Objective To investigate the effect of pathologically elevated-cyclic stretch induced by hypertension on mitochondrial biogenesis of vascular smooth muscle cells (VSMCs), and the role of PGC1α in this process. Methods The Flexcell-5000T stretch loading system in vitro was applied to VSMCs with a frequency of 1. 25 Hz and an amplitude of 5% or 15% to simulate the mechanical environment under normal physiological or hypertensive pathological conditions respectively. Western blotting and qPCR were used to detect the expression of PGC1α, citrate synthase and mitochondrial DNA (mtDNA) copy number in VSMCs under normal physiological or hypertensive pathological conditions. VSMCs were treated with PGC1α specific activator ZLN005 to promote PGC1α expression or specific interfering fragment siRNA to inhibit PGC1α expression in order to detect the effect on citrate synthase and mtDNA copy number. Results Compared with 5% physiological cyclic stretch, 15% pathologically elevated-cyclic stretch significantly suppressed the expression of PGC1α, citrate synthase and mtDNA copy number in VSMCs. Compared with control group, the protein expression of PGC1α was significantly decreased and increased respectively. When VSMCs transfected with PGC1α siRNA or incubated PGC1α activator ZLN005, the expression of citrate synthase and mtDNA copy number were also significantly down regulated and up-regulated in VSMCs accordingly. Under physiological cyclic stretch conditions, the protein level of PGC1α was significantly down-regulated by PGC1α siRNA, which also significantly down-regulated citrate synthase expression and mtDNA copy number. The protein expression of PGC1α was significantly up-regulated by ZLN005, which also enhanced the expression of citrate synthase and mtDNA copy number. Conclusions The pathological cyclic stretch induced by hypertension significantly down-regulated the expression of citrate synthase and mtDNA copy number via suppressing the expression of PGC1α, resulting in mitochondrial dysfunction of VSMCs. PGC1α may be a potential therapeutic target molecule to alleviate the progression of hypertension.

Objective To investigate the effects of cyclic stretch on migration of MC3T3-E1 cells and its related mechanism. Methods The strain loading system was used to stretch MC3T3-E1 cells cultured in vitro with 15% amplitude, to simulate the mechanical condition in vivo. The wound healing assay was used to detect the migration of MC3T3-E1 cells. Western blotting was used to test Runx2 expression. RNA interfering was used to decrease Runx2 expression. Results Cyclic mechanical stretch with 15% amplitude, 1.25 Hz frequency and lasting for 24 hours could promote the migration of MC3T3-E1 cells and increase the expression level of Runx2. Runx2 interference inhibited the migration of MC3T3-E1 cells in static culture condition. Interference with Runx2 expression in MC3T3-E1 cells could partially reduce the positive effect of cyclic mechanical stretch on cell migration. Conclusions Cyclic stretch can promote the migration of MC3T3-E1 cells, and Runx2 may play an important role in this process. This study provides experimental basis for finding innovative clinical treatment method to promote fracture healing.

To report a typical case of Morvan syndrome with positive anti-leucine rich glioma-inactivated 1(LGI1) and contactin-associated protein 2 (CASPR2) antibodies in serum and cerebrospinal fluid. A 39-years-old female initially presented weakness of extremeties. The main symptoms included paroxysmal limb pain, wheezing, itching, muscle twitching, epilepsy, hypomnesia, dysphoria, apathy, intractable insomnia, salivation and sweating. Tests of electrolytes found hypokalemia (2.7-3.1 mmol/L) and hyponatremia (130-136 mmol/L). Arterial blood gas analysis showed hypoxemia (oxygen saturation 50%-70%). Total thyroxine (TT4) was elevated to 207 nmol/L with positive thyroid peroxidase antibody (TPO-Ab) and thyroglobulin antibody (TG-Ab). LGI1and CASPR2 antibodies (CBA method) were positive in both serum and cerebrospinal fluid, and the remaining antibodies related to autoimmune encephalitis and paraneoplastic syndrome were negative. Head MRI was almost normal, while mild abnormalities were found in electroencephalogram. Electromyography showed slightly increased voltage of left quadriceps motor unit potential. After treated with corticosteroids, IVIG and mycophenolate mofetil, the patient completely improved. Cognitive function scores recovered from MoCA/MMSE (16/24) to MoCA/MMSE (26/29). Positivity of LGI1/CASPR2 antibodies both in serum/cerebrospinal fluid are rarely seen in patients with Morvan syndrome. Steroids and immunosuppressants are suggested for treatment as early as possible.

Objective To explore the role of adenosine monophosphate-activated protein kinase (AMPK), a key regulator of cellular energy metabolism, in vascular smooth muscle cell (VSMC) migration in response to physiological cyclic stretch. Methods The Flexcell-5000T mechanical loading system was applied with a physiological cyclic stretch at 10% amplitude and 1.25 Hz frequency to primary rat VSMCs, to simulate mechanical stimulation of VSMCs in vivo. The protein expression of p-AMPK in VSMCs was detected by Western blotting, and VSMC migration was detected by wound healing test. Results Compared with the static group, physiological cyclic stretch loading for 24 h significantly decreased the area of wound healing, indicating that physiological cyclic stretch inhibited VSMC migration. The protein expression of p-AMPK in VSMCs was increased significantly after physiological cyclic stretch loading for 3 h, and was decreased significantly after 24 h. Under physiological cyclic stretch loading conditions, incubating AMPK inhibitor could significantly reduce the protein expression of p-AMPK after 3 h, and promote VSMC migration after 24 h; incubating AMPK activator AICAR under static conditions significantly increased the protein expression of p-AMPK after 3 h, and weakened VSMC migration after 24 h. Conclusions Physiological cyclic stretch inhibits VSMC migration by increasing the protein expression of p-AMPK, indicating that VSMC migration regulated by physiological cyclic stretch is of great significance for maintaining vascular homeostasis.

Objective:To establish a tissue based assay and in-house cell based assay combined system to screen anti-metabotropic glutamate receptor 1 antibodies in a case of previously idiopathic encephalitis with prominent cerebellar ataxia and make the final diagnosis, and to summarize and analyze clinical characteristics and treatment response of the disease.Methods:A middle-aged woman admitted to Department of Neurology, People's Liberation Army General Hospical Accredited to the Sixth Medical Center in January 9, 2020, who presented with acute dizziness, unsteady gait and developed head titubation, repeated language and calculation impairment was reported. The patient′s serum and cerebrospinal fluid were firstly tested with commercial kits for conventional neural antibodies.Then samples were incubated with rat hippocampus, cerebellum and human embryonic kidney 293 cells transfected with metabotropic glutamate receptor 1 plasmid to screen extra antibodies by indirect immunofluorescence method. By reviewing literature, physical functions of metabotropic glutamate receptor 1 and clinical features of anti-metabotropic glutamate receptor 1 antibodies associated encephalitis were summarized.Results:The patient was neural antibodies negative with commercial kits. Further investigation showed neuropil staining pattern after her serum and cerebral spinal fluid were incubated with rat brain slices. The characteristic "Medusa head" staining pattern of Purkinje cells in cerebellum was also noticed. Along with her previous head titubation symptom, an in-house cell based assay using human embryonic kidney 293 cells transfected with metabotropic glutamate receptor 1 plasmid was developed and proved the existence of anti-metabotropic glutamate receptor 1 antibodies. The final diagnosis of anti-metabotropic glutamate receptor 1 antibodies associated encephalitis was made. One-year follow-up revealed her serum antibodies titers dramatically decreased and cerebrospinal fluid antibodies were negative after using steroids and intravenous immunoglobulin, but still left prominent cerebellum atrophy and severe ataxia.Conclusions:Anti-metabotropic glutamate receptor 1 antibodies may cause acute encephalitis. Cerebellar ataxia and head titubation are characteristic symptoms of metabotropic glutamate receptor 1 autoimmunity. The response to immunotherapies is limited and patients may have severe neurological deficits.

Objective To investigate the synergistic effects of pathologically elevated cyclic stretch and platelet-derived microvesicles (PMVs) on migration of vascular smooth muscle cells (VSMCs) and the potential role of calcium in this process. Methods The FX-5000T strain loading system was used to apply cyclic stretch to VSMCs with magnitudes of 5% and 15%, which simulated physiological and hypertensive situation respectively in vitro; wound healing assay was used to analyze VSMCs migration; Ca2+-free medium was used to remove extracellular calcium; 2-APB (an antagonist of IP3R) was used to inhibit the release of intercellular stored calcium; GSK219 (an antagonist of TRPV4) and Nifedipine (an inhibitor of L-type voltage-gated calcium channel) were applied to block the activity of respective calcium channel; thrombin was used to stimulate platelets in vitro which simulated the hypertensive activation of PMVs in vivo. ResultsCompared with 5% cyclic stretch, 15% cyclic stretch significantly promoted VSMC migration. Removal of extracellular calcium inhibited VSMCs migration, but the application of GSK219 and Nifedipine did not affect the migration up-regulated by 15% cyclic stretch; while 2-APB which inhibited the release of intracellular stored calcium could also repress VSMCs migration under 15% cyclic stretch. PMVs further promoted VSMC migration under 15% cyclic stretch condition, and both extracellular calcium and intercellular stored calcium were involved in this process. Conclusions Both intracellular and extracellular calcium play important roles in VSMC migration induced by 15% cyclic stretch, and PMVs synergistically participate in the above process. The study is aimed to provide new mechanobiological insights into the molecular mechanism and clinical targets of vascular remodeling in hypertension.

Objective To investigate the effect of cyclic stretch on adhesion of vascular smooth muscle cells (VSMCs) with platelet-derived microparticles (PMPs), and the role of PMPs in VSMC autophagy. Methods Cyclic stretch with the magnitude of 5% (simulating physiological mechanical stretch) or 15% (simulating pathological mechanical stretch) was subjected to VSMCs in vitro by using FX-5000T cyclic stretch loading system, and the adhesion of PMPs in VSMCs was detected by using flow cytometry. Immunofluorescence was used to detect the expression of autophagy microtubule associated protein light chain 3 (LC3) after 24 h stimulation with PMPs. Western blotting was used to detect the expression of autophagy related protein (Atg) in VSMCs after 24 h stimulation by PMPs. Results Compared with 5% cyclic stretch, 15% cyclic stretch significantly increased the adhesion ability of VSMCs with PMPs. Immunofluorescence and Western blotting result revealed that PMPs stimulation significantly increased the expression of autophagy marker protein LC3 in VSMCs. Furthermore, the protein expressions of Atg5, Atg7 and Atg12 were all significantly increased in VSMCs stimulated with PMPs. Conclusions High cyclic stretch may enhance the autophagy of VSMCs by promoting the adhesion of PMPs, which will subsequently increase the expressions of Atg5, Atg7, Atg12 and LC3.

Objective:To investigate the clinical application of carbon nanoparticles mapping lymph nodes in curative resection for colorectal carcinoma.Methods:Patients diagnosed with colorectal cancer before operation and undergoing radical surgery with intact postoperative pathological data in the Sixth Affiliated Hospital, Sun Yat-sen University from March 2016 to March 2018 were included in this retrospective case-control study. Those who were diagnosed with ileus, recurrent carcinoma or underwent emergency operation were excluded. A total of 1421 cases were included, with 156 cases in the carbon nanoparticles mapping group and 1265 cases in the control group. Using 1∶3 case control matching based on gender, weight, TNM staging and neoadjuvant chemotherapy, 145 and 435 cases were finally recruited in the carbon nanoparticles mapping group and control group, respectively. Patients in the carbon nanoparticles mapping group underwent preoperative colonoscopy with carbon nanoparticles submucosal injection 2.4 (1.0 - 14.0) days before operation. Carbon nanoparticles of 0.25 ml was injected at 4 points (3, 6, 9 and 12 o'clock each) 0.5-1.0 cm around the tumor. The number of eliminated lymph node, number of positive lymph node and positive rate between the two groups were compared, and the number of eliminated lymph node in different subgroups of T stage, N stage, TNM stage and neoadjuvant chemotherapy was analyzed and compared.Results:After case control matching, total number of eliminated lymph nodes in the carbon nanoparticles mapping group was significantly higher than that in the control group (22.2±11.2 vs. 19.0±9.5, t=3.025, P=0.003). However, no statistically significant differences were found in the number of positive lymph node and lymph node positive rate between two groups (all P>0.05). Subgroup analysis showed that as compared to the control group, total number of eliminated lymph nodes in the carbon nanoparticles mapping group was significantly higher in T3 stage subgroup (median: 22 vs. 18, Z=2.435, P=0.015), N0 stage subgroup (median: 20.5 vs. 17.5, Z=2.772, P=0.006), TNM II stage subgroup (median: 23.5 vs. 19.0, Z=2.654, P=0.008) and neoadjuvant chemotherapy (median: 22.5 vs. 13.0, Z=3.287, P=0.001), while compared to the control group, the number of positive lymph node (median: 4.0 vs. 6.5, Z=-2.530, P=0.011) and the lymph node metastasis degree (median: 16% vs. 31%, Z=-2.862, P=0.004) were lower in the carbon nanoparticles mapping group in N2 subgroup. Conclusion:Carbon nanoparticles mapping lymph nodes can effectively enhance the number of eliminated lymph nodes in curative resection for colorectal cancer.

Objective:To investigate the clinical application of carbon nanoparticles mapping lymph nodes in curative resection for colorectal carcinoma.Methods:Patients diagnosed with colorectal cancer before operation and undergoing radical surgery with intact postoperative pathological data in the Sixth Affiliated Hospital, Sun Yat-sen University from March 2016 to March 2018 were included in this retrospective case-control study. Those who were diagnosed with ileus, recurrent carcinoma or underwent emergency operation were excluded. A total of 1421 cases were included, with 156 cases in the carbon nanoparticles mapping group and 1265 cases in the control group. Using 1∶3 case control matching based on gender, weight, TNM staging and neoadjuvant chemotherapy, 145 and 435 cases were finally recruited in the carbon nanoparticles mapping group and control group, respectively. Patients in the carbon nanoparticles mapping group underwent preoperative colonoscopy with carbon nanoparticles submucosal injection 2.4 (1.0 - 14.0) days before operation. Carbon nanoparticles of 0.25 ml was injected at 4 points (3, 6, 9 and 12 o'clock each) 0.5-1.0 cm around the tumor. The number of eliminated lymph node, number of positive lymph node and positive rate between the two groups were compared, and the number of eliminated lymph node in different subgroups of T stage, N stage, TNM stage and neoadjuvant chemotherapy was analyzed and compared.Results:After case control matching, total number of eliminated lymph nodes in the carbon nanoparticles mapping group was significantly higher than that in the control group (22.2±11.2 vs. 19.0±9.5, t=3.025, P=0.003). However, no statistically significant differences were found in the number of positive lymph node and lymph node positive rate between two groups (all P>0.05). Subgroup analysis showed that as compared to the control group, total number of eliminated lymph nodes in the carbon nanoparticles mapping group was significantly higher in T3 stage subgroup (median: 22 vs. 18, Z=2.435, P=0.015), N0 stage subgroup (median: 20.5 vs. 17.5, Z=2.772, P=0.006), TNM II stage subgroup (median: 23.5 vs. 19.0, Z=2.654, P=0.008) and neoadjuvant chemotherapy (median: 22.5 vs. 13.0, Z=3.287, P=0.001), while compared to the control group, the number of positive lymph node (median: 4.0 vs. 6.5, Z=-2.530, P=0.011) and the lymph node metastasis degree (median: 16% vs. 31%, Z=-2.862, P=0.004) were lower in the carbon nanoparticles mapping group in N2 subgroup. Conclusion:Carbon nanoparticles mapping lymph nodes can effectively enhance the number of eliminated lymph nodes in curative resection for colorectal cancer.

Objective To investigate the role of microRNA-214-3p (miR-214-3p) in differentiation and proliferation of endothelial progenitor cells (EPCs) induced by cyclic stretch. Methods EPCs were exposed to cyclic stretch at physiological level (with the magnitude of 5%, at a constant frequency of 1.25 Hz) for 24 h by FX-5000T Strain Unit. miRNAs array was performed to identify the expression profiling of miRNAs. Real-time PCR was used to examine the expression levels of miRs. The expression of vascular smooth muscle cells (VSMCs) markers in EPCs was detected by real-time PCR. EPC proliferation was detected by BrdU ELISA assay. After EPCs were transfected with miR-214-3p inhibitor (IN) to knockdown expression of miR-214-3p, the level of VSMC markers expression and EPC proliferation was detected. Results Cyclic stretch significantly decreased miR-214-3p expression, depressed EPC differentiation toward VSMCs, and increased EPCs proliferation. Similarly, transfection with the miR-214-3p inhibitor led to the decreased expression of VSMC markers under static station. Meanwhile, miR-214-3p down-regulation promoted EPC proliferation significantly. Conclusions Physiological cyclic stretch could down-regulate the expression of miR-214-3p in EPCs, depress EPC differentiation towards VSMC and promote EPC proliferation eventually. Therefore, the research findings provide a potential therapeutic strategy for treating vessel injuries.