Objective To investigate the protective effect and mechanism of Danshen Tongluo Jiedu Decoction medicated serum for hypoxia/reoxygenation rat myocardial microvascular endothelial cells(CMECs)by regulating MALAT1.Methods Rats CMECs cells were cultured in vitro to establish a model of hypoxia/reoxygenation damaged cells,and were transfected overexpressing/silencing blank MALAT1 slow virus,cells were divided into overexpressed blank + TCM group,overexpressed MALAT1 + TCM group,overexpressed MALAT1 group,silenced blank + TCM group,silenced MALAT1 group,and silenced MALAT1 + TCM group.They were cultured with corresponding serum separately.Beclin-1 protein expression was detected by immunofluorescence method,and SRPK1,SRSF1,VEGF and Bax protein expressions were detected by Western blot,MALAT1,SRPK1 and SRSF1 mRNA expressions were detected by RT-PCR.Results Compared with the overexpressed blank + TCM group,Beclin-1 protein expression increased in the overexpressed MALAT1 + TCM group,the protein expressions of SRPK1,SRSF1 and Bax significantly increased(P<0.05,P<0.01),VEGF protein expression significantly decreased(P<0.01),while MALAT1,SRPK1 and SRSF1 mRNA expressions significantly increased(P<0.05,P<0.01).Compared with the overexpressed MALAT1 group,the protein expression of Beclin-1 in overexpressed MALAT1 + TCM group decreased,the expressions of SRPK1,SRSF1 and Bax protein significantly decreased(P<0.01,P<0.05),the expression of VEGF protein significantly increased(P<0.01),the mRNA expressions of MALAT1,SRPK1 and SRSF1 significantly decreased(P<0.05).Compared with the silenced blank + TCM group,the protein expression of Beclin-1 in silenced MALAT1 + TCM group decreased,the expressions of SRPK1,SRSF1 and Bax protein significantly decreased(P<0.01),while the expression of VEGF protein significantly increased(P<0.01),the mRNA expressions of MALAT1,SRPK1 and SRSF1 significantly decreased(P<0.01).Compared with the silenced MALAT1 group,the protein expression of Beclin-1 in silenced MALAT1 + TCM group decreased,the expressions of SRPK1,SRSF1 and Bax protein significantly decreased(P<0.05),the expression of VEGF protein significantly increased(P<0.01),the mRNA expressions of MALAT1,SRPK1 and SRSF1 significantly decreased(P<0.01,P<0.05).Conclusion Upregulation of MALAT1 expression can promote autophagy in hypoxia/reoxygenation model CMECs,while Danshen Tongluo Jiedu Decoction medicated serum can inhibit MALAT1 expression,thus inhibiting autophagy and promoting angiogenesis,and the mechanism may be related to the downregulation of SRPK1 and SRSF1 expressions.

Objective To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells(hiPSCs-NECs)into functional midbrain dopaminergic progenitor cells(DAPs).Methods HiPSCs were cultured in mTeSRTM medium containing DMH1(10 μmol/L),SB431542(10 μmol/L),SHH(200 ng/mL),FGF8(100 ng/mL),purmorphamine(2 μmol/L),CHIR99021(3 μmol/L),and N2(1%)for 12 days to induce their differentiation into primitive neuroepithelial cells(NECs).The hiPSCs-NECs were digested with collagenase IV and then cultured in neurobasal medium supplemented with 1%N2,2%B27-A,BDNF(10 ng/mL),GDNF(10 ng/mL),AA,TGF-β,cAMP,and 1%GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632,and the culture medium was changed the next day to remove Y27632.Continuous induction was performed until day 28 to obtain DAPs.Results Human iPSCs expressed the pluripotency markers OCT4,SOX2,Nanog,and SSEA1 and were positive for alkaline phosphatase staining.The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2,nestin,and PAX6.In digested hiPSCs-NECs,the addition of 5 μmol/L Y27632 significantly promoted survival of the adherent cells,increased cell viability and the proportion of S-phase cells(P<0.01),and reduced the rate of apoptotic cells(P<0.05).On day 28 of induction,the obtained cells highly expressed the specific markers of DAPS(TH,FOXA2,NURR1,and Tuj1).Conclusion Treatment with Y27632(5 μmol/L)for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation,which indirectly enhances the efficiency of cell differentiation.

