OBJECTIVE To prepare the β -cyclodextrin （ β -CD） inclusion complex with volatile oil from Qianghuo qushi qingwen granules， and to characterize and quantitatively analyze the inclusion complex. METHODS The comprehensive scores calculated by inclusion rate and inclusion compound yield were used as indicators for screening the inclusion method. The single-factor experiments and Box-Behnken response surface experiments were used to op timize the inclusion conditions， with the above comprehensive score as response value， and taking the ratio of β -CD to volatile oil， inclusion temperature and inclusion time as indexes. The volatile oil inclusion complex of Qianghuo qushi qingwen granules was prepared according to the determined optimal process， followed by validation. Ultraviolet （UV）-visible spectroscopy， thin-layer chromatography （TLC）， and microscopic imaging were also performed. Ultra-high performance liquid chromatography was used to determine the contents of perillaldehyde， pogostone and atractylodin. RESULTS The saturation aqueous solution method was adopted. The optimal inclusion process conditions were as follows： the ratio of β -CD to volatile oil was 7.5∶1， the inclusion temperature was 40 ℃， and the inclusion time was 2.2 h. In three verification experiments， the average inclusion rate was 72.32%， the average yield of inclusion compound was 74.45%， the average comprehensive score was 72.96 points， and the relative error with the predicted value （74.15 points） of the model was 1.61%. UV-visible spectroscopy， TLC and microscopic imaging showed that β -CD and volatile oil successfully formed a new inclusion complex. The average contents of perillaldehyde， pogostone and atractylodin were 4.498 2， 0.814 9， 0.905 7 mg/g， respectively， with RSDs of 0.31%， 0.56% and 0.63% （ n ＝3）. CONCLUSIONS A stable and feasible preparation process of the volatile oil inclusion complex of Qianghuo qushi qingwen granules is successfully established.

ObjectiveBased on the theory of "gut-prostate" axis， this study explored the effects and mechanisms of Zishen Tongguan formula and Cinnamomi Cortex in the formula in treating rats with chronic non-bacterial prostatitis（CNP） by detecting the levels of inflammatory factors， and the composition and structure of intestinal flora in CNP rats. MethodsEight out of 42 SD rats were randomly selected as the normal group， and the remaining rats were injected with carrageenan to prepare the CNP model. After successful modeling， 32 rats were randomly divided into the model group， Ningmitai capsule group（0.50 g·kg^-1）， Zishen Tongguan formula group（2.00 g·kg^-1）， and the Phellodendri Chinensis Cortex-Anemarrhenae Rhizoma pair group（PCC-AR group， 2.00 g·kg^-1）， with 8 rats in each group. The administered groups were given the corresponding medicinal solution by gavage， and the normal and model groups were intragastrically administered with an equal volume of normal saline， once a day for 14 consecutive days. The prostate tissues of rats were collected and subjected to hematoxylin-eosin（HE） staining and Masson staining to observe the pathological changes of the tissues in each group. Enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of related inflammatory factors in rat serum， and 16S rDNA sequencing was used to analyze the abundance and diversity changes of gut microbiota before and after administration， and species difference analysis was performed. ResultsAll the administered groups could alleviate the inflammatory symptoms of CNP rats， increase the expression levels of anti-inflammatory factors and decrease the expression levels of pro-inflammatory factors， with the most sIgnificant effect observed in the Zishen Tongguan formula group. Compared with the normal group， the expression levels of interleukin（IL）-8， hypersensitive C-reactive protein（hs-CRP）， immunoglobulin（Ig）M， secretory IgA （sIgA）， and inducible nitric oxide synthase（iNOS） were sIgnificantly increased in the model group（P<0.01）. Compared with the model group， the expression levels of the above inflammatory factors in all administered groups were significantly reduced（P<0.01）. When compared with the PCC-AR group， the Zishen Tongguan formula group showed a significant decrease in transforming growth factor（TGF）-β₁ expression level（P<0.05） and a significant increase in IgM expression level（P<0.01）. The results of gut microbiota analysis showed that， compared with the PCC-AR group， at the order level， the Zishen Tongguan formula group significantly reduced the relative abundance of conditional pathogens such as Bacteroidales， Acidaminococcales， Rhodospirillales， Clostridiales， and Elusimicrobiales（P<0.01）. And at the genus level， the Zishen Tongguan formula group significantly decreased the relative abundance of pathogenic microbiota such as Lachnospira and Bacteroides（P<0.01） and significantly increased the relative abundances of beneficial microbiota such as Ruminococcus and Lactobacillus（P<0.01）. ConclusionZishen Tongguan formula can reduce the level of harmful intestinal bacteria， increase the level of beneficial intestinal bacteria， down-regulate the expression of serum inflammatory factors， and the small amount of Cinnamomi Cortex in the formula may play a key role in the treatment of CNP with this formula.

ObjectiveBased on the theory of "gut-prostate" axis， this study explored the effects and mechanisms of Zishen Tongguan formula and Cinnamomi Cortex in the formula in treating rats with chronic non-bacterial prostatitis（CNP） by detecting the levels of inflammatory factors， and the composition and structure of intestinal flora in CNP rats. MethodsEight out of 42 SD rats were randomly selected as the normal group， and the remaining rats were injected with carrageenan to prepare the CNP model. After successful modeling， 32 rats were randomly divided into the model group， Ningmitai capsule group（0.50 g·kg^-1）， Zishen Tongguan formula group（2.00 g·kg^-1）， and the Phellodendri Chinensis Cortex-Anemarrhenae Rhizoma pair group（PCC-AR group， 2.00 g·kg^-1）， with 8 rats in each group. The administered groups were given the corresponding medicinal solution by gavage， and the normal and model groups were intragastrically administered with an equal volume of normal saline， once a day for 14 consecutive days. The prostate tissues of rats were collected and subjected to hematoxylin-eosin（HE） staining and Masson staining to observe the pathological changes of the tissues in each group. Enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of related inflammatory factors in rat serum， and 16S rDNA sequencing was used to analyze the abundance and diversity changes of gut microbiota before and after administration， and species difference analysis was performed. ResultsAll the administered groups could alleviate the inflammatory symptoms of CNP rats， increase the expression levels of anti-inflammatory factors and decrease the expression levels of pro-inflammatory factors， with the most sIgnificant effect observed in the Zishen Tongguan formula group. Compared with the normal group， the expression levels of interleukin（IL）-8， hypersensitive C-reactive protein（hs-CRP）， immunoglobulin（Ig）M， secretory IgA （sIgA）， and inducible nitric oxide synthase（iNOS） were sIgnificantly increased in the model group（P<0.01）. Compared with the model group， the expression levels of the above inflammatory factors in all administered groups were significantly reduced（P<0.01）. When compared with the PCC-AR group， the Zishen Tongguan formula group showed a significant decrease in transforming growth factor（TGF）-β₁ expression level（P<0.05） and a significant increase in IgM expression level（P<0.01）. The results of gut microbiota analysis showed that， compared with the PCC-AR group， at the order level， the Zishen Tongguan formula group significantly reduced the relative abundance of conditional pathogens such as Bacteroidales， Acidaminococcales， Rhodospirillales， Clostridiales， and Elusimicrobiales（P<0.01）. And at the genus level， the Zishen Tongguan formula group significantly decreased the relative abundance of pathogenic microbiota such as Lachnospira and Bacteroides（P<0.01） and significantly increased the relative abundances of beneficial microbiota such as Ruminococcus and Lactobacillus（P<0.01）. ConclusionZishen Tongguan formula can reduce the level of harmful intestinal bacteria， increase the level of beneficial intestinal bacteria， down-regulate the expression of serum inflammatory factors， and the small amount of Cinnamomi Cortex in the formula may play a key role in the treatment of CNP with this formula.

OBJECTIVE To analyze the quantity transfer rule in the processing of Astragalus membranaceus before and after moistening-soaking and steaming-soaking followed by cutting. METHODS Three batches of A. membranaceus decoction pieces processed through moistening-soaking and steaming-soaking followed by cutting were prepared. The HPLC overlapping fingerprints of A. membranaceus and its decoction pieces were established through the Similarity Evaluation System of Chromatographic Fingerprints of TCM （2012 edition）. Combined with the previous qualitative analysis results， the common peaks were identified， the changes of common peak area were analyzed， and the principal component analysis was carried out. The contents of calycosin-7-glucoside， astragaloside Ⅰ and astragaloside Ⅳ in A. membranaceus and its decoction pieces were determined by HPLC， and the content differences of each component in different samples were compared. RESULTS The results of fingerprint analysis showed that 17 common peaks were identified. After steaming-soaking and moistening-soaking of A. membranaceus， the proportion of common peak area in the decoction pieces changed compared with the original medicine （for example， in A. membranaceus steaming-soaking decoction pieces， the proportion of peak area of malonyl calycosin-7-glucoside and malonyl astragaloside Ⅰ decreased， while the proportion of peak area of calycosin-7-glucoside increased）. The results of principal component analysis showed that A. membranaceus， and its decoction pieces after moistening-soaking and steaming-soaking followed by cutting were all clustered into one category respectively. The results of content determination showed that， compared with A. membranaceus， the average content of calycosin-7-glucoside in A. membranaceus moistening-soaking decoction pieces was significantly reduced （P＜0.05）； the average contents of calycosin-7-glucoside and astragaloside Ⅳ in A. membranaceus steaming- soaking decoction pieces were significantly increased （P＜0.05）； there was no significant difference in the average content of astragaloside Ⅳ in A. membranaceus moistening-soaking decoction pieces and astragaloside Ⅰ in the two decoction pieces （P＞ 0.05）. CONCLUSIONS There are differences in the quantity transfer rules of A. membranaceus before and after moistening-soaking and steaming-soaking followed by cutting. Steaming-soaking followed by cutting may make the transformation of unstable components （such as malonyl calycosin-7-glucoside and malonyl astragaloside Ⅰ） more complete.

BACKGROUND: The protective effect of mesenchymal stem cells (MSCs) on cardiac ischemia-reperfusion (I/R) injury has been widely reported. Dental pulp-derived mesenchymal stem cells (DP-MSCs) have therapeutic effects on various diseases, including diabetes and cirrhosis. This study aimed to determine the therapeutic effects of DP-MSCs on I/R injury and elucidate the underlying mechanism. METHODS: Myocardial I/R injury model mice were treated with DP-MSCs or a miR-19a-3p mimic. The infarct volume, fibrotic area, pyroptosis, inflammation level, and cardiac function were measured. Cardiomyocytes exposed to hypoxia-reoxygenation were transfected with the miR-19a-3p mimic, miR-19a-3p inhibitor, or negative control. Pyroptosis and protein expression in the interferon regulatory factor 8/mitogen-activated protein kinase (IRF-8/MAPK) pathway were measured. RESULTS: DP-MSCs protected cardiac function in cardiac I/R-injured mice and inhibited cardiomyocyte pyroptosis. The upregulation of miR-19a-3p protected cardiac function, inhibited cardiomyocyte pyroptosis, and inhibited IRF-8/MAPK signaling in cardiac I/R-injured mice. DP-MSCs inhibited cardiomyocyte pyroptosis and the IRF-8/MAPK signaling by upregulating the miR-19a-3p levels in cardiomyocytes injured by I/R. CONCLUSION DP-MSCs protected cardiac function by inhibiting cardiomyocyte pyroptosis through miR-19a-3p under I/R conditions.
Animals ; MicroRNAs/metabolism* ; Pyroptosis/genetics* ; Mesenchymal Stem Cells/metabolism* ; Myocytes, Cardiac/cytology* ; Mice ; Male ; Mice, Inbred C57BL ; Dental Pulp/cytology* ; Myocardial Reperfusion Injury/therapy* ; MAP Kinase Signaling System/physiology*

OBJECTIVES: To investigate the protective effect of Yiqi Yangyin Huazhuo Tongluo Formula (YYHT) against high glucose-induced injury in mouse renal podocytes (MPC5 cells) and the possible mechanism. METHODS: Adult Wistar rats were treated with 19, 38, and 76 g/kg YYHT or saline via gavage for 7 days to prepare YYHT-medicated or blank sera for treatment of MPC5 cells cultured in high glucose (30 mmol/L) prior to transfection with a miR-21a-5p inhibitor or a miR-21a-5p mimic. The changes in miR-21a-5p expressions and the mRNA levels of FoxO1, PINK1, and Parkin in the treated cells were detected with qRT-PCR, and the protein levels of nephrin, podocin, FoxO1, PINK1, and Parkin were detected with Western blotting. Autophagic activity in the cells were evaluated with MDC staining. The effect of miR-21a-5p mimic on FoxO1 transcription and the binding of miR-21a-5p to FoxO1 were examined with luciferase reporter gene assay and radioimmunoprecipitation assay. RESULTS: MPC5 cells exposed to high glucose showed significantly increased miR-21a-5p expression, lowered expressions of FoxO1, PINK1, and Parkin1 mRNAs, and reduced levels of FoxO1, PINK1, parkin, nephrin, and podocin proteins and autophagic activity. Treatment of the exposed cells with YYHT-medicated sera and miR-21a-5p inhibitor both significantly enhanced the protein expressions of nephrin and podocin, inhibited the expression of miR-21a-5p, increased the mRNA and protein expressions of FoxO1, PINK1 and Parkin, and upregulated autophagic activity of the cells. Transfection with miR-21a-5p mimic effectively inhibited the transcription of FoxO1 and promoted the binding of miR-21a-5p to FoxO1 in MPC5 cells, and these effects were obviously attenuated by treatment with YYHT-medicated sera. CONCLUSIONS YYHT-medicated sera alleviate high glucose-induced injury in MPC5 cells by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy.
Animals ; MicroRNAs/genetics* ; Podocytes/pathology* ; Drugs, Chinese Herbal/pharmacology* ; Autophagy/drug effects* ; Rats, Wistar ; Protein Kinases/metabolism* ; Rats ; Forkhead Box Protein O1 ; Mice ; Mitochondria/drug effects* ; Ubiquitin-Protein Ligases/metabolism* ; Glucose ; Diabetic Nephropathies ; Male ; Membrane Proteins/metabolism* ; Intracellular Signaling Peptides and Proteins

Osteoporosis is a prevalent skeletal condition characterized by reduced bone mass and strength, leading to increased fragility. Buqi-Tongluo (BQTL) decoction, a traditional Chinese medicine (TCM) prescription, has yet to be fully evaluated for its potential in treating bone diseases such as osteoporosis. To investigate the mechanism by which BQTL decoction inhibits osteoclast differentiation in vitro and validate these findings through in vivo experiments. We employed MTS assays to assess the potential proliferative or toxic effects of BQTL on bone marrow macrophages (BMMs) at various concentrations. TRAcP experiments were conducted to examine BQTL's impact on osteoclast differentiation. RT-PCR and Western blot analyses were utilized to evaluate the relative expression levels of osteoclast-specific genes and proteins under BQTL stimulation. Finally, in vivo experiments were performed using an osteoporosis model to further validate the in vitro findings. This study revealed that BQTL suppressed receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and osteoclast resorption activity in vitro in a dose-dependent manner without observable cytotoxicity. The inhibitory effects of BQTL on osteoclast formation and function were attributed to the downregulation of NFATc1 and c-fos activity, primarily through attenuation of the MAPK, NF-κB, and Calcineurin signaling pathways. BQTL's inhibitory capacity was further examined in vivo using an ovariectomized (OVX) rat model, demonstrating a strong protective effect against bone loss. BQTL may serve as an effective therapeutic TCM for the treatment of postmenopausal osteoporosis and the alleviation of bone loss induced by estrogen deficiency and related conditions.
Animals ; NFATC Transcription Factors/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Ovariectomy ; Osteoclasts/metabolism* ; Female ; Osteogenesis/drug effects* ; Rats, Sprague-Dawley ; Rats ; NF-kappa B/genetics* ; Osteoporosis/genetics* ; Signal Transduction/drug effects* ; Bone Resorption/genetics* ; Cell Differentiation/drug effects* ; Humans ; RANK Ligand/metabolism* ; Mitogen-Activated Protein Kinases/genetics* ; Transcription Factors

Objective To investigate the protective effect of Yiqi Yangyin Huazhuo Tongluo Formula(YYHT)against high glucose-induced injury in mouse renal podocytes(MPC5 cells)and the possible mechanism.Methods Adult Wistar rats were treated with 19,38,and 76 g/kg YYHT or saline via gavage for 7 days to prepare YYHT-medicated or blank sera for treatment of MPC5 cells cultured in high glucose(30 mmol/L)prior to transfection with a miR-21a-5p inhibitor or a miR-21a-5p mimic.The changes in miR-21a-5p expressions and the mRNA levels of FoxO1,PINK1,and Parkin in the treated cells were detected with qRT-PCR,and the protein levels of nephrin,podocin,FoxO1,PINK1,and Parkin were detected with Western blotting.Autophagic activity in the cells were evaluated with MDC staining.The effect of miR-21a-5p mimic on FoxO1 transcription and the binding of miR-21a-5p to FoxO1 were examined with luciferase reporter gene assay and radioimmunoprecipitation assay.Results MPC5 cells exposed to high glucose showed significantly increased miR-21a-5p expression,lowered expressions of FoxO1,PINK1,and Parkin1 mRNAs,and reduced levels of FoxO1,PINK1,parkin,nephrin,and podocin proteins and autophagic activity.Treatment of the exposed cells with YYHT-medicated sera and miR-21a-5p inhibitor both significantly enhanced the protein expressions of nephrin and podocin,inhibited the expression of miR-21a-5p,increased the mRNA and protein expressions of FoxO1,PINK1 and Parkin,and upregulated autophagic activity of the cells.Transfection with miR-21a-5p mimic effectively inhibited the transcription of FoxO1 and promoted the binding of miR-21a-5p to FoxO1 in MPC5 cells,and these effects were obviously attenuated by treatment with YYHT-medicated sera.Conclusion YYHT-medicated sera alleviate high glucose-induced injury in MPC5 cells by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy.

Objective To observe the effects of electroacupuncture on the motor function and mitochondrial dynamics of skeletal muscle of SAMP8 mice;To explore the mechanism of electroacupuncture in improving the motor dysfunction of Alzheimer disease(AD)from the perspective of mitochondrial dynamics.Methods Totally 18 SAMP8 mice were divided into model group and electroacupuncture group,with 9 mice in each group,and the SAMR1 mice with the same age were set as control group."Baihui","Dazhui"and"Shenshu"were selected in the electroacupuncture group,and electroacupuncture was performed daily for 20 min,8 d as a course of treatment.Each course of treatment was separated by 2 d,for a total of 3 courses of treatment.The model group and the control group were not intervened.The motor function of mice was tested by grip strength test,suspension test,hind limb extension test and Morris water maze experiment.The morphology and structure of gastrocnemius were observed by HE staining,ATP content in gastrocnemius was determined by colorimetry,the mRNA expression of optic atrophy 1(OPA1),mitofusin 2(MFN2)and dynamin-related protein 1(DRP1)in gastrocnemius were detected by real-time quantitative PCR,the expressions of OPA1,MFN2 and DRP1 in gastrocnemius were detected by Western blot.Results Compared with the control group,the grip strength,the score in suspension test,and the average speed and maximum speed of Morris water maze experiment of mice in model group significantly decreased(P<0.01);the arrangement of fibers in the gastrocnemius muscle tissue was disordered,the gaps become wider,and the distribution of nuclei was uneven;the ATP content in the gastrocnemius muscle tissue was significantly decreased(P<0.01),the mRNA and protein expressions of OPA1 and MFN2 were significantly decreased(P<0.01),and the expression of DRP1 mRNA and protein significantly increased(P<0.01).Compared with the model group,the grip strength,the score in suspension test,and the average speed and maximum speed of Morris water maze experiment in electroacupuncture group significantly increased(P<0.01);the arrangement of gastrocnemius muscle tissue was relatively neat,the gaps become narrower,and the distribution of nuclei was more uniform;the ATP content in gastrocnemius muscle tissue significantly increased(P<0.01),while the mRNA and protein expressions of OPA1 and MFN2 significantly increased(P<0.05,P<0.01),the expression of DRP1 mRNA and protein significantly decreased(P<0.01).Conclusion Electroacupuncture can improve the skeletal muscle morphological structure and motor dysfunction of SAMP8 mice,and the mechanism may be related to the correction of skeletal muscle mitochondrial dynamic imbalance and the increase of skeletal muscle ATP content.

Ferroptosis is an iron-dependent form of regulated cell death,which is distinct from apoptosis,ne-crosis,and pyroptosis.Recent studies have found that activators of ferroptosis,such as Erastin,can activate autophagy-re-lated proteins,induce the formation of autophagosomes,and ultimately release ferric ions to mediate ferroptosis.This pro-cess,called ferritinophagy,is initiated by the binding of an autophagic cargo receptor protein,nuclear receptor coactivator 4(NCOA4),to iron-laden ferritin.The transfer of NCOA4-ferritin to the lysosome by ferritinophagy results in the proteoly-sis of ferritin,and,in turn,the release of its iron content and lipid-reactive oxygen species(ROS)accumulation.Ferritin-ophagy has been closely associated with central nervous system disorders,circulatory system diseases,and cancer.Fur-thermore,the regulation mechanism of ferritinophagy is also a hot topic in the study of iron-dependent cell death process.With the in-depth study of ferritinophagy,great progress has been made in the study of key components of ferritinophagy as well as its molecular mechanisms and processes.However,a comprehensive summary of the methods for detecting ferritin-ophagy is still unclear.To further deepen the understanding of ferritinophagy and its detection methods,this review focus-es on the concept,characteristics,methods,and precautions during detection of ferritinophagy.This review provided ex-perimental reference for subsequent researchers and promoting the progress of research related to ferritinophagy.