Objective To explore the impact of Metformin(Met)on the balance of type 17 helper T cells(Th17)/regulatory T cells(Treg)in DM rats by regulating the C-C motif chemokine ligand 2(CCL2)-C-C motif chemokine receptor 2(CCR2)axis.Methods A total of 50 SD rats were randomly divided into normal control(NC)group,model(Mod)group,Met(100 mg/kg)group,Met+empty vector(Met+oe-NC)group,and Met+CCL2 overexpression(Met+oe-CCL2)group,with 10 rats in each group.The changes of FBG evaluated in each group,and the pathological changes of pancreatic islet tissue were assessed using hematoxylin-eosin(HE)staining followed by histological scoring.The ratio of Th17/Treg in peripheral blood was detected by flow cytometry.ELISA was used to measure the serum levels of IL-17,TNF-α,TGF-β and IL-10.Immunofluorescence was used to detect the proportion of Th17 and Treg in pancreatic tissue.RT-PCR and Western blot were used to detect the mRNA and protein expression of ROR-γt,forkhead box P3(FOXP3),CCL2 and CCR2 in pancreatic tissue.Results The islet structure was intact and the boundary was clear,and there was no pathological damage in the NC group.In the Mod group,the islet tissue exhibited significant pathological damage,characterized by disrupted architecture,reduced volume,and blurred boundaries with surrounding tissues.Additionally,the cells in the islet displayed marked disorganization and substantial loss.Compared with the Mod group,the pathological damage of the islet tissue was alleviated in the Met and Met+oe-NC group.Compared with the Met group,the pathological damage was aggravated in the Met+oe-CCL2 group.Compared with the NC group,the levels of FBG,Th17/Treg ratio,IL-17,TNF-α,Th17 infiltration rate,ROR-γt,CCL2,and CCR2 mRNA and protein expression were higher(P<0.05),while TGF-β,IL-10,Treg infiltration rate,FOXP3 mRNA and protein expression were lower in the Mod group(P<0.05).Compared with the Mod group,the levels of FBG,Th17/Treg ratio,IL-17,TNF-α,Th17 infiltration rate,ROR-γt,CCL2,and CCR2 mRNA and protein were lower(P<0.05),while TGF-β,IL-10,Treg infiltration rate,FOXP3 mRNA and protein expression were higher in the Met and Met+oe-NC groups(P<0.05).Compared with the Met group,the levels of FBG,Th17/Treg ratio,IL-17,TNF-α,Th17 infiltration rate,ROR-γt,CCL2,and CCR2 mRNA and protein were higher(P<0.05),and TGF-β,IL-10,Treg infiltration rate,FOXP3 mRNA and protein expression were higher in the Met+oe-CCL2 group(P<0.05).Conclusions Metformin can promote Th17/Treg cell balance drift towards Treg direction by inhibiting the activation of CCL2-CCR2 signal axis,thereby preventing the occurrence and development of inflammatory reactions,alleviating pancreatic islet damage,and improving the glucose metabolism in DM rats.

Objective To explore the impact of Metformin(Met)on the balance of type 17 helper T cells(Th17)/regulatory T cells(Treg)in DM rats by regulating the C-C motif chemokine ligand 2(CCL2)-C-C motif chemokine receptor 2(CCR2)axis.Methods A total of 50 SD rats were randomly divided into normal control(NC)group,model(Mod)group,Met(100 mg/kg)group,Met+empty vector(Met+oe-NC)group,and Met+CCL2 overexpression(Met+oe-CCL2)group,with 10 rats in each group.The changes of FBG evaluated in each group,and the pathological changes of pancreatic islet tissue were assessed using hematoxylin-eosin(HE)staining followed by histological scoring.The ratio of Th17/Treg in peripheral blood was detected by flow cytometry.ELISA was used to measure the serum levels of IL-17,TNF-α,TGF-β and IL-10.Immunofluorescence was used to detect the proportion of Th17 and Treg in pancreatic tissue.RT-PCR and Western blot were used to detect the mRNA and protein expression of ROR-γt,forkhead box P3(FOXP3),CCL2 and CCR2 in pancreatic tissue.Results The islet structure was intact and the boundary was clear,and there was no pathological damage in the NC group.In the Mod group,the islet tissue exhibited significant pathological damage,characterized by disrupted architecture,reduced volume,and blurred boundaries with surrounding tissues.Additionally,the cells in the islet displayed marked disorganization and substantial loss.Compared with the Mod group,the pathological damage of the islet tissue was alleviated in the Met and Met+oe-NC group.Compared with the Met group,the pathological damage was aggravated in the Met+oe-CCL2 group.Compared with the NC group,the levels of FBG,Th17/Treg ratio,IL-17,TNF-α,Th17 infiltration rate,ROR-γt,CCL2,and CCR2 mRNA and protein expression were higher(P<0.05),while TGF-β,IL-10,Treg infiltration rate,FOXP3 mRNA and protein expression were lower in the Mod group(P<0.05).Compared with the Mod group,the levels of FBG,Th17/Treg ratio,IL-17,TNF-α,Th17 infiltration rate,ROR-γt,CCL2,and CCR2 mRNA and protein were lower(P<0.05),while TGF-β,IL-10,Treg infiltration rate,FOXP3 mRNA and protein expression were higher in the Met and Met+oe-NC groups(P<0.05).Compared with the Met group,the levels of FBG,Th17/Treg ratio,IL-17,TNF-α,Th17 infiltration rate,ROR-γt,CCL2,and CCR2 mRNA and protein were higher(P<0.05),and TGF-β,IL-10,Treg infiltration rate,FOXP3 mRNA and protein expression were higher in the Met+oe-CCL2 group(P<0.05).Conclusions Metformin can promote Th17/Treg cell balance drift towards Treg direction by inhibiting the activation of CCL2-CCR2 signal axis,thereby preventing the occurrence and development of inflammatory reactions,alleviating pancreatic islet damage,and improving the glucose metabolism in DM rats.

ObjectiveTo develop the Evidence Grading Scale for Ancient classical prescriptions in Traditional Chinese medicine， assess its reliability and validity， and apply it in practice to provide multi-source evidence for clinical practice guidelines development. MethodsLiterature retrieval was conducted to extract and screen existing evaluation dimensions， then the initial items were summarized using thematic analysis. Experts in the clinical medicine， medical history and literature participated in the Delphi questionnaire survey to evaluate and refine the items. An expert consensus meeting was conducted to finalize the included items， refine the method for items evaluation and evidence grading. The evidence quality rating scale for ancient classical traditional Chinese medicine （TCM） prescriptions was then established and tested for reliability and validity. ResultsThrough literature review， extraction， screening and summarization， a total of 3 dimensions and 12 initial items were formed. Questionnaires were sent to 69 experts to evaluate the initial items， with a questionnaire response rate of 100% and an expert authority coefficient of 0.92. All 12 items were retained for they had importance scores above 4. The Evidence Grading Scale on Ancient classical prescriptions in Traditional Chinese medicine includes 3 dimensions with 12 items. The 3 dimensions includes ancient evidence， inheritance status， and modern application. Each dimension contains 4 items， and each item has a full score of 5 points. The evidence was rated as high-level， moderate-level， and low-level according to the final scores. The content validity index （CVI） of the 12 items was ＞0.9， the average CVI of the scale was 0.98， and the intraclass correlation coefficient （ICC） was 0.90. ConclusionThe Evidence Grading Scale on Ancient classical prescriptions in Traditional Chinese medicine has good reliability and validity， which is practical for use in the development of TCM clinical guidelines and can better support clinical decision-making.

The proportion of casualties caused by blast injuries is increasing year by year,with those induced by shock waves being the main life-threatening factor.Because of the different propagation laws of the explosive shock waves in various environments,the waves will result in different damages on the person in the space.So,clarifying the impacts of different work conditions on the blast injuries will provide important supports for the assessment of the explosive power of weapons and ammunition and for the guidance for personal protective equipment selection in the process of actual combat.In this article,we discuss the rules in the shock wave-induced injuries in the free field,underwater,confined and semi-confined spaces,identify the shortcomings in the current research on blast injuries in different environments,summarize the current states of the research and application,and finally,bring forward to the key technologies and development directions that need to be solved in the subsequent research in order to provide a reference basis for future research on the injuries caused by explosive shock waves.

Objective:To investigate the effects of dual vascularized tissue-engineered bone constructed by vascular bundles and endothelial progenitor cells (EPCs) on repair of large bone defects and vascular regeneration.Methods:EPCs were seeded on the demineralized bone matrix (DBM) scaffolds and cultured for 6 days. The attachment and morphology of EPCs on DBM scaffolds were observed by electron microscopy. Next, the radial artery was implanted into a vascular groove opened inside the DBM-EPCs composite scaffolds. Finally, models of a large segmental bone defect were constructed using the radii from 18 New Zealand white rabbits. The rabbits were randomly divided into 4 groups using a simple random sampling method: DBM group, DBM+EPCs group, DBM+vascular bundle group, and DBM+EPCs+vascular bundle group. The DBM group and DBM+EPCs group shared the same rabbits so that transplantations were conducted into the left and right forearms respectively; the DBM+vascular bundle group and DBM+EPCs+vascular bundle group also shared the same rabbits so that transplantations were conducted into the left and right forearms respectively. Consequently, there were 9 experimental sites in each group. X-ray examination and gross morphological observation were performed to evaluate the bone regeneration in the experimental rabbits in each group at 4, 8, and 12 weeks after surgery, and CD31 immunofluorescence staining was used to evaluate the vascular regeneration. Micro-CT was used to analyze bone tissue parameters and reconstruct the three-dimensional structures of the defects site at 12 weeks after surgery.Results:Compared with the DBM, DBM+EPCs and DBM+vascular bundle groups, the DBM+EPCs+vascular bundle group showed new bone tissue crawling on the scaffold surface at 4 weeks after surgery, almost complete healing of the bone defect area at 8 weeks, and forming of a complete and dense bone bridge and appearance of a bone marrow cavity at 12 weeks. Micro-CT data at 12 weeks after surgery showed regular arrangement of the trabeculae, significantly improved mineralization, and increased thickness of the bone cortex in the DBM+EPCs+vascular bundle group. Additionally, in the DBM+EPCs+vascular bundle group, the number of microvessels was significantly higher than that in the other groups at 4, 8, 12 weeks after surgery ( P<0.05), and the angiogenesis and bone tissue regeneration were particularly prominent at 12 weeks after surgery. The number of CD31 cells in the DBM+EPCs+vascular bundle group increased significantly more than that in the DBM, DBM+EPCs and DBM+vascular bundle groups ( P<0.05). Conclusion:As the dual vascularized tissue-engineered bone constructed by vascular bundles and EPCs can significantly promote bone tissue regeneration and angiogenesis, it may be a potential therapeutic strategy for repair of large bone defects.

Objective To investigate the regulation of macrophage polarization and foaminess by homocysteine(Hcy)and its potential underlying mechanisms.Methods ELISA and flow cytometry were used to detect the effect of Hcy treatment on the polarization of macrophages.The contents of various forms of intracellular cholesterol were detected,and the effects of Hcy on intracellular lipid accumulation and ox-LDL uptake were evaluated by oil red O staining and Dil-oxLDL.RT-qPCR and Western blot were used to detect mRNA and pro-tein expression of key genes modified by N6-methyladenosine(m6 A).Results Hcy promoted M1 polarization of macrophages and ox-LDL-induced foam macrophages and promoted ox-LDL uptake as well as intracellular lipid accumulation.In addition,Hcy upregulated methyltransferase like 3(METTL3)expression,and the tendency of Hcy to promote macrophage M1 polarization and foaminess was markedly reduced after inhibition or knockdown of METTL3 expression.Conclusions Hcy significantly promotes macrophage M1 polarization and foaminess,an effectthat may be attenuated by METTL3 silencing.

Objective:To investigate the effects of dual vascularized tissue-engineered bone constructed by vascular bundles and endothelial progenitor cells (EPCs) on repair of large bone defects and vascular regeneration.Methods:EPCs were seeded on the demineralized bone matrix (DBM) scaffolds and cultured for 6 days. The attachment and morphology of EPCs on DBM scaffolds were observed by electron microscopy. Next, the radial artery was implanted into a vascular groove opened inside the DBM-EPCs composite scaffolds. Finally, models of a large segmental bone defect were constructed using the radii from 18 New Zealand white rabbits. The rabbits were randomly divided into 4 groups using a simple random sampling method: DBM group, DBM+EPCs group, DBM+vascular bundle group, and DBM+EPCs+vascular bundle group. The DBM group and DBM+EPCs group shared the same rabbits so that transplantations were conducted into the left and right forearms respectively; the DBM+vascular bundle group and DBM+EPCs+vascular bundle group also shared the same rabbits so that transplantations were conducted into the left and right forearms respectively. Consequently, there were 9 experimental sites in each group. X-ray examination and gross morphological observation were performed to evaluate the bone regeneration in the experimental rabbits in each group at 4, 8, and 12 weeks after surgery, and CD31 immunofluorescence staining was used to evaluate the vascular regeneration. Micro-CT was used to analyze bone tissue parameters and reconstruct the three-dimensional structures of the defects site at 12 weeks after surgery.Results:Compared with the DBM, DBM+EPCs and DBM+vascular bundle groups, the DBM+EPCs+vascular bundle group showed new bone tissue crawling on the scaffold surface at 4 weeks after surgery, almost complete healing of the bone defect area at 8 weeks, and forming of a complete and dense bone bridge and appearance of a bone marrow cavity at 12 weeks. Micro-CT data at 12 weeks after surgery showed regular arrangement of the trabeculae, significantly improved mineralization, and increased thickness of the bone cortex in the DBM+EPCs+vascular bundle group. Additionally, in the DBM+EPCs+vascular bundle group, the number of microvessels was significantly higher than that in the other groups at 4, 8, 12 weeks after surgery ( P<0.05), and the angiogenesis and bone tissue regeneration were particularly prominent at 12 weeks after surgery. The number of CD31 cells in the DBM+EPCs+vascular bundle group increased significantly more than that in the DBM, DBM+EPCs and DBM+vascular bundle groups ( P<0.05). Conclusion:As the dual vascularized tissue-engineered bone constructed by vascular bundles and EPCs can significantly promote bone tissue regeneration and angiogenesis, it may be a potential therapeutic strategy for repair of large bone defects.

Cognitive dysfunction caused by blast traumatic brain injury (bTBI) is a serious neurological disease with high incidence, serious condition and poor prognosis. bTBI can lead to a series of symptoms such as short-term memory loss, inattention or multi-tasking difficulties. In severe cases, bTBI can develop into Alzheimer′s disease, which has a great impact on patients′ normal work and life. At present, researches on cognitive dysfunction caused by bTBI mainly involve model construction, pathogenesis, pathophysiological changes, diagnosis and treatment, etc., and the molecular mechanism of its occurrence remains to be further studied. Under normal physiological conditions, the release of excitatory and inhibitory neurotransmitters, the release and uptake of Ca 2+, oxidation and antioxidant systems, and the promotion and inhibition of apoptosis are in a dynamic balance. bTBI disturbs the balance, which will lead to the damage of nerve cells at the molecular level, thus resulting in the occurrence of cognitive dysfunction. To this end, the authors summarized the aspects of excitatory toxicity and Ca 2+homeostasis disorder, oxidative stress, inflammation and edema, apoptosis, etc., and reviewed the research progress on the molecular mechanism of cognitive dysfunction caused by bTBI, so as to provide a reference for the treatment and rehabilitation of cognitive dysfunction in patients with bTBI.

Objective To investigate the effects of high-definition transcranial direct current stimulation(HD-tDCS)on the modulation of the H-reflex and M-wave during ankle dorsiflexion-plantar flexion fatigue tasks to provide direction for the application of HD-tDCS in mitigating neuromuscular fatigue.Methods Twenty healthy young male participants were recruited and randomly assigned to either the real stimulation or sham stimulation group,with 10 participants in each group.The intervention consisted of a 5-day single-blind HD-tDCS application(duration:20 min;intensity:2 mA;target:Cz).Baseline measurements of the H-reflex and M-wave under resting conditions,M-wave during maximal voluntary isometric contraction(MVIC)of the dorsiflexor muscle,and MVIC torque of the dorsiflexor and plantar flexor muscles were obtained.An ankle dorsiflexion fatigue task was performed to determine the time to achieve fatigue for the task.The same fatigue task was repeated and evaluated one day after the intervention.A repeated-measures two-factor(stimulation condition x pre/post fatigue)analysis of variance(ANOVA)was used to analyze the effects of independent variables on the mechanical properties of the muscles and α-motoneuron conduction characteristics.Results After fatigue,voluntary activation(VA),maximal H-reflex(Hmax),maximal M-wave(Mmax),and dorsiflexor and plantar flexor MVIC torques in both groups were significantly reduced compared with pre-fatigue levels(P＜0.05).However,compared to the real stimulation group,the sham stimulation group showed a more significant decline in VA and plantar flexor MVIC torque(P＜0.05).Conclusions A continuous 5-day HD-tDCS intervention can effectively increase α-motoneuron activity at the spinal segment.It can also exert an inhibitory effect on reducing information transmission capacity at the peripheral neuromuscular junction under the ankle dorsi-plantarflexion fatigue task.

Background and purpose:In 2016 the National Cancer Institute(NCI)decided stopping to use NCI-60 cell lines for drug screening,suggesting that tumor cell lines were losing their value as a tool for drug discovery and basic research.The reason for NCI-60 cells'retirement'was that the preclinical studies based on traditional cellular and animal models did not obtain the corresponding expected efficacy in clinical trials.Since the major cancer behaviors,such as proliferation and metastasis,are fundamentally altered with long-term culture,the tumor cell lines are not representative of the characteristics of cancer in patients.Currently,scientists hope to create a new cancer model that are derived from fresh patient samples and tagged with details about their clinical past.Our purpose was to create patient-derived breast cancer primary cell lines as new cancer model for drug screening and basic research.Methods:Breast cancer tissues were collected in the Department of Breast Surgery,Affiliated Hospital of Guizhou Medical University.The collection of tumor tissue samples was approved by the Ethics Committee of the Affiliated Hospital of Guizhou Medical University(approval number:2022 ethics No.313),and the collection and use of tumor tissues complied with the Declaration of Helsinki.The primary breast cancer cell lines were isolated from the patient's breast cancer tissues and cultured in BCMI medium.After the cells proliferated,the media were replaced with DEME medium.Cell line STR genotyping was done to determine cell-specific genetic markers and identification.Clone formation assay and transplantation assay were done to analyze the ability of breast cancer primary cell lines to form tumors.Results:We created 6 primary breast cancer cell lines.The 6 primary breast cancer cell lines from the patients were tagged with the definitively clinicopathological features,clinical diagnosis,therapeutic regimens,clinical effectiveness and prognostic outcomes.The STR genotyping assays identified the genetic markers and determined the identities of the 6 primary breast cancer cell lines.Clone formation assays and transplantation assay showed that the proliferative capacities of the patient-derived primary breast cancer cell lines were significantly greater compared with the conventional breast cancer cell lines.Conclusion:We created a panel of 6 patient-derived primary breast cancer cell lines as new cancer model for drug screening and basic research in breast cancer.