Objective:To study the role of special microbial communities in the development of periodontitis in type 2 diabetes melli-tus(T2DM)patients.Methods:40 subjects aged 20-70 years were included and divided into 3 groups:moderate to severe periodon-titis with T2DM(SP.T2DM,n=15),moderate to severe periodontitis group(SP,n=15)and normal healthy group(N,n=10).The basic information,periodontal clinical indicators and blood sugar of the subjects were recorded.Subgingival plaque samples were col-lected,DNA samples of the plaque were extracted,and sequenced by Illumina NovaSeq6000 platform.The microbial diversity,eco-logical characteristics and functions of the plaque were analyzed by Uparse,SPSS and other softwares.Results:481 species in 22 phyla,30 classes,73 orders,129 families and 265 genera were obtained from the samples.Beta polymorphism analysis showed that the species composition of CP.T2DM group and CP group was similar.Alpha polymorphism analysis showed that the species richness and evenness in CP.T2DM group and CP group were higher than those in N group(P＜0.01).Venn diagram analysis showed that the species richness of the plaque in CP.T2DM group was the highest,followed by CP group and the lowest in N group.At the genus lev-el,Klebsiella and Bifidobacterium in CP.T2DM group were larger than those in CP group and N group(P＜0.05),and between group CP and N,P＞0.05.At the species level,the Capnocytophaga leadbetteri in CP.T2DM group was higher than that in CP group and N group(P＜0.05),between group CP and N,P＞0.05;There were some differences in the microbial community structure of subgingival plaque among the 3 groups.The species richness of subgingival flora in patients with CP and T2DM was higher than that in patients with CP and healthy people.Conclusion:The increase of Klebsiella,Bifidobacterium and Capnocytophaga leadbetter in subgingival flora of patients with moderate and severe periodontitis may be related to the development of T2DM.

Objective To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells(hiPSCs-NECs)into functional midbrain dopaminergic progenitor cells(DAPs).Methods HiPSCs were cultured in mTeSRTM medium containing DMH1(10 μmol/L),SB431542(10 μmol/L),SHH(200 ng/mL),FGF8(100 ng/mL),purmorphamine(2 μmol/L),CHIR99021(3 μmol/L),and N2(1%)for 12 days to induce their differentiation into primitive neuroepithelial cells(NECs).The hiPSCs-NECs were digested with collagenase IV and then cultured in neurobasal medium supplemented with 1%N2,2%B27-A,BDNF(10 ng/mL),GDNF(10 ng/mL),AA,TGF-β,cAMP,and 1%GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632,and the culture medium was changed the next day to remove Y27632.Continuous induction was performed until day 28 to obtain DAPs.Results Human iPSCs expressed the pluripotency markers OCT4,SOX2,Nanog,and SSEA1 and were positive for alkaline phosphatase staining.The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2,nestin,and PAX6.In digested hiPSCs-NECs,the addition of 5 μmol/L Y27632 significantly promoted survival of the adherent cells,increased cell viability and the proportion of S-phase cells(P<0.01),and reduced the rate of apoptotic cells(P<0.05).On day 28 of induction,the obtained cells highly expressed the specific markers of DAPS(TH,FOXA2,NURR1,and Tuj1).Conclusion Treatment with Y27632(5 μmol/L)for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation,which indirectly enhances the efficiency of cell differentiation.

OBJECTIVES: To investigate the role of mitochondrial autophagy disorder caused by deletion of E3 ubiquitin ligase Parkin in neuroinflammation in a mouse model of MPTP-induced Parkinson's disease (PD). METHODS: Wild-type (WT) male C57BL/6 mice and Parkin^-/- mice were given intraperitoneal injections with MPTP or PBS for 5 consecutive days, and the changes in motor behaviors of the mice were observed using open field test. The effects of Parkin deletion on PD development and neuroinflammation were evaluated using immunofluorescence and Western blotting. The changes of the PINK 1/Parkin signaling pathway in the midbrain substantia nigra of the mice were examined to explore the molecular mechanism of Parkin-mediated regulation of mitochondrial autophagy and its effect on neuroinflammation in PD mice. RESULTS: Compared with their WT counterparts, the Parkin^-/- mice with MPTP injections exhibited significant impairment of motor function with decreased TH⁺ neurons, increased α-synuclein (α-syn) accumulation, and increased numbers of GFAP⁺ and I-ba1⁺ cells in the midbrain substantia nigra. Parkin deletion obviously affected PINK1/Parkin-mediated mitochondrial autophagy to result in significantly increased mtDNA and upregulated expressions of STING and NLRP3 inflammatosomes in the midbrain substantia nigra of MPTP-treated transgenic mice. CONCLUSIONS Parkin deletion causes mitochondrial autophagy disorder to accelerate PD progression and exacerbates neuroinflammation in mice by affecting the PINK1/Parkin signaling pathway, suggesting the important role of Parkin in early pathogenesis of PD.
Animals ; Ubiquitin-Protein Ligases/genetics* ; Mice ; Mice, Inbred C57BL ; Male ; Parkinson Disease/genetics* ; Protein Kinases/genetics* ; Mitochondria/metabolism* ; Disease Models, Animal ; Autophagy ; Signal Transduction ; Neuroinflammatory Diseases/metabolism* ; Mice, Knockout ; alpha-Synuclein/metabolism* ; Substantia Nigra/metabolism* ; Mitophagy ; Disease Progression

ObjectiveTo observe the effect of Hedysari Radix polysaccharide （HRP） on the Janus kinase 2 （JAK2）/signal transducer and activator of transcription protein 3 （STAT3） signaling pathway in diabetic nephropathy db/db mice. MethodFifty db/db mice were randomly divided into model group， irbesartan group （irbesartan suspension， 22.75 mg·kg^-1）， and high-， medium-， and low-dose HRP groups （HRP suspension， 200， 100， 50 mg·kg^-1） according to the body weight， with 10 mice in each group. Another 10 C57BL/6 mice were assigned to the normal group. The mice were treated with corresponding drugs by gavage， while those in the normal group and the model group received distilled water at 5 mL·kg^-1. The mice in the six groups were administered once a day by gavage for 12 consecutive weeks. The uric acid （UA）， triglycerides （TG）， and total cholesterol （TC） were detected. Periodic acid-Schiff （PAS） staining and Masson staining were used to observe the pathological changes in kidney tissues. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to detect the protein and mRNA expression levels of JAK2， STAT3， suppressor of cytokine signaling 3 （SOCS3）， and tumor necrosis factor-α （TNF-α） in the kidney. ResultAfter 12 weeks of treatment， compared with the normal group， the model group showed significant pathological ultrastructural changes in kidney tissues and increased UA， TG， and TC levels （P<0.01）. Compared with the model group， the high- and medium-dose HRP groups and the irbesartan group showed improvement in pathological ultrastructure of kidney tissues and reduced UA， TG， and TC levels （P<0.05， P<0.01）. Compared with the normal group， the model group showed a decrease in SOCS3 protein and mRNA expression levels and an increase in JAK2， STAT3， and TNF-α protein and mRNA expression levels （P<0.01）. Compared with the model group， the high- and medium-dose HRP groups and the irbesartan group showed an increase in SOCS3 protein and mRNA expression levels and a decrease in JAK2， STAT3， and TNF-α protein and mRNA expression levels （P<0.05， P<0.01）. ConclusionHRP can alleviate renal damage in diabetic nephropathy to a certain extent， and its mechanism may be related to the inhibition of the activation of the JAK2/STAT3 signaling pathway.

