectively analyzed.By studying all patients' preoperative clinical data,we give all the patients the RoSCo score,then divided all the patients into the RoSCo low group (3-4),RoSCo moderate group (5-6),RoSCo high group (7-8).Respectively study the association between the RoSCo score and Clavein score,operative time,intraoperative blood loss,warm ischemia time and length of hospital stay after laparoscopic partial nephrectomy.Results The RoSCo score was associated with postoperative complications of laparoscopic partial nephrectomy (P ＜ 0.05) and was more accurate than the RENAL score alone.There were significant differences in bleeding,warm ischemia time,hospital stay and operation time between the low,middle and high groups of RoSCo (P ＜ 0.05).Conclusion The RoSCo scoring system can be used to assess the complications of laparoscopic partial nephrectomy.

Objective To investigate the therapeutic effect of high intensity focused ultrasound (HIFU) ablation combined with homemade liquid fluorocarbon nanoparticles on cervical cancer in nude mice.Methods The cell experiment was divided into three groups:a,control group;b,HIFU group;c, HIFU + PFB nanoparticle group,and the viability of cells was detected using CCK-8 reagent.The mice were also divided into three groups:A,0.9% NaCI group;B,HIFU + 0.9% NaCI group;C,HIFU + PFB nanoparticle group. The tumors were removed and underwent triphenyl tetrazolium chloride(TTC) staining,and the necrosis area was measured.Histopathological changes of the tumors were examined by light microscopy.Results After HIFU irradiation,the viability rate of group c was (40.5 ±9.7)%,it was lower than that of group b (77.7 ±8.5)% (P <0.05) and that of group a(100 ±4.8)% (P <0.05). TTC staining of tumor showed a large scale of necrotic tissue in group C.The necrosis ratio of the three groups was 0%,(34.14±12.2)% and (65.97 ±25.1)%,respectively (P <0.05).HE staining showed karyorrhexis or an absence of nuclei in group B and group C,which demonstrated the coagulation necrosis. Conclusions HIFU ablation combined with liquid fluorocarbon nanoparticles can effectively treat the xenograft model of the human cervical carcinoma in nude mice.