Osteoporosis(OP) is a senile bone disease characterized by an imbalance between bone remodeling and bone formation. Targeting pathogenesis of kidney deficiency, spleen deficiency, and blood stasis, Bushen Jianpi Huoxue Decoction has a significant effect on the treatment of OP by tonifying kidney, invigorating spleen, and activating blood circulation. MicroRNA(miRNA) and the anti-apoptotic protein B-cell lymphoma-2-like protein 1(BCL2L1) are closely related to bone cell metabolism. Therefore, in this study, the binding of miR-140-5p to BCL2L1 was detected by dual luciferase assay and polymerase chain reaction(PCR). After silencing or overexpressing miR-140-5p, the apoptosis, autophagy, and osteogenic function of human fetal osteoblast cell line 1.19(HFOB1.19) were observed by flow cytometry and Western blot. Bushen Jianpi Huoxue Decoction-containing serum was prepared by intragastric administration of Bushen Jianpi Huoxue Decoction in rats. Different concentrations of Bushen Jianpi Huoxue Decoction-containing serum were used to treat HFOB1.19 with or without miR-140-5p mimic. The expression of osteogenic proteins in each group was observed, and the role of miR-140-5p/BCL2L1 in apoptosis and autophagy of HFOB1.19 was studied, along with the effect of Bushen Jianpi Huoxue Decoction on these processes. As indicated by the dual luciferase assay, miR-140-5p bound to BCL2L1. Flow cytometry and Western blot showed that miR-140-5p promoted apoptosis and inhibited autophagy in HFOB1.19. After intervention with high, medium, and low doses of Bushen Jianpi Huoxue Decoction-medicated serum, compared with the miR-140-5p NC group, the expression of osteocalcin(OCN), osteopontin(OPN), Runt-related transcription factor 2(RUNX2), and transforming growth factor beta 1(TGF-β1) decreased in the miR-140-5p mimic group, while the expression of bone morphogenetic protein 2(BMP2) showed no significant difference under high-dose intervention. Therefore, miR-140-5p/BCL2L1 can promote apoptosis and inhibit autophagy in HFOB1.19. Bushen Jianpi Huoxue Decoction can affect the osteogenic effect of miR-140-5p through BMP2.
MicroRNAs/metabolism* ; Autophagy/drug effects* ; Apoptosis/drug effects* ; Humans ; Drugs, Chinese Herbal/administration & dosage* ; Animals ; Cell Line ; bcl-X Protein/metabolism* ; Osteoblasts/metabolism* ; Rats ; Osteoporosis/physiopathology* ; Male ; Rats, Sprague-Dawley ; Osteogenesis/drug effects*

This paper aimed to explore the effects of Duzhong Decoction(DZD)-containing serum on the proliferation and osteoblast differentiation of MC3T3-E1 cells through L-type voltage-gated calcium channels(L-VGCCs). L-VGCCs inhibitors, nifedipine and verapamil, were used to block L-VGCCs in osteoblasts. MC3T3-E1 cells were divided into a control group, a low-dose DZD-containing serum(L-DZD) group, a medium-dose DZD-containing serum(M-DZD) group, a high-dose DZD-containing serum(H-DZD) group, a nifedipine group, a H-DZD + nifedipine group, verapamil group, and a H-DZD + verapamil group. The CCK-8 method was used for cell proliferation analysis, alkaline phosphatase(ALP) assay kits for intracellular ALP activity measurement, Western blot for protein expression level in cells, real-time fluorescence quantitative PCR technology for intracellular mRNA expression level determination, fluorescence spectrophotometer for free Ca~(2+) concentration determination in osteoblasts, and alizarin red staining(ARS) for mineralized nodule formation in osteoblasts. The experimental results show that compared to the control group, DZD groups can promote MC3T3-E1 cell proliferation, ALP activity, and mineralized nodule formation, increase intracellular Ca~(2+) concentrations, and upregulate the protein expression of bone morphogenetic protein 2(BMP2), collagen Ⅰ(COL1), α2 subunit protein of L-VGCCs(L-VGCCα2), and the mRNA expression of Runt-related transcription factor 2(RUNX2), and BMP2. After blocking L-VGCCs with nifedipine and verapamil, the intervention effects of DZD-containing serum were inhibited to varying degrees. Both nifedipine and verapamil could inhibit ALP activity, reduce mineralized nodule areas, and downregulate the expression of bone formation-related proteins. Moreover, the effects of DZD-containing serum on increasing MC3T3-E1 cell proliferation, osteoblast differentiation, and Ca~(2+) concentrations, upregulating the mRNA expression of osteoprotegerin(OPG) and protein expression of phosphorylated protein kinase B(p-Akt) and phosphorylated forkhead box protein O1(p-FOXO1), and upregulating phosphatase and tensin homolog(PTEN) expression were reversed by nifedipine. The results indicate that DZD-containing serum can increase the Ca~(2+) concentration in MC3T3-E1 cells to promote bone formation, which may be mediated by L-VGCCs and the PTEN/Akt/FoxO1 signaling pathway, providing a new perspective on the mechanism of DZD in treating osteoporosis.