Objective To explore the effect of exercise preconditioning on inflammatory response in ischemic stroke brain tissue which mediated by miR-146a in extracellular vesicles in rats with middle cerebral artery oc-clusion(MCAO),and its mechanism.Methods Sixty 6-week-old male Sprague-Dawley rats were randomly divid-ed into a non-exercise group and an exercise group.The non-exercise group was further divided into a sham-operation control group(C,n=15)and an MCAO model group(M,n=15),while the exercise group was further di-vided into an exercise only group(E,n=15)and an exercise plus MCAO model group(EM,n=15).Rats in the E and EM groups underwent 8 weeks of treadmill exercise,6 days per week,30 minutes per day.Then rats in the M and EM groups received MCAO to induce ischemic stroke,while the C and E groups underwent a sham surgery.Twenty-four hours after the surgery,neurobehavioral tests were performed.Plasma was collected to ex-tract extracellular vesicles,and brain tissue was collected to measure the volume of cerebral infarction by using the triphenyl tetrazolium chloride(TTC)staining.Moreover,the Nissl staining was conducted to observe neuronal and Nissl body.Mean while,the content of miR-146a in plasma extracellular vesicles and brain tissue was mea-sured by using the quantitative polymerase chain reaction(qPCR),and the expression of TNF receptor associat-ed factor 6(TRAF6),nuclear factor kappa-B(NF-κB)and tumor necrosis factor-α(TNF-α)in brain tissues were determined using Western blotting.The targeting relationship between miR-146a and TRAF6 was detected by using the dual luciferase reporter gene assay.Results(1)The neurological behavioral scores of the EM and M groups were higher than those of the C group(P<0.01 and P<0.01),with that of the EM group lower than the M group(P<0.01).(2)TTC staining showed that the infarct volume of the EM and M groups was larger than that of the other two groups(P<0.01 and P<0.01),with that of the EM group smaller than the M group(P<0.01).(3)Nissl staining results showed that the neuronal arrangement was loose,the number of neurons re-duced,and the Nissl bodies were lightly stained and decreased in the M group compared with the C and E groups.Moreover,compared with the M group,the number of neurons and Nissl bodies increased in the EM group.(4)The qPCR analysis showed that the expression of miR-146a in the plasma-derived exosomes and brain tissues of the EM and M groups decreased compared with the C group(P<0.05 and P<0.01),with that of the EM group higher than the M group(P<0.05).(5)According to Western blotting,compared with the C group,the expression levels of TRAF6,NF-κB,and TNF-α proteins increased significantly(P<0.05 and P<0.01),with that of group EM signfiicantly lower than group M(P<0.05 and P<0.05).(6)Dual-luciferase report-er gene assay showed that miR-146a had a specific binding site with TRAF6.Conclusion Eight weeks of exer-cise preconditioning reduces the infarct area and the extent of brain damage,which may be mediated by miR-146a via exosomes,increasing the expression of miR-146a in brain tissue,targeting TRAF6,negatively regulat-ing TRAF6/NF-κB,and reducing the expression of TNF-α,thus alleviating the inflammatory response in brain tissue and exerting a protective effect on ischemic brain injury.

Diabetic nephropathy （DN）， as one of the common chronic microvascular complications of diabetes， has become the main cause of renal failure in end-stage renal disease in China， increasing the risks of renal dialysis and kidney transplantation in diabetes patients. It is the leading cause of death in people with diabetes. The latest research on DN has focused on the gene level. microRNAs （miRNAs） are a family of endogenous accessible short-chain non-coding RNA molecules. By acting on a particular target， they activate or inhibit its mediated signaling pathways and related molecules， playing an important role in the occurrence and development of DN. They have become microeconomic factors for the prevention and treatment of DN. Traditional Chinese medicine （TCM） has a long history in the diagnosis and treatment of DN and has unique advantages such as significant curative effect and few side effects. A large number of studies have proved that TCM can target miRNA to affect multiple signaling pathways， participate in the regulation of inflammatory response， pyroptosis， mesenchymal transdifferentiation， and other pathological changes， and delay the further development of DN. Therefore， this study discusses the biogenesis mechanism of miRNA and its action mechanism in disease-related signaling pathways based on TCM diagnosis and treatment approaches from the perspective of miRNA， and summarizes the effect of TCM targeting miRNA on the disease-related signaling pathways and on DN. Thus， this study is expected to provide a theoretical reference for exploring the progress of TCM intervention in DN from the perspective of genes.

