Objective To investigate the effect of miR-129-1-3p on the osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs)and its potential mechanism.Methods Negative control group,the miR-129-1-3p mimic group,the miR-129-1-3p inhibitor group and the corresponding negative control were constructed and transfected into hBMSCs.The formation of calcium-mineralized nodules was observed by alkaline phosphatase staining and alizarin red S staining.The expression levels of miR-129-1-3p and osteogenic differentiation markers were detected by qRT-PCR.Western blot detected the protein expressions of bone mor-phogenetic protein 2(BMP2),SMAD1 and p-SMAD1.Results After transfection,the expression level of miR-129-1-3p in mimic group was significantly increased(P＜0.05),the number of mineralized nodules was significantly decreased,and the expression levels ofBMP2,Runt-related transcription factor 2(RUNX2),osteocalcin(OCN)mRNA were significantly down-regulated(P＜0.05).BMP2 and p-SMAD1 protein were also significantly down-regulated(P＜0.05)compared with Mimic-NC group.The expression levels of BMP2,RUNX2,OCN mRNA were significantly up-regulated in inhibitor group(P＜0.05)compared with Inhibitor-NC group.BMP2 and p-SMAD1 protein were significantly up-regulated in inhibitor group(P＜0.05).Conclusion miR-129-1-3p can inhibit the osteo-genic differentiation of human bone marrow mesenchymal stem cells by suppressing BMP2/SMAD1 signaling pathway.

Objective To investigate the effect of miR-129-1-3p on the osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs)and its potential mechanism.Methods Negative control group,the miR-129-1-3p mimic group,the miR-129-1-3p inhibitor group and the corresponding negative control were constructed and transfected into hBMSCs.The formation of calcium-mineralized nodules was observed by alkaline phosphatase staining and alizarin red S staining.The expression levels of miR-129-1-3p and osteogenic differentiation markers were detected by qRT-PCR.Western blot detected the protein expressions of bone mor-phogenetic protein 2(BMP2),SMAD1 and p-SMAD1.Results After transfection,the expression level of miR-129-1-3p in mimic group was significantly increased(P＜0.05),the number of mineralized nodules was significantly decreased,and the expression levels ofBMP2,Runt-related transcription factor 2(RUNX2),osteocalcin(OCN)mRNA were significantly down-regulated(P＜0.05).BMP2 and p-SMAD1 protein were also significantly down-regulated(P＜0.05)compared with Mimic-NC group.The expression levels of BMP2,RUNX2,OCN mRNA were significantly up-regulated in inhibitor group(P＜0.05)compared with Inhibitor-NC group.BMP2 and p-SMAD1 protein were significantly up-regulated in inhibitor group(P＜0.05).Conclusion miR-129-1-3p can inhibit the osteo-genic differentiation of human bone marrow mesenchymal stem cells by suppressing BMP2/SMAD1 signaling pathway.

ObjectiveTo identify the pharmacodynamic material basis of Ruyi Zhenbaowan in relieving neuropathic pain by integrating the calculation of biological network proximity and pharmacokinetic characterization. MethodThe interaction network of "drug candidate target-related gene of disease" was constructed by Cytoscape 3.8.2， and the average shortest path value of each drug putative target acting on neuropathic pain-related genes in this network was calculated by Pesca 3.8.0 tool so as to evaluate the network proximity between them， and screen prescription candidate targets with strong intervention efficiency and their corresponding potential effect components. After that， plasma and cerebrospinal fluid samples were collected from rats after administration of Ruyi Zhenbaowan at set time points， and the contents of potential effect components in samples was quantified by ultra performance liquid chromatography-quadrupole-ion trap mass spectrometry（UPLC-Q-TRAP/MS）， and drug concentration-time curves were plotted， then the pharmacokinetic parameters were calculated by DAS 2.1.1. ResultBy evaluating the network proximity between candidate targets and neuropathic pain-related genes in the interaction network， a total of 40 putative targets of Ruyi Zhenbaowan with strong intervention effects on neuropathic pain-related genes， such as estrogen receptor 1（ESR1）， cyclic adenosine monophosphate（cAMP）-dependent protein kinase catalytic subunit alpha（PRKACA） and protein kinase B1 （Akt1）， and 10 corresponding potential effect components， such as glycyrrhizic acid and betulinic acid， were obtained. Pharmacokinetic characterization showed that among the 10 potential effect components， gallic acid， apigenin-7-O-glucuronide， glycyrrhizic acid and apigenin were well absorbed and metabolized in plasma and cerebrospinal fluid， with long onset time and good bioavailability. ConclusionFrom the perspective of efficacy-target-constituent-pharmacokinetic， this study analyzes the main effective materials of Ruyi Zhenbaowan， such as glycyrrhizic acid， gallic acid， apigenin-7-O-glucuronide and apigenin， which have a high exposure in plasma or cerebrospinal fluid and have a strong intervention effect on neuropathic pain. The related results provide reliable experimental evidences for clarifying the material basis and developing quality standards of Ruyi Zhenbaowan.

ObjectiveTo identify the chemical constituents of Ruyi Zhenbaowan in vitro and in vivo. MethodThe chemical constituents of Ruyi Zhenbaowan were identified based on UHPLC-Q Exactive Orbitrap HRMS. A total of 12 male SD rats were randomized into two groups： control （pure water） and Ruyi Zhenbaowan （1.8 g·kg^-1）. The rats were administrated with the suspension of Ruyi Zhenbaowan or pure water by gavage. After 1.5 h， the plasma and cerebrospinal fluid were collected. Chromatographic separation was performed on a Waters ACQUITY UPLC BEH C₁₈ column （2.1 mm × 150 mm， 1.7 μm） with a mixture of 0.1% formic acid aqueous solution （A） and acetonitrile （B） as the mobile phase. Gradient elution was carried out according to the procedure of 0~15 min，97%~80%A；15~30 min ，80%~60%A；30~40 min，60%~30%A；40~45 min，30%~5%A. The ion source was electrospray ionization， and scan range was m/z 100-1 500. The prototype components and the components in the plasma and cerebrospinal fluid were analyzed qualitatively by scanning in positive and negative ion modes and identified by comparison with the data in published literature and the information of standard substances. ResultA total of 126 chemical constituents were identified from the 80% methanol solution of Ruyi Zhenbaowan， and 14 and 7 prototype constituents were detected in the plasma and the cerebrospinal fluid， respectively. In addition， the fragmentation rules of apigenin， apigenin-7-O-glucuronide， galangin， liquiritin， piperine， glycyrrhizic acid， eugenol， gallic acid， and cholic acid were deduced. ConclusionThis study achieved rapid multicomponent characterization and identification of Ruyi Zhenbaowan in vitro and in vivo， providing theoretical support for exploring active substances and performing quality control.l.

BACKGROUND:Alzheimer's disease is a degenerative neurological disorder characterized primarily by cognitive impairment.Acupuncture is a kind of traditional Chinese medicine therapy for treating Alzheimer's disease,but its mechanism is not yet clear. OBJECTIVE:To observe the effects of electroacupuncture with"Zhi San Zhen"on the Notch signaling pathway,β-amyloid protein(Aβ)and synaptic plasticity in 5xFAD mice. METHODS:Sixteen male,6-month-old 5xFAD mice,SPF-grade,were randomly divided into the electroacupuncture with"Zhi San Zhen"group(electroacupuncture group)and the model group,with eight mice in each group.Eight SPF-grade,male,6-month-old C57BL/6 mice were used as the wild control(wild)group.The electroacupuncture group received electroacupuncture with"Zhi San Zhen"intervention,5 times a week for 4 consecutive weeks.The model group and the wild group did not receive electroacupuncture intervention.The Morris water maze was used to preliminarily assess their learning and memory abilities.Thioflavin S staining was performed to detect Aβ plaque deposition.Western blot and real-time quantitative polymerase chain reaction(RT-qPCR)were used to measure the expression levels of transmembrane receptor protein Notch-1,Notch 1 intracellular domain(NICD),hairy and enhancer of split 1(Hes 1),hairy and enhancer of split 5(Hes 5),synaptophysin(SYN),postsynaptic density protein-95(PSD-95),and Aβ. RESULTS AND CONCLUSION:Compared with the model group,the wild group and the electroacupuncture group showed shortened escape latency,increased platform crossing times,and longer target quadrant dwell time(P<0.05).Compared with the wild group,the model group had significantly increased deposition of Aβ plaques,while electroacupuncture with"Zhi San Zhen"inhibited the deposition of Aβ plaques in the hippocampus of 5xFAD mice(P<0.05).Compared with the wild group,the model group had decreased mRNA levels of SYN,PSD-95,Notch 1,NICD,Hes 1,and Hes 5 in the hippocampal tissue of mice,and increased mRNA levels of Aβ(P<0.05).Electroacupuncture with"Zhi San Zhen"increased the mRNA levels of SYN,PSD-95,Notch 1,NICD,Hes 1,and Hes 5 in the hippocampal tissue,and decreased the mRNA level of Aβ(P<0.05).Compared with the Wild group,the model group had decreased protein expression levels of SYN,PSD-95,Notch 1,NICD,Hes 1,and Hes 5 in the hippocampal tissue of mice,and increased protein expression levels of Aβ(P<0.05).Electroacupuncture with"Zhi San Zhen"upregulated the protein expression levels of SYN,PSD-95,Notch 1,NICD,Hes 1,and Hes 5,and inhibited the protein expression of Aβ(P<0.05).To conclude,electroacupuncture with"Zhi San Zhen"can improve the learning and memory abilities of 5xFAD mice,possibly by inhibiting the deposition of Aβ protein and activating the Notch signaling pathway in the hippocampus to enhance synaptic plasticity.

Objective To establish the animal model of comorbidity of knee osteoarthritis(KOA)and hypertension with pattern of liver and kidney deficiency and evaluate its characteristics of comorbidity and pattern.Methods Wistar-Kyoto(WKY)rats and spontaneously hypertensive rats(SHR)were assigned to the WKY control group(control group),hypertension combined with KOA sham-operation group(sham-operation group),hypertension combined with KOA group(model group),hypertension combined with KOA and liver-kidney deficiency pattern group(LKD group).The animal model of KOA combined with hypertension was prepared by anterior cruciate ligament transection(ACLT)in spontaneously hypertensive rats.ACLT combined with intramuscular injection of hydrocortisone was performed to prepare an animal model of KOA with liver-kidney deficiency(LKD)pattern type,combined with hypertension.Then,the related indexes of LKD syndrome were detected in turn,including the contents of thyroid stimulating hormone(TSH),testosterone(T),corticosterone(CORT),adrenocorticotropic hormone(ACTH)in serum,and enzyme activities of alanine transaminase(ALT),aspartate transaminase(AST),and alkaline phosphatase(ALP)in serum,the mass ratio of liver,kidney,spleen,thymus to the brain,body weight,anal temperature,activity situation,and emotion.Systolic blood pressure,diastolic blood pressure,and other blood pressure-related indices were also detected.The levels of serum tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β),plantar mechanical pain sensitivity threshold,weight difference score of both hind limbs,hind limb joint swelling,and quadruped gait parameters were also measured.Furthermore,hematoxylin-eosin,safranine-fast green,and Masson staining were performed to observe pathological changes,cartilage degeneration,and bone destruction of the knee joint,and the microstructure parameters of the tibia were detected by Micro-CT imaging.Results Compared to the model group,the contents of serum TSH,ACTH,T,CORT and the mass ratio of the kidney,spleen,and thymus to the brain in the LKD group decreased(P<0.05).Compared to the control group,the systolic blood pressure and diastolic blood pressure of the other three groups increased significantly(P<0.05).Compared to the sham-operation group,serum TNF-α and IL-1β levels increased,plantar mechanical pain threshold decreased,weight difference score of both hind limbs and joint swelling of the affected limb increased(P<0.05),and gait parameters(e.g.,gait length and standing time of the affected limbs)became abnormal in the model and LKD groups.Simultaneously,the cartilage surface defect of the rat knee joint was severe,the arrangement of the surface chondrocytes was altered,the cartilage layer became thinner,the muscle fibers increased,and the cartilage ossification was severe.Furthermore,the relative volume,thickness,and number of trabeculae of the knee joint decreased significantly(P<0.05).Conclusion The rat model established in this study is consistent with the clinical characteristics of integrated traditional Chinese and Western medicine in patients with comorbidities of hypertension and KOA with liver and kidney deficiency pattern.This rat model can characterize the typical symptoms of liver and kidney deficiency pattern.It has typical pathological changes in knee cartilage and subchondral bone tissues and can maintain a stable range of high systolic and diastolic blood pressure.It also explore the scientific connotation of simultaneous treatment of different diseases in traditional Chinese medicine,revealing the therapeutic mechanism and developing new drugs.

The correspondence between prescription and syndrome is the advantage and characteristic of traditional Chinese medicine(TCM)treatment.However,the pathogenesis of clinical diseases is complex and the condition is changeable,and the clinical application is difficult to achieve the maximum effect under the existing cognition of the correspondence between prescription and syndrome.In this paper,the five categories of physiological characteristics,pathological characteristics,constitution,syndrome,and disease of the longitudinal classification of"TCM state"are introduced into the correspondence of prescription and syndrome.Under the vertical perspective of"TCM state",the theoretical connotation of the correspondence between prescription and syndrome is interpreted as"correspondence between prescription and state",namely correspondence of"prescription-physiological characteristics",correspondence of"prescription-pathological characteristics",correspondence of"prescription-constitution",correspondence of"prescription-syndrome",and correspondence of"prescription-disease".It is hoped to accurately grasp the corresponding connotation of the correspondence between prescription and syndrome,in order to deepen the clinical thinking mode of TCM.

Objective By investigating the retention and fatigue performance of polyetheretherketone(PEEK)clasps with increased proportions of clasp arm engaging the undercut,an innovative method to improve PEEK clasps'performance was proposed and verified.Methods Three groups of PEEK clasps(n=10/group)with the terminal 1/3,2/3 and whole of their retentive arms engaging the 0.75 mm undercut were designed and fabricated respectively along with a group of cobalt-chrome clasps(n=10)with their terminal 1/3 of retentive arms engaging the 0.25 mm undercut.Retentive forces were recorded initially and every 1 500 cycles for a total of 15 000 in-sertion and removal fatigue cycles.Optical scanning and software analysis were applied to evaluate the deformation of the clasps by root mean square(RMS)after fatigue test.The inner surface morphology of clasps'arms was observed by scanning electron microscopy.Re-sults PEEK clasps with the whole of their retentive arms engaging the undercut exhibited highest mean retentive force(9.24±1.78)Namong other PEKK clasps,which was slightly lower than that of cobalt-chrome clasps(11.88±2.05)N.The retentive force of each group turned out to be reduced after fatigue cycles and cobalt-chrome clasps showed the greatest reduction(38.38%).The RMS of the PEEK clasps groups were of no statistical difference(P=0.111)and were lower than that of cobalt-chrome clasps(105.47±10.82)μm.Evidence of surface abrasion was observed on all groups of clasps especially on the section that engaged the undercut.Conclusion Increasing the proportion of PEEK clasp arm engaging the undercut effectively improved the retentive force and satisfied clinical re-quirements after fatigue cycles.PEEK clasps exhibited greater retention stability than cobalt-chrome clasps.It is feasible to improve the performance of PEEK clasps by increasing the proportion of clasp arm engaging the undercut.

In order to meet the fertility needs of infertile patients as much as possible during the pandemic of COVID-19 Omicron strain, this expert group reviewed global and domestic literature, combined with the current epidemic situation and prevention and control policies, fully considered the service procedures and characteristics of assisted reproductive institutions and sperm banks in China, and then put forward specific recommendations on institutional prevention and control management, and treatment and surgical management of COVID-19 infected patients, so as to guide and standardize the related practices in assisted reproductive institutions and sperm banks. In the practical application process, assisted reproductive institutions and sperm banks in various provinces and cities should develop personalized institutional prevention and control measures and patient management procedures based on local prevention and control requirements and the actual situation of their own institutions.

In order to meet the fertility needs of infertile patients as much as possible during the pandemic of COVID-19 Omicron strain, this expert group reviewed global and domestic literature, combined with the current epidemic situation and prevention and control policies, fully considered the service procedures and characteristics of assisted reproductive institutions and sperm banks in China, and then put forward specific recommendations on institutional prevention and control management, and treatment and surgical management of COVID-19 infected patients, so as to guide and standardize the related practices in assisted reproductive institutions and sperm banks. In the practical application process, assisted reproductive institutions and sperm banks in various provinces and cities should develop personalized institutional prevention and control measures and patient management procedures based on local prevention and control requirements and the actual situation of their own institutions.