BACKGROUND:Shujin Jiannao Prescription is an empirical formula for the treatment of cerebral palsy in Dongzhimen Hospital,Beijing University of Chinese Medicine,with clear clinical efficacy,but the specific mechanism needs to be elucidated. OBJECTIVE:To explore the possible mechanism of Shujin Jiannao Prescription in treating cerebral palsy. METHODS:Sixty-four 7-day-old Sprague-Dawley rats were randomly divided into a normal group(n=12)and a model group(n=52).An animal model was established by the Rice-Vannucci method.After successful modeling,52 model rats were randomly divided into control model group(n=12),minocycline group,and the low-,medium-,and high-dose groups of the Shujin Jiannao Prescription(n=10 per group).Rats in the minocycline group were given 40 mg/kg·d minocycline by gavage;rats in the low-,medium,and high-dose groups were given 4,8,and 16 g/kg·d Shujin Jiannao Prescription granules by gavage,respectively;and rats in the normal group and control model group were given an equal dose of normal saline by gavage.Medication in each group was given once a day for 1 week.The rats in each group were evaluated behaviorally using suspension test,abnormal involuntary movement score,and Bederson score.The pathological changes of the cerebral cortex were observed by hematoxylin-eosin staining.The levels of tumor necrosis factor α,interleukin 1β,and interleukin 10 in the cerebral cortex were determined using ELISA.The positive expressions of Janus kinase 2(JAK2),phosphorylated Janus kinase 2(p-JAK2),phosphorylated signal transducer and activator of transcription 3(p-STAT3)in the cerebral cortex were detected using immunohistochemistry.The protein expression levels of JAK2,p-JAK2,and p-STAT3 were detected using western blot. RESULTS AND CONCLUSION:Compared with the normal group,the suspension test score and involuntary movement score were decreased in the control model group(P＜0.01 or P＜0.05).The pathological results showed structural disruption of nerve cells,formation of large numbers of vacuoles,cell swelling,and increased intercellular space in the control model group.In addition,the expressions of tumor necrosis factor α and interleukin 1β in the cerebral cortex were significantly increased(P＜0.01),the expression of interleukin 10 was decreased(P＜0.05),and the protein expressions of JAK2,p-JAK2,and p-STAT3 in the cerebral cortex were significantly increased(P＜0.01)in the control model group compared with the normal group.Compared with the model group,minocycline and Shujin Jiannao Prescription at each dose could improve the behavioral indexes of rats(P＜0.01 or P＜0.05)and ischemic-hypoxic pathological changes were attenuated,with only a small amount of necrotic nerve cells and a few vacuoles,and reduced intercellular space.Moreover,the expressions of tumor necrosis factor α and interleukin 1β in the cerebral cortex were decreased in each drug group compared with the control model group(P＜0.05),while the protein expressions of JAK2,p-JAK2,and p-STAT3 in the cerebral cortex were significantly decreased(P＜0.01).The most obvious improvement was observed in the high-dose Shujin Jiannao Prescription group.To conclude,Shujin Jiannao Prescription can inhibit inflammation in the cerebral cortex of rats with hypoxic-ischemic brain injury.The mechanism may be related to the regulation of the JAK2/STAT3 signaling pathway.

AIM:To investigate the effects of γ-synuclein on autophagy and apoptosis of colon cancer cells in-duced by endoplasmic reticulum stress,as well as the protective effect on the cells.METHODS:Gene expression profile chip analysis was performed to compare the cDNA expression profiles between human colon cancer HCT116 cells with γ-synu-clein knockdown and HCT116 cells with control siRNA,and to identify potential molecules related to autophagy and apop-tosis.In colon cancer cell lines,the functional effects of γ-synuclein on autophagy and apoptosis induced by thapsigargin(TG),an endoplasmic reticulum stress-inducing agent,were systematically explored by conducting immunofluorescence staining,Western blot,CCK-8 assay,flow cytometry,and transmission electron microscopy.Western blot was used to de-tect the expression of γ-synuclein protein,autophagy-related proteins[microtubule-associated protein 1 light chain 3(LC3),beclin-1,autophagy-related protein 5(ATG5)and ATG7],and apoptosis-related proteins[poly(ADP-ribose)polymerase(PARP),pro-caspase-3,and pro-caspase-9].To further analyze the mechanism of γ-synuclein in regulating autophagy and apoptosis,extracellular signal-regulated kinase(ERK)inhibitor PD98059,ERK inhibitor SP600125 and c-Jun N-terminal kinase(JNK)activator anisomycin were applied separately to test HCT116 cells transfected with γ-synu-clein siRNA.Subsequently,autophagy proteins,apoptosis proteins,and ERK and JNK pathway-related proteins were de-tected by Western blot.RESULTS:The TG-induced autophagy of colon cancer cells mainly occurred at the early stage(0～24 h),and apoptosis mainly occurred at the late stage(36～48 h).Endoplasmic reticulum stress up-regulated the ex-pression of γ-synuclein in colon cancer cells,which was associated with enhanced autophagy.γ-Synuclein promoted au-tophagy by activating ERK and JNK pathways at the early stage(0～24 h),and inhibited apoptosis by blocking JNK path-ways at the late stage(24～48 h)to protect HCT116 cells.In our model,γ-synuclein was observed to play a critical role in the transition from endoplasmic reticulum stress-induced autophagy to apoptosis.CONCLUSION:In the context of endo-plasmic reticulum stress,γ-synuclein promotes autophagy and inhibits apoptosis by regulating ERK and JNK signaling pathways,thus protecting colon cancer cells.This provides a potential idea for anti-tumor therapy.

Objective: Sialidosis type 2 has variants that are both catalytically inactive (severe), while sialidosis type 1 has at least one catalytically active (mild) variant. This study aimed to discuss the structural changes associated with these variants in a newly reported family carrying N-acetyl-α-neuraminidase-1 (NEU1) variants and explore the clinical characteristics of different combinations of variants in sialidosis type 1. Methods: First, whole-exome sequencing and detailed clinical examinations were performed on the family. Second, structural analyses, including assessments of energy, flexibility and polar contacts, were conducted for several NEU1 variants, and a sialidase activity assay was performed. Third, previous NEU1 variants were systematically reviewed, and the clinical characteristics of patients in the severe-mild and mild-mild groups with sialidosis type 1 were analyzed. Results: We report a novel family with sialidosis type 1 and the compound heterozygous variants S182G and V143E. The newly identified V143E variant was predicted to be a mild variant through structural analysis and was confirmed by a sialidase activity assay. Cherry-red spots were more prevalent in the severe-mild group, and ataxia was more common in the mild-mild group. Impaired cognition was found only in the severe-mild group. Moreover, patients with cherry-red spots and abnormal electroencephalographies and visual evoked potentials had a relatively early age of onset, whereas patients with myoclonus had a late onset. Conclusion Changes in flexibility and local polar contacts may be indicators of NEU1 pathogenicity. Sialidosis type 1 can be divided into two subgroups according to the variant combinations, and patients with these two subtypes have different clinical characteristics.

ObjectiveTo explore the mechanism of Tangbikang granules （TBK） against diabetic peripheral neuropathy （DPN） based on network pharmacology and in-vivo experiment. MethodThe active components in medicinals of TBK and their target genes were searched from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）. The active components of the medicinals which are not included in TCMSP were searched from previous research. After the analysis of drug-likeness by SwissADME， the target genes of them were predicted with SwissTargetPrediction. DPN-related target genes were retrieved from GeneCards. The common targets of the disease and the prescription were the hub genes of TBK against DPN， which were uploaded to Metascape for Gene Ontology （GO） term enrichment and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis. High-sugar and high-fat diet and low-dose streptozotocin （STZ， ip） were employed to induce diabetes in rats， and then the model rats were respectively treated with low-dose （0.625 g·kg^-1）， medium-dose （1.25 g·kg^-1）， and high-dose （2.5 g·kg^-1） TBK for 12 weeks. Sensory nerve conduction velocity （SNCV） was evaluated. After hematoxylin and eosin （HE） staining， the sciatic nerve was observed under light microscope to examine the nerve damage. Real-time PCR was performed to detect the gene expression of adenosine monophosphate-activated protein kinase （AMPK） pathway-related targets in rat sciatic nerve， and Western blot to measure the protein expression of AMPK and phosphorylated （p）-AMPK in rat sciatic nerve. ResultThe main active components of TBK， such as quercetin， kaempferol， β-sitosterol， leech pteridine A， stigmasterol， and baicalein were screened out， mainly acting on interleukin-6 （IL-6）， tumor necrosis factor （TNF）， protein kinase B （Akt）， JUN， and HSP90AA1 and signaling pathways such as AMPK， nuclear factor-κB （NF-κB）， and Janus kinase/signal transducer and activator of transcription （JAK/STAT）. Molecular docking results showed that β-sitosterol and stigmasterol had high binding affinity with IL-6， TNF， JUN， and HSP90AA1. As for the animal experiment， compared with the normal group， model group had low SNCV of sciatic nerve （P<0.01）， disordered and loose myelinated nerve fibers with axonotmesis and demyelinization， low mRNA expression of AMPKα， AMPKβ， peroxisome proliferator-activated receptor γ coactivator-1α （PGC-1α）， Sirtuin 3 （SirT3）， mitochondrial transcription factor A （TFAM）， and low p-AMPK/AMPK ratio in sciatic nerve （P<0.05， P<0.01）. Compared with the model group， TBK of the three doses raised the SNCV （P<0.01）， restored nerve morphology and nerve compactness， and increased the mRNA expression of AMPKα， AMPKβ， PGC-1α， SirT3， and TFAM （P<0.05， P<0.01）. The ratio of p-AMPK/AMPK in the high-dose and medium-dose TBK groups was higher than that in the model group （P<0.01）， while the protein expression in the low-dose TBK group was insignificantly different from that in the model group. ConclusionTBK exerts therapeutic effect on DPN through multiple pathways and targets. The mechanism is that it activates and regulates AMPK/PGC-1α/SirT3 signaling， which lays a basis for further study of TBK in the treatment of DPN.

Objective Taking 41 kinds of Chinese herbal medicines commonly used in Meizhou Hakka as the research object,their inhibitory activities against α-glucosidase and α-amylase were screened and the enzyme inhibition types of the species with the strongest activities were explored.Methods The inhibitory activities of 41 commonly used Hakka herbs in Meizhou against α-glucosidase and α-amylase were evaluated by the p-Nitrophenyl a-D-mannopyranoside(pNPG)method and the 3,5-Dinitrosalicylic acid(DNS)method,using the inhibitory rate of half(IC50)as an index.The inhibitory activity of 95%ethanol extracts of 41 Chinese herbal medicines commonly used in Meizhou Hakka on α-glucosidase and α-amylase were analysed.The enzymatic kinetics method and Lineweaver-Burk curve were used to analyze the inhibitory type of the most active species.Results The results showed that 40 Chinese herbal medicines commonly used in Meizhou Hakka had α-glucosidase inhibitory activity,and 23 medicines had α-amylase inhibitory activity,among which Psychotria asiatica Wall.showed the strongest inhibitory activity with the IC50 values aganist α-glucosidase and α-amylase of 0.17±0.001 mg·mL-1 and 0.09±0.001 mg·mL-1,respectively.The inhibition types were reversible competitive inhibition and reversible non-competitive inhibition,respectively.Conclusion The Psychotria asiatica Wall.Chinese herbal medicines commonly used in Meizhou Hakka has significant inhibitory effect on the activity of glucose metabolism enzymes,which has potential value for further research and development on the prevention and treatment of diabetes mellitus.

Objective:To observe the expression levels of glial fibrillary acidic protein (GFAP), β-tubulin Ⅲ and synaptophsin, and explore the role of tripartite synapse in the mechanism of central nervous system (CNS) injury and the neuroprotective effect of chondroitin sulfate (CS).Methods:One month old clean grade, 48 female Sparague-Dawley rats and 48 male Sparague-Dawley rats, were randomly divided into 8 groups according to body weight (90 - 120 g) by random number table method, with 12 rats in each group, half male and half female. These rats were fed with water containing different concentrations of sodium fluoride (NaF) [ < 0.5 mg/L (control, CN), 10.0 mg/L (low dose fluoride, LF) and 50.0 mg/L (high dose fluoride, HF)]. Some rats were fed directly for 185 days (CN, LF and HF groups). In addition, rats of CN + normal saline (NS), LF + NS, HF + NS groups and LF + CS, HF + CS groups, were intraperitoneally injected with NS or 0.66 mg/kg CS for 5 consecutive days after 180 days of feeding. After the experiment, the pathological changes of hippocampal CA4 of brain tissue in each group were observed by hematoxylin eosin staining under light microscope, and the expression and distribution of GFAP, β-tubulin Ⅲ and synaptophsin in hippocampal CA4 of rats were detected by immunohistochemistry, the expression of GFAP, β-tubulin Ⅲ and synaptophsin at protein level in hippocampus of rats were detected by Western blotting.Results:Under light microscope, eosinophilic change, loss and irregular arrangement of neuron in the hippocampal CA4 were observed in LF, HF, LF + NS and HF + NS groups. The morphology of LF + CS and HF + CS groups was not significantly changed compared with CN group, but was significant changed compared with LF, HF, LF + NS and HF + NS groups. Immunohistochemical results showed that the rates of positive area of GFAP, β-tubulin Ⅲ and synaptophsin in female and male rats in LF and HF groups were significantly decreased than those in CN group ( P < 0.05); the positive area rates of female and male rats in LF + CS and HF + CS groups were higher than those in LF and HF groups, respectively ( P < 0.05). Western blotting results showed that the proten expression levels of GFAP, β-tubulin Ⅲ and synaptophsin of female and male rats in LF and HF groups (LF group: 0.90 ± 0.09, 0.82 ± 0.08, 1.43 ± 0.14, 0.92 ± 0.02, 1.21 ± 0.15, 0.87 ± 0.02, HF group: 0.58 ± 0.14, 0.73 ± 0.03, 0.63 ± 0.06, 0.67 ± 0.03, 0.87 ± 0.04, 0.70 ± 0.05) were lower than those in CN group (1.24 ± 0.08, 1.09 ± 0.10, 2.64 ± 0.30, 1.54 ± 0.09, 1.72 ± 0.10, 1.13 ± 0.06, P < 0.05). Conclusions:The tripartite synapse and extracellular matrix may take part in pathogenesis of the damages of CNS results from chronic fluorosis; CS may reduce the injury to a certain extent.

Objective To investigate the correlation between vital capacity, pulmonary ventilation and morphological parameters among children living in different altitude areas, so as to provide a reference basis for the development of prevention and control strategies for high altitude illness. Methods From January 2019 to June 2020, primary and secondary school students aged 7 to 15 years old were randomly selected from three different altitude areas, which were Xining (2 260m, low altitude group), Haixi (2 900m, medium altitude group), and Yushu (4 493m, high altitude group), respectively. The vital capacity, pulmonary ventilation and morphological parameters of the selected children were recorded. Results The vital capacity, pulmonary ventilation and morphological parameters showed statistically significant difference among three groups (P<0.05). The vital capacity and pulmonary ventilation were positively correlated with lung volume, but negatively correlated with lung density and lung artery diameter (P<0.05). Logistic regression analysis showed that there were three factors affecting children's vital capacity and lung ventilation: mean lung density, total lung transverse diameter, and total lung volume (P<0.05). Conclusion The monitoring of lung morphological indexes, mean lung density, total lung transverse diameter, and total lung volume can effectively judge children's lung function, and have certain value in the prevention and treatment of related high-altitude illness.

Objective To study the mechanism of central nervous system (CNS) injury in chronic fluorosis and the neuroprotective effect of chondroitin sulfate (CS).Methods Forty-eight female Sprague-Dawley rats weighting 90-120 g were divided into 8 groups according to body weight by random number table,6 rats in each group:control group,drinking tap water freely;low dose and high dose fluoride groups,freely drinking tap water with fluoride content of 10 and 50 mg/L,respectively;control + normal saline (NS),low dose fluoride + NS,and high dose fluoride + NS groups,each group was fed for 180 d,and treated with intraperitoneal injection of 0.66 mg/kg NS for 5 d (once a day);low dose fluoride + CS and high dose fluoride + CS groups,each group was fed for 180 d,0.66 mg/kg CS was injected intraperitoneally for 5 d (once a day).All groups were fed standard nutritive animal feed for 185 d and dissected for brain tissue.The pathologic change was observed after hematoxylin-eosin (HE)staining;the expression levels of phosphorylated extracellular signal-regulated protein kinase 1/2 (phospho-Erk1/2)and glutamate receptors 1,2 (GluR1,GluR2) in the brain cortex were detected by immunohistochemistry;the protein levels of Erk1/2,phospho-Erk1/2,GluR1,and GluR2 in the brain cortex were detected by Western blotting.Results Brain cortex of all rats in the fluoride groups showed eosinophilic degeneration,loss and disordered arrangement of neurons,and the brain morphological changes in each fluoride + CS groups were significantly improved compared with those in the fluoride groups.Immunohistochemistry results showed that compared with the control group [(0.44 ± 0.09)％,(1.49 ± 0.05)％,(2.51 ± 0.54)％],the expression levels of phospho-Erk1/2 [(1.47 ±0.09)％,(1.03 ± 0.05)％],and GluR2 [(2.37 ± 0.06)％,(3.38 ± 0.12)％] in the low dose and high dose fluoride groups were increased,and the expression levels of GluR1 [(1.49 ± 0.02)％,(0.99 ± 0.19)％] were decreased (P ＜ 0.05).Western blotting results showed that compared with the control group (1.00 ± 0.12,1.76 ± 0.33),the protein levels of Erk1/2 (3.10 ± 0.76,1.99 ± 0.01) and phospho-Erk1/2 (3.27 ± 0.25,2.67 ± 0.05) in low dose and high dose fluoride groups were significantly increased (P ＜ 0.05);compared with low dose fluoride group,the protein levels of Erk1/2,and phospho-Erk1/2 (1.30 ± 0.31,2.20 ± 0.34) in low dose fluoride + CS group decreased significantly (P ＜0.05).Compared with control group (1.86 ± 0.47,1.17 ± 0.27),the protein levels of GluR1 (1.09 ± 0.26,0.61 ± 0.14) in low dose and high dose fluoride groups decreased significantly,while the protein level of GluR2 (1.99 ± 0.42,3.38 ±0.27) increased significantly (P ＜ 0.05);compared with low dose and high dose fluoride groups,the protein levels of GluR2 in low dose fluoride + CS and high dose fluoride + CS groups (1.53 ± 0.41,2.65 ± 0.32) decreased significantly (P ＜ 0.05).The protein level of phospho-Erk1/2 was negatively correlated with GluR1 protein level (r =-0.975,-0.991,P ＜ 0.05) in low dose and high dose fluoride groups,and it was positively correlated with the protein level of GluR2 (r =0.986,0.993,P ＜ 0.05).Conclusion The CNS injury caused by chronic fluorosis may be related to GluR1 and GluR2 activated Erk1/2 signaling pathway,and CS has certain protection to the injury.

Objective To investigate the effect of periosteum deficiency on the treatment of segmental bone defect of lower extremity by Ilizarov bone transport Technique.Methods From April 2009 to April 2016,data of 54 cases with lower limb segmental bone defect who were treated by Ilizarov bone transport technique were retrospectively analyzed.According to intraoperative observation of the distribution of periosteum at the osteotomy site,all cases were divided into two groups.There were 10 cases in absent periosteum group,including 6 males and 4 females,aged from 23 to 57 years old (mean,38.20±9.90 years old).There were 3 cases locating at femur,and 7 cases at tibia.4 cases had non-infective bone defect,while the other 6 had infective bone defect.Segmental bone defect ranged from 4.0 to 14.0 cm (mean,6.85±2.87 cm).There were 44 cases in normal periosteum group,including 32 males and 12 females,aged from 22 to 65 years old (mean,38.90±10.10 years old).There were 9 cases locating at femur and 35 cases at tibia.12 cases had non-infective bone defect,while 32 cases had infective bone defect.Segmental bone defect ranged from 4.0 to 9.0 cm (mean,6.09±1.54 cm).Visual analogue scale (VAS) was used to estimate the pain during bone transport.The distraction index,bone healing index,external fixator index was collected to evaluate the distraction osteogenesis and mineralization.The Paley method was utilized to appraisal the function of proximal joints in bone transport.Results All the 54 cases were followed up for 24-48 months,with an average of 28.50±5.70 months.All skin and soft tissue defects were satisfied healed.All limbs were restored to their original lengths,and bone healing was eventually achieved.There were no statistically significant differences in the distraction index,bone healing index,external fixator index,VAS between absent periosteum group (12.29± 1.04 d/cm,39.00±3.96 d/cm,51.25±3.69 d/cm,3.30± 1.77 score) and normal periosteum group (11.67±0.96 d/cm,38.07±4.22 d/cm,49.74±4.25 d/cm,3.36± 1.66 score)(P ＞ 0.05).Paley adjacent joint function evaluation showed no statistically difference between absent periosteum group [good rate 100％(10/10)] and normal periosteum group [good rate 97.7％ (43/44)] (P=0.901).The overall incidence of complications was 20.0％ (2/10) in the absence of periosteum group,with 1 case of mild infection and 1 cases of tissue contracture incarcerated at the docking site.The overall incidence of complications was 27.3％ (12/44) in the normal of periosteum group,with 2 cases of mild infection,3 cases of poor limb strength alignment,6 cases of tissue contracture incarcerated at the docking site and 1 case of severe foot drop deformity.There was no statistically significant difference in the overall incidence of complications between the two groups (P=0.636).Conclusion The absence of periosteum at the epiphyseal osteotomy site hasno significant effect on Ilizarov bone transport technique in the treatment of segmental bone defects of lower limbs in bone regeneration and healing,mineralization and bone remodeling,and does not increase the incidence of complications.

OBJECTIVE: The periaqueductal gray matter (PAG), a small midbrain structure, presents dysfunction in migraine. However, the precise neurological mechanism is still not well understood. Herein, the aim of this study was to investigate the texture characteristics of altered PAG in episodic migraine (EM) patients based on high resolution brain structural magnetic resonance (MR) images. MATERIALS AND METHODS: The brain structural MR images were obtained from 18 normal controls (NC), 18 EM patients and 16 chronic migraine (CM) patients using a 3T MR system. A PAG template was created using the International Consortium Brain Mapping 152 gray matter model, and the individual PAG segment was developed by applying the deformation field from the structural image segment to the PAG template. A grey level co-occurrence matrix was used to calculate the texture parameters including the angular second moment (ASM), contrast, correlation, inverse difference moment (IDM) and entropy. RESULTS: There was a significant difference for ASM, IDM and entropy in the EM group (998.629 ± 0.162 × 10−3, 999.311 ± 0.073 × 10−3, 916.354 ± 0.947 × 10−5) compared to that found in the NC group (998.760 ± 0.110 × 10−3, 999.358 ± 0.037 × 10−3 and 841.198 ± 0.575 × 10−5) (p < 0.05). The entropy was significantly lower among the patients with CM (864.116 ± 0.571 × 10−5) than that found among patients with EM (p < 0.05). The area under the receiver operating characteristic curve was 0.776 and 0.750 for ASM and entropy in the distinction of the EM from NC groups, respectively. ASM was negatively related to disease duration (DD) and the Migraine Disability Assessment Scale (MIDAS) scores in the EM group, and entropy was positively related to DD and MIDAS in the EM group (p < 0.05). CONCLUSION: The present study identified altered MR image texture characteristics of the PAG in EM. The identified texture characteristics could be considered as imaging biomarkers for EM.
Biomarkers ; Brain ; Brain Mapping ; Entropy ; Gray Matter ; Humans ; Magnetic Resonance Imaging ; Mesencephalon ; Migraine Disorders ; Periaqueductal Gray ; ROC Curve