Objective To clone and express Chlamydia trachomatis (Ct) heat shock protein 60 (hsp60) gene. Methods The hsp60 gene fragment was amplified from Ct chromosomal DNA by PCR. After purification and digestion with enzymes Sal I and Not I , the hsp60 gene fragment was inserted into the compatible site of prokaryotic expression vector pET-28a. The constructed recombinant plasmid was identified by PCR, restriction enzyme cleavage and sequencing, then, it was transfected into an expression strain Escherichia coli BL21 (DE3). The expression of fusion protein was induced by isopropy-β-D- thiogalactoside (IPTG) in the host bacteria, and the expressed product was identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western-blot. Results PCR and restriction enzymes cleavage analysis confirmed that the hsp60 gene was successfully cloned into the recombinant plasmid. DNA sequencing showed that the sequence of cloned gene was fully consistent with the published sequence in Genebank. As revealed by SDS-PAGE, the size of expressed fusion protein approximated 60 kilodaltons, and Western-blot confirmed the expressed product to be the expected protein. The final concentration of fusion protein was 17.85 mg/L with a purity of more than 90%. Conclusions A recombinant expression plasmid pET-28a-hsp60 is successfully constructed in this study, and soluble hsp60 protein is expressed by the recombinant plasmid-transfected E. coli.

ObjectiveTo systematically evaluate the efficacy and safety of Tianmeng Oral Liquid in the treatment of hyperprolactinemia caused by antipsychotics in female patients with schizophrenia， and to provide references for intervention of antipsychotic-induced hyperprolactinemia patients. MethodsEnglish databases （PubMed， Cochrane Library， PsycINFO and Embase） and Chinese databases （CNKI and Wanfang） were systematically searched， with a search time frame of the period from database creation to September 16， 2022. Randomized controlled studies （RCTs） on adjunctive Tianmeng Oral Liquid in the treatment of hyperprolactinemia caused by antipsychotics in female patients with schizophrenia were included. Three investigators independently screened the literatures and extracted data according to PICOS principles and evaluated the quality of including literatures， and the Meta-analysis was performed by using RevMan 5.3. ResultsA total of 3 articles including 256 female schizophrenic patients with hyperprolactinemia were included. Meta-analysis results showed that at the treatment endpoint， the improvement of hyperprolactinemia symptoms reported a significant superiority of the intervention group than the control group （RR=1.73， 95% CI： 1.07~2.79， P˂0.05）. Similarly， the significant superiority of the intervention group than the control group were also found in reducing serum prolactin levels （WMD=-55.17， 95% CI： -68.16~-42.18， P<0.01） and Positive and Negative Syndrome Scale （PANSS） total scores were lower in the intervention group than in the control group （WMD=-7.36， 95% CI： -8.94~-5.77， P<0.01）. ConclusionTianmeng Oral Liquid may help improve clinical efficacy and reduce serum prolactin levels in female schizophrenia patients with hyperprolactinemia. It may improve psychiatric symptoms in female patients with schizophrenia.

ObjectiveTo investigate the mechanism of neferine（Nef） in promoting vascular regeneration against cerebral ischemia through modulation of mitochondrial calcium uniporter（MCU） ion channel. MethodTaking the area of subintestinal vessels in microvascular deficiency zebrafish as an index， the vascular regenerative efficacy of Nef was evaluated， and the median effective concentration（EC₅₀） was calculated. Rats were randomly divided into a sham operation group， a model group， a positive drug group（butylphthalide， 6 mg·kg^-1）， and Nef low， medium， and high dose groups（0.125， 0.625， 3.125 μg·kg^-1）. Except for the sham operation group， the middle cerebral artery occlusion（MCAO） model was established in other groups. After modeling， the groups were administered the corresponding dose of drugs by gavage， while the sham operation and model groups received equal volumes of saline， once a day for 7 consecutive days. Neurobehavioral scores were assessed for each group of rats， and the infarct rate of ischemic brain tissue was calculated by 2，3，5-triphenyltetrazolium chloride（TTC） staining. The regional cerebral blood flow（rCBF） of each group was measured using a speckle contrast imaging. Immunofluorescence and Western blot were conducted to detect the expression of vascular endothelial growth factor（VEGF）， platelet endothelial cell adhesion molecule-1（CD31）， and hypoxia-inducible factor-1α（HIF-1α） proteins in each group. Human umbilical vein endothelial cells（HUVECs） were divided into the normal group， model group， positive drug group（astragaloside Ⅳ， 10 μmol·L^-1）， and Nef group （32 nmol·L^-1）. In the verification of mitochondrial protection of Nef and its mechanism in promoting vascular regeneration， the spermine（MCU agonist） and Nef+spermine group were added. HUVECs model of oxygen-glucose deprivation（OGD） was established in all groups except the normal group， the cell viability was assessed using 3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyltetrazolium bromide（MTT） assay， and cell migration ability was evaluated through scratch and tube formation assays. Fluorescent probes（Rhod-2 AM， Fluo-3 AM， JC-1， Calcein AM） and a cellular energy metabolism analyzer were used to analyze the mitochondrial protective effects of Nef. Molecular docking was performed to predict the binding ability of Nef with MCU and HIF-1α， and Western blot was used to detect the effects of Nef on the protein expressions of MCU， B-cell lymphoma-2 associated X protein（Bax）， Caspase-3 and HIF-1α in the OGD model HUVECs. ResultThe results of vascular regeneration in microvascular deficiency zebrafish showed that compared to the normal group， the area of subintestinal vessels in the model group significantly decreased（P<0.01）. Compared to the model group， different concentrations of Nef could significantly increase the area of subintestinal vessels（P<0.01）， with the maximum tolerated concentration of 10.24 μmol·L^-1 and the EC₅₀ of 0.23 μmol·L^-1. Anti-cerebral ischemia results on MCAO rats showed that compared to the sham operation group， the model group had a significant decrease in rCBF and a significant increase in infarct rate， while CD31 expression significantly decreased（P<0.01）， and VEGF and HIF-1α protein expressions significantly increased（P<0.05）. Compared to the model group， the treated groups showed significant increases in rCBF， significant reductions in infarct volume， and significant increases in CD31， VEGF， and HIF-1α protein expression（P<0.01）. Cell experiment results showed that compared to the normal group， the model group had decreased cell viability and migration ability， increased intracellular Ca²⁺ and mitochondrial Ca²⁺ levels， reduced mitochondrial permeability transition pore（MPTP） opening， and decreased mitochondrial energy metabolism capability， with increased expressions of MCU， Bax， Caspase-3 and HIF-1α proteins（P<0.05， P<0.01）. Compared to the model group， the Nef group showed increased cell viability and migration ability， decreased intracellular Ca²⁺ and mitochondrial Ca²⁺ levels， increased MPTP opening， enhanced mitochondrial energy metabolism capability， decreased expressions of MCU， Bax and Caspase-3 proteins， and increased HIF-1α protein expression（P<0.05， P<0.01）. ConclusionNef can stabilize mitochondrial membrane potential and inhibit mitochondrial apoptosis. By down-regulating the expression of MCU， it suppresses the activation of intracellular Bax and Caspase-3 while activating the HIF-1α signaling pathway， enhancing the expression of VEGF and CD31， thereby promoting vascular regeneration to treat ischemic brain injury.