ObjectiveTo observe the effect of Hedysarum polysaccharides （HPS） on the Wnt/β-catenin signal pathway in db/db mice with diabetic nephropathy. MethodFifty db/db mice were randomly divided into model group， irbesartan group， and high， middle， and low-dose HPS experimental groups according to their body mass， with 10 mice in each group， and another 10 C57BL/6 mice were selected as a normal group. The normal group and the model group were given 5 mL·kg^-1·d^-1 distilled water， the irbesartan group was given 22.75 mg·kg^-1·d^-1 irbesartan suspension， and the high， middle， and low-dose HPS experimental groups were given 200， 100， and 50 mg·kg^-1·d^-1 HPS suspensions， respectively. The mice in the 6 groups were given intragastric administration once a day for 12 weeks. The general state， blood glucose （GLU）， 24 h urine protein （UTP）， blood creatinine （SCr）， and urea nitrogen （BUN） of mice in each group were determined. The pathological changes in the kidney tissue were observed by hematoxylin-eosin staining （HE）. The protein and mRNA expression levels of Wnt1， β-catenin， glycogen synthesis kinase-3β （GSK-3β）， and phosphorylated GSK-3β （p-GSK-3β） in the kidney were detected by Western blot and real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultAfter treatment for 12 weeks， as compared with the normal group， the general state of mice in the model group was worse and the pathological ultrastructural lesions of kidney tissues were obvious. The levels of GLU， 24 h UTP， SCr， and BUN in the model group increased （P<0.01）. As compared with the model group， the general state and renal pathological ultrastructure of mice in the high and middle-dose HPS groups were improved to some extent， and the levels of SCr， BUN， and 24 h UTP in the high and middle-dose HPS groups decreased （P<0.05，P<0.01）. As compared with the normal group， the expression levels of Wnt1， β-catenin， GSK-3β， and p-GSK-3β protein and mRNA in the model group were higher （P<0.01）， while the expression levels of Wnt1， β-catenin， GSK-3β， and p-GSK-3β protein and mRNA in the high and middle-dose HPS groups were lower than those in the model group （P<0.05，P<0.01）. ConclusionHPS can alleviate the renal injury of diabetic nephropathy to some extent， and its mechanism may be related to the inhibition of the activation of the Wnt/β-catenin signal pathway.

Objective:To evaluate the airway and voice quality improvement in patients with bilateral vocal fold paralysis (BVFP) who underwent selective laryngeal reinnervation surgery.Methods:From January 2012 to December 2016, a retrospective study was conducted in 39 patients with BVFP who underwent selective laryngeal reinnervation surgery in Department of Otorhinolaryngology Head and Neck Surgery, the First Affiliated Hospital of Navy Medical University. All patients were examined by videostroboscopy, vocal function assessment, laryngeal electromyography and pulmonary function test before and after the surgery, and followed up for at least 2 years to evaluate the efficacy and safety of the surgery.Wilcoxon signed rank test was used to analyze the G score and VHI-10 score data. Paired t-test was used to analyze acoustic parameters, MPT values and pulmonary function parameters. Results:Postoperative infection and hemorrhage occurred in one patient separately.Videostroboscopic videos showed that at 4-8 months postoperatively, vocal folds in 35 patients achieved moderate or severe abduction during inspiration, 2 patients only achieved mild abduction, 2 patients showed no abduction,while all patients achieved adduction in bilateral vocal cords during phonation. The recovery rate of moderate-to-severe abduction was 89.7% (35/39), and these patients were decannulated successfully. At 12 months after operation, G score and VHI-10 score were significantly lower than those before operation ( P<0.05), and the acoustic parameters jitter, shimmer, HNR and MPT were significantly improved ( P<0.05). Most of the parameters of the pulmonary function test at 3 months postoperatively returned to the normal reference level, while the maximum inspiratory pressure (PImax) at 12 months after operation was still slightly lower than the normal level, but it was significantly improved compared with preoperative value ( P<0.05). The EMG data at 12 months postoperatively showed full interference potentials in 37 patients in bilateral posterior cricoarytenoid muscles during inspiration, and full interference potentials in bilateralthyroarytenoid muscles during phonation. Obvious misdirected regeneration electric activitieswere found in two of them. Potentials in posterior cricoarytenoid muscle were weak in 2 cases with poor abduction. During long-term follow-up, only one case showed decreased abduction, but did not affect respiratory function. Conclusions:The selective laryngeal reinnervation procedure applied in the present study can restore physiological motion of vocal cords. The success rate was high, the curative effect was stable, and the complications were rare. It is worth of promotion.