Animals ; Osteoblasts/metabolism* ; Cell Proliferation/drug effects* ; Cell Differentiation/drug effects* ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Calcium Channels, L-Type/genetics* ; Alkaline Phosphatase/genetics* ; Serum/chemistry* ; Cell Line ; Osteogenesis/drug effects* ; Bone Morphogenetic Protein 2/genetics*

This study aims to explore the mechanism of Buyang Huanwu Decoction(BHD) in promoting angiogenesis after oxygen-glucose deprivation/reoxygenation(OGD/R) of mouse brain microvascular endothelial cell line(brain-derived Endothelial cells.3, bEnd.3) based on the caveolin-1(Cav1)/Yes-associated protein 1(YAP1)/hypoxia-inducible factor-1α(HIF-1α) signaling pathway. Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to analyze the blood components of BHD. The cell counting kit-8(CCK-8) method was used to detect the optimal intervention concentration of drug-containing serum of BHD after OGD/R injury of bEnd.3. The lentiviral transfection method was used to construct a Cav1 silent stable strain, and Western blot and polymerase chain reaction(PCR) methods were used to verify the silencing efficiency. The control bEnd.3 cells were divided into a normal group(sh-NC control group), an OGD/R model + blank serum group(sh-NC OGD/R group), and an OGD/R model + drug-containing serum group(sh-NC BHD group). Cav1 silent cells were divided into an OGD/R model + blank serum group(sh-Cav1 OGD/R group) and an OGD/R model + drug-containing serum group(sh-Cav1 BHD group). The cell survival rate was detected by the CCK-8 method. The cell migration ability was detected by a cell migration assay. The lumen formation ability was detected by an angiogenesis assay. The apoptosis rate was detected by flow cytometry, and the expression of YAP1/HIF-1α signaling pathway-related proteins in each group was detected by Western blot. Finally, co-immunoprecipitation was used to verify the interaction between YAP1 and HIF-1α. The results showed astragaloside Ⅳ, formononetin, ferulic acid, and albiflorin in BHD can all enter the blood. The drug-containing serum of BHD at a mass fraction of 10% may be the optimal intervention concentration for OGD/R-induced injury of bEnd.3 cells. Compared with the sh-NC control group, the sh-NC OGD/R group showed significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, significantly increased cell apoptotic rate, significantly lowered phosphorylation level of YAP1 at S127 site, and significantly elevated nuclear displacement level of YAP1 and protein expression of HIF-1α, vascular endothelial growth factor(VEGF), and vascular endothelial growth factor receptor 2(VEGFR2). Compared with the same type of OGD/R group, the sh-NC BHD group and sh-Cav1 BHD group had significantly increased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly decreased cell apoptotic rate, a further decreased phosphorylation level of YAP1 at S127 site, and significantly increased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. Compared with the sh-NC OGD/R group, the sh-Cav1 OGD/R group exhibited significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly increased cell apoptotic rate, a significantly increased phosphorylation level of YAP1 at S127 site, and significantly decreased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. Compared with the sh-NC BHD group, the sh-Cav1 BHD group showed significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly increased cell apoptotic rate, a significantly increased phosphorylation level of YAP1 at the S127 site, and significantly decreased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. YAP1 protein was present in the protein complex precipitated by the HIF-1α antibody, and HIF-1α protein was also present in the protein complex precipitated by the YAP1 antibody. The results confirmed that the drug-containing serum of BHD can increase the activity of YAP1/HIF-1α pathway in bEnd.3 cells damaged by OGD/R through Cav1 and promote angiogenesis in vitro.
Drugs, Chinese Herbal/pharmacology* ; Animals ; Mice ; Signal Transduction/drug effects* ; Glucose/metabolism* ; Caveolin 1/genetics* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; YAP-Signaling Proteins ; Oxygen/metabolism* ; Endothelial Cells/metabolism* ; Cell Line ; Adaptor Proteins, Signal Transducing/genetics* ; Neovascularization, Physiologic/drug effects* ; Cell Hypoxia/drug effects* ; Angiogenesis

BACKGROUND: The use of inserted sham acupuncture as a placebo in randomized controlled trials (RCTs) is controversial, because it may produce specific effects that cause an underestimation of the effect of acupuncture treatment. OBJECTIVE: This systematic survey investigates the magnitude of insert-specific effects of sham acupuncture and whether they affect the estimation of acupuncture treatment effects. SEARCH STRATEGY: PubMed, Embase and Cochrane Central Register of Controlled Trials were searched to identify acupuncture RCTs from their inception until December 2022. INCLUSION CRITERIA: RCTs that evaluated the effects of acupuncture compared to sham acupuncture and no treatment. DATA EXTRACTION AND ANALYSIS: The total effect measured for an acupuncture treatment group in RCTs were divided into three components, including the natural history and/or regression to the mean effect (controlled for no-treatment group), the placebo effect, and the specific effect of acupuncture. The first two constituted the contextual effect of acupuncture, which is mimicked by a sham acupuncture treatment group. The proportion of acupuncture total effect size was considered to be 1. The proportion of natural history and/or regression to the mean effect (PNE) and proportional contextual effect (PCE) of included RCTs were pooled using meta-analyses with a random-effect model. The proportion of acupuncture placebo effect was the difference between PCE and PNE in RCTs with non-inserted sham acupuncture. The proportion of insert-specific effect of sham acupuncture (PIES) was obtained by subtracting the proportion of acupuncture placebo effect and PNE from PCE in RCTs with inserted sham acupuncture. The impact of PIES on the estimation of acupuncture's treatment effect was evaluated by quantifying the percentage of RCTs that the effect of outcome changed from no statistical difference to statistical difference after removing PIES in the included studies, and the impact of PIES was externally validated in other acupuncture RCTs with an inserted sham acupuncture group that were not used to calculate PIES. RESULTS: This analysis included 32 studies with 5492 patients. The overall PNE was 0.335 (95% confidence interval [CI], 0.255-0.415) and the PCE of acupuncture was 0.639 (95% CI, 0.567-0.710) of acupuncture's total effect. The proportional contribution of the placebo effect to acupuncture's total effect was 0.191, and the PIES was 0.189. When we modeled the exclusion of the insert-specific effect of sham acupuncture, the acupuncture treatment effect changed from no difference to a significant difference in 45.45% of the included RCTs, and in 40.91% of the external validated RCTs. CONCLUSION The insert-specific effect of sham acupuncture in RCTs represents 18.90% of acupuncture's total effect and significantly affects the evaluation of the acupuncture treatment effect. More than 40% of RCTs that used inserted sham acupuncture would draw different conclusions if the PIES had been controlled for. Considering the impact of the insert-specific effect of sham acupuncture, caution should be taken when using inserted sham acupuncture placebos in RCTs. Please cite this article as: Luo XC, Liu JL, Yao MH, Chen YM, Fan AY, Liang FR, Zhao JP, Zhao L, Zhou X, Zhong XY, Yang JH, Li B, Zhang Y, Sun X, Li L. Specific effect of inserted sham acupuncture and its impact on the estimation of acupuncture treatment effect in randomized controlled trials: A systematic survey. J Integr Med. 2025; 23(6):630-640.
Acupuncture Therapy/methods* ; Humans ; Randomized Controlled Trials as Topic ; Placebo Effect ; Placebos ; Treatment Outcome

OBJECTIVE: To evaluate the clinical efficacy and safety of Yangxue Qingnao Pills (YXQNP) combined with amlodipine in treating patients with grade 1 hypertension. METHODS: This is a multicenter, randomized, double-blind, and placebo-controlled study. Adult patients with grade 1 hypertension of blood deficiency and Gan (Liver)-yang hyperactivity syndrome were randomly divided into the treatment or the control groups at a 1:1 ratio. The treatment group received YXQNP and amlodipine besylate, while the control group received YXQNP's placebo and amlodipine besylate. The treatment duration lasted for 180 days. Outcomes assessed included changes in blood pressure, Chinese medicine (CM) syndrome scores, symptoms and target organ functions before and after treatment in both groups. Additionally, adverse events, such as nausea, vomiting, rash, itching, and diarrhea, were recorded in both groups. RESULTS: A total of 662 subjects were enrolled, of whom 608 (91.8%) completed the trial (306 in the treatment and 302 in the control groups). After 180 days of treatment, the standard deviations and coefficients of variation of systolic and diastolic blood pressure levels were lower in the treatment group compared with the control group. The improvement rates of dizziness, headache, insomnia, and waist soreness were significantly higher in the treatment group compared with the control group (P<0.05). After 30 days of treatment, the overall therapeutic effects on CM clinical syndromes were significantly increased in the treatment group as compared with the control group (P<0.05). After 180 days of treatment, brachial-ankle pulse wave velocity, ankle brachial index and albumin-to-creatinine ratio were improved in both groups, with no statistically significant differences (P>0.05). No serious treatment-related adverse events occurred during the study period. CONCLUSIONS Combination therapy of YXQNP with amlodipine significantly improved symptoms such as dizziness and headache, reduced blood pressure variability, and showed a trend toward lowering urinary microalbumin in hypertensive patients. These findings suggest that this regimen has good clinical efficacy and safety. (Registration No. ChiCTR1900022470).
Humans ; Amlodipine/adverse effects* ; Drugs, Chinese Herbal/adverse effects* ; Male ; Female ; Hypertension/complications* ; Middle Aged ; Treatment Outcome ; Drug Therapy, Combination ; Adult ; Blood Pressure/drug effects* ; Double-Blind Method ; Aged ; Antihypertensive Agents/adverse effects*

OBJECTIVE: To evaluate the efficacy and safety of Shexiang Tongxin Dropping Pill (STDP) in treating stable angina patients with phlegm-heat and blood-stasis syndrome by exercise duration and metabolic equivalents. METHODS: This multicenter, randomized, double-blind, placebo-controlled clinical trial enrolled stable angina patients with phlegm-heat and blood-stasis syndrome from 22 hospitals. They were randomized 1:1 to STDP (35 mg/pill, 6 pills per day) or placebo for 56 days. The primary outcome was the exercise duration and metabolic equivalents (METs) assessed by the standard Bruce exercise treadmill test after 56 days of treatment. The secondary outcomes included the total angina symptom score, Chinese medicine (CM) symptom scores, Seattle Angina Questionnaire (SAQ) scores, changes in ST-T on electrocardiogram and adverse events (AEs). RESULTS: This trial enrolled 309 patients, including 155 and 154 in the STDP and placebo groups, respectively. STDP significantly prolonged exercise duration with an increase of 51.0 s, compared to a decrease of 12.0 s with placebo (change rate: -11.1% vs. 3.2%, P<0.01). The increase in METs was significantly greater in the STDP group than in the placebo group (change: -0.4 vs. 0.0, change rate: -5.0% vs. 0.0%, P<0.01). The improvement of total angina symptom scores (25.0% vs. 0.0%), CM symptom scores (38.7% vs. 11.8%), reduction of nitroglycerin consumption (100.0% vs. 11.3%), and all domains of SAQ, were significantly greater with STDP than placebo (all P<0.01). The changes in Q-T intervals at 28 and 56 days from baseline were similar between the two groups (both P>0.05). Twenty-five participants (16.3%) with STDP and 16 (10.5%) with placebo experienced AEs (P=0.131), with no serious AEs observed. CONCLUSION STDP could improve exercise tolerance in patients with stable angina and phlegm-heat and blood stasis syndrome, with a favorable safety profile. (Registration No. ChiCTR-IPR-15006020).
Humans ; Double-Blind Method ; Drugs, Chinese Herbal/adverse effects* ; Male ; Female ; Middle Aged ; Angina, Stable/physiopathology* ; Aged ; Syndrome ; Treatment Outcome ; Placebos ; Tablets

Learning-associated functional plasticity at hippocampal synapses remains largely unexplored. Here, in a single session of reward-based trace conditioning, we examine learning-induced synaptic plasticity in the dorsal CA1 hippocampus (dCA1). Local field-potential recording combined with selective optogenetic inhibition first revealed an increase of dCA1 synaptic responses to the conditioned stimulus (CS) induced during conditioning at both Schaffer collaterals to the stratum radiatum (Rad) and temporoammonic input to the lacunosum moleculare (LMol). At these dCA1 inputs, synaptic potentiation of CS-responding excitatory synapses was further demonstrated by locally blocking NMDA receptors during conditioning and whole-cell recording sensory-evoked synaptic responses in dCA1 neurons from naive animals. An overall similar time course of the induction of synaptic potentiation was found in the Rad and LMol by multiple-site recording; this emerged later and saturated earlier than conditioned behavioral responses. Our experiments demonstrate a cued memory-associated dCA1 synaptic plasticity induced at both Schaffer collaterals and temporoammonic pathways.
Animals ; CA1 Region, Hippocampal/physiology* ; Male ; Association Learning/physiology* ; Neuronal Plasticity/physiology* ; Cues ; Memory/physiology* ; Synapses/physiology* ; Conditioning, Classical/physiology* ; Excitatory Postsynaptic Potentials/physiology* ; Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors* ; Rats ; Optogenetics

Purpose: Cancer has become a significant major public health concern, making the discovery of new cancer markers or therapeutic targets exceptionally important. Elevated expression of tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) expression has been observed in certain types of cancer. This project aims to investigate the function of TNFRSF12A in tumors and the underlying mechanisms. Materials and Methods: Various websites were utilized for conducting the bioinformatics analysis. Tumor cell lines with stable knockdown or overexpression of TNFRSF12A were established for cell phenotyping experiments and subcutaneous tumorigenesis in BALB/c mice. RNA-seq was employed to investigate the mechanism of TNFRSF12A. Results: TNFRSF12A was upregulated in the majority of cancers and associated with a poor prognosis. Knockdown TNFRSF12A hindered the colorectal cancer progression, while overexpression facilitated malignancy both in vitro and in vivo. TNFRSF12A overexpression led to increased nuclear factor кB (NF-κB) signaling and significant upregulation of baculoviral IAP repeat containing 3 (BIRC3), a transcription target of the NF-κB member RELA, and it was experimentally confirmed to be a critical downstream factor of TNFRSF12A. Therefore, we speculated the existence of a TNFRSF12A/RELA/BIRC3 regulatory axis in colorectal cancer. Conclusion TNFRSF12A is upregulated in various cancer types and associated with a poor prognosis. In colorectal cancer, elevated TNFRSF12A expression promotes tumor growth, potentially through the TNFRSF12A/RELA/BIRC3 regulatory axis.