Effect of Guizhi Fuling Capsule on Apoptosis of Myeloma Cells Through Mitochondrial Apoptosis Pathway.

OBJECTIVE: To observe the effects of Guizhi Fuling Capsule (GZFLC) on myeloma cells and explore the mechanisms. METHODS: MM1S and RPMI 8226 cells were co-cultured with different concentrations of serum and the cell experiments were divided into negative (10%, 20% and 40%) groups, GZFLC (10%, 20%, and 40%) groups and a control group. Cell counting kit-8 (CCK-8) assays and flow cytometry were used to detect the viability and apoptosis levels of myeloma cells. The effects on mitochondria were examined by reactive oxygen specie (ROS) and tetrechloro-tetraethylbenzimidazol carbocyanine iodide (JC-1) assays. Western blot was used to detect the expression of B cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), cleaved caspase-3, -9, cytochrome C (Cytc) and apoptotic protease-activating factor 1 (Apaf-1). RPMI 8226 cells (2 × 107) were subcutaneously inoculated into 48 nude mice to study the in vivo antitumor effects of GZFLC. The mice were randomly divided into four groups using a completely randomized design, the high-, medium-, or low-dose GZFLC (840, 420, or 210 mg/kg per day, respectively) or an equal volume of distilled water, administered daily for 15 days. The tumor volume changes in and survival times of the mice in the GZFLC-administered groups and a control group were observed. Cytc and Apaf-1 expression levels were detected by immunohistochemistry. RESULTS: GZFLC drug serum decreased the viability and increased the apoptosis of myeloam cells (P<0.05). In addition, this drug increased the ROS levels and decreased the mitochondrial membrane potential (P<0.01). Western blot showed that the Bcl-2/Bax ratios were decreased in the GZFLC drug serum-treated groups, whereas the expression levels of cleaved caspase-3, -9, Cytc and Apaf-1 were increased (all P<0.01). Over time, the myeloma tumor volumes of the mice in the GZFLC-administered groups decreased, and survival time of the mice in the GZFLC-administered groups were longer than that of the mice in the control group. Immunohistochemical analysis of tumor tissues from the mice in the GZFLC-administered groups revealed that the Cytc and Apaf-1 expression levels were increased (P<0.05). CONCLUSION: GZFLC promoted apoptosis of myeloma cells through the mitochondrial apoptosis pathway and significantly reduced the tumor volumes in mice with myeloma, which prolonged the survival times of the mice.

[Platelet-rich plasma vs corticosteroid for treatment of rotator cuff tendinopathy:a Meta-analysis].

OBJECTIVE: To explore clinical effects regrarding functional recovery, pain relief, and range of motion of shoulder of platelet-rich plasma (PRP) injection and corticosteroid(CS) injection in treating rotator cuff tendinopathy. METHODS: Randomized controlled trials (RCT) of PRP injection and CS injection in Cochrane Library, EMBASE(Excerpta Medica Database), PebMed, China knowledge Network(CNKI) and Wanfang database were searched from building database to April 20, 2022. According to inclusion and exclusion criteria, literature screening, data extraction and quality evaluation were carried out between two independent researchers, and extracted data were statistically analyzed by Review Manager 5.4.1 software. Short-term (3-6 weeks), medium-term (8-12 weeks) and long-term (≥24 weeks) visual analogue score (VAS), American Shoulder and Elbow Surgeons (ASES) score, Xi'an Western Ontario Rotator Cuff Index (WORC) and shoulder range of motion (ROM) were compared between two groups. RESULTS: Totally 7 RCT were included with 379 patients, 188 patients in PRP group and 191 patients in CS group. Meta analysis results showed there were no significant difference in VAS, ASES and WORC between short-term group and medium-term group(P>0.05). During long-term follow-up, there were significant differences in ASES score[MD=7.1, 95%CI(2.06, 12.14), P=0.006] and VAS [MD=-1.55, 95%CI(-2.65, 0.55), P=0.002]. There was no significant difference in shoulder ROM between two groups(P>0.05). CONCLUSION: For patients with shoulder cuff tendon disease, there are no significant difference in pain relief and functional recovery during short and medium-term follow-up period. However, RPR injection showed advantages over corticosteroid injection in terms of functional recovery and pain relief during long-term follow-up. There is no significant difference in shoulder range of motion between two groups during the whole follow-up period.

Paeoniflorin Ameliorates BiPN by Reducing IL6 Levels and Regulating PARKIN-Mediated Mitochondrial Autophagy.

Background: Bortezomib-induced peripheral neuropathy (BiPN) is a common complication of multiple myeloma (MM) treatment that seriously affects the quality of life of patients. The purpose of the present study was to explore the therapeutic effect of paeoniflorin on BiPN and its possible mechanism. Methods: ELISA was used to measure the level of interleukin-6 (IL6) in the plasma of MM patients, and bioinformatics analysis was used to predict the mechanism underlying the effect of paeoniflorin on peripheral neuropathy. Cell and animal models of BiPN were constructed to evaluate mitochondrial function by measuring cell viability and mitochondrial quality and labeling mitochondria with MitoTracker Green. Nerve injury in mice with BiPN was assessed by behavioral tests, evaluation of motor nerve conduction velocity, hematoxylin-eosin (HE) staining, electron microscopy and analysis of the levels of reactive oxygen species (ROS). Western blotting and immunohistochemistry (IHC) were used to assess the expression of autophagy-related proteins. Results: In MM patients, IL6 levels were positively correlated with the degree of PN. The results of bioinformatics analysis suggested that paeoniflorin ameliorated PN by altering inflammation levels and mitochondrial autophagy. Paeoniflorin increased PC12 cell viability and mitochondrial autophagy levels, alleviated mitochondrial damage, and reduced IL6 levels. In addition, paeoniflorin effectively improved the behavior of mice with BiPN, relieved sciatic nerve injury in mice, increased the expression of LC3II/I, beclin-1, and Parkin in sciatic nerve cells, and increased the expression of LC3B and Parkin in the nerve tissue. Conclusion: The present study confirmed that paeoniflorin significantly ameliorated peripheral neuropathy (PN) caused by bortezomib, possibly by reducing IL6 levels to regulate PARKIN-mediated mitochondrial autophagy and mitochondrial damage.

Exosomal circ-CACNG2 promotes cardiomyocyte apoptosis in multiple myeloma via modulating miR-197-3p/caspase3 axis.

Multiple myeloma (MM) secreted exosomes are essential in MM-related complications such as osteolytic bone lesions and renal failure, but their role and underlying mechanism in cardiac complications has not yet been clarified. Here, we investigated the effects of U266 (a MM cell line) exosomes (U266-exo) on regulating the viability, cell cycle, oxidative stress and apoptosis of H9C2 cells and the role of circ-CACNG2 in these effects. We found that U266-exo coculture significantly inhibited viability and promoted apoptosis of H9C2 cells, and serum exosomes of MM patients harbored high level of circ-CACNG2. The clinical data analyses indicated that circ-CACNG2 was an independent prognostic and diagnostic indicator of MM-related cardiac complications. Also, in vitro experiments showed that circ-CACNG2 inhibited viability and promoted apoptosis of H9C2 cells. RIPA, pull-down assays, dual-luciferase reporter assays, and RNA FISH assays revealed that miR-197-3p could bind to circ-CACNG2 and caspase3 directly. Rescue experiments proved that circ-CACNG2 can increase the expression of caspase3 by binding to and decreasing the expression of miR-197-3p. In conclusion, MM-exosomes could inhibit cardiomyocyte viability and promote apoptosis partially through circ-CACNG2/miR-197-3p/caspase3 axis.

Baicalin regulates autophagy to interfere with small intestinal acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is the main complication of and cause of death after allogeneic hematopoietic stem cell transplantation. Baicalin can protect the small intestinal epithelial cells of rats against TNF-α-induced injury and alleviate enteritis-related diarrhea. To verify whether baicalin can protect the small intestinal mucosal barrier by regulating abnormal autophagy and interfering with intestinal aGVHD, a mouse model of aGVHD was established. CB6F1 micewere intravenously injected with a suspension of mononuclear cells derived from BALB/c donor mouse bone marrow and splenic tissue after treatment with 60Co X-rays. After treatment with different doses of baicalin for 15 days, the survival time, serum TNF-α and IL-10 levels, and autophagy markers levels in the intestine were assessed. A cell model of intestinal barrier dysfunction was also used to verify the effect of baicalin. The results showed that baicalin significantly prolonged the survival time, significantly reduced the aGVHD pathology score and clinical score by decreasing the TNF-α level with increasing the IL-10 level compared with the control. Transmission electron microscopy examination showed that baicalin treatment increased the number of autophagic vacuoles and led to the recovery of mitochondrial structures in the intestinal mucosal epithelial cells of mice and in Caco-2 cells. Western blotting results showed that baicalin treatment enhanced autophagy in vivo by regulating the AMPK/mTOR autophagy pathway. Similar results were observed in vitro in Caco-2 cells. Furthermore, the effect of baicalin was reduced after combination treatment with the autophagy inhibitor 3-methyladenine(3-MA). Baicalin can decrease the severity of small intestinal aGVHD by regulating autophagy by influencing imbalances in inflammatory cytokine levels and mucosal barrier damage, thus baicalin may have potential as a new treatment for aGVHD.

lnc-AC145676.2.1-6-3 plays an important role in intestinal acute graft-versus-host disease through the regulation of interleukin-1ß.

INTRODUCTION: Acute graft-versus-host disease (aGVHD) is one of the major complications of allogeneic hematopoietic stem cell transplantation, and the liver, skin, and gastrointestinal tract are the main target organs. The most common type is intestinal aGVHD. Long noncoding RNAs (lncRNAs) have coregulatory functions and participate in a variety of intracellular regulatory processes. We investigated the expression of lncRNAs and their mechanisms in the development of aGVHD. METHODS: The participants included 15 patients with aGVHD and 4 healthy controls (HCs). To generate profiles of abnormally expressed lncRNAs, peripheral blood mononuclear cell (PBMC) lncRNAs from four patients and four HCs were validated by high-throughput sequencing and quantitative real-time-PCR (qRT-PCR). A number of databases, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, miRanda, TargetScan, and Metascape, were used for bioinformatics analysis. Bioinformatics analysis indicated that overexpression of lnc-AC145676.2.1-6-3 might induce aGVHD via the interleukin (IL)-1ß axis and a downstream miRNA. After the higher levels of lnc-AC145676.2.1-6-3 in other patients were confirmed by qRT-PCR, serum IL-1ß, IL-6, and tumor necrosis factor-α were measured by enzyme linked immunosorbent assays. RESULTS: In our study, a large number of lncRNAs were found in PBMCs of patients with intestinal aGVHD, and bioinformatics analysis showed that the upregulated lncRNA lnc-AC145676.2.1-6-3 probably affected the progression of intestinal aGVHD by regulating the hsa-miR-3064-5p/IL-1ß axis. In addition, the changes in lncRNA expression levels were positively correlated with the clinical characteristics of intestinal aGVHD. CONCLUSION: Our results suggest that lncRNAs in PBMCs may become new biomarkers and therapeutic targets for intestinal aGVHD.

A novel circRNA-miRNA-mRNA network revealed exosomal circ-ATP10A as a biomarker for multiple myeloma angiogenesis.

The importance of angiogenesis in multiple myeloma (MM) is unquestionable; however, to date, the success of antiangiogenic therapies has been fairly limited. Exosomal circular RNAs (circRNAs) have been proven to be pivotal players in angiogenesis in various cancers. Nevertheless, their role in MM remains unknown. Therefore, we aimed to identify differentially expressed circRNAs in peripheral blood exosomes from MM patients and explore their diagnostic and prognostic values. We screened 2,052 circRNAs with significant differential expression between MM patients and healthy controls via high-throughput sequencing. qRT-PCR confirmed that the expression of circ-ATP10A was significantly increased in MM patients. The bioinformatics analyses suggested that circ-ATP10A can act as a microRNA (miRNA) sponge and regulate the expression of downstream vascular endothelial growth factor-B (VEGFB), hypoxia-inducible factor-1alpha (HIF1A), platelet-derived growth factor subunit A (PDGFA), and fibroblast growth factor (FGF). The immunohistochemical results indicated that the circ-ATP10A level was positively correlated with the protein levels of VEGFB and marrow microvessel density (MVD) in MM patients, and the receiver operating characteristic (ROC) curve, area under the ROC curve (AUC) and Kaplan-Meier survival curve analyses confirmed it as a prognostic biomarker. Collectively, our study indicates that exosomal circ-ATP10A is a valuable prognostic biomarker in MM and may promote MM angiogenesis by targeting hsa-miR-6758-3p/hsa-miR-3977/hsa-miR-6804-3p/hsa-miR-1266-3p/hsa-miR-3620-3p and modulating their downstream mRNAs, such as VEGFB, HIF1A, PDGF, and FGF.

Exosomal circRNA as a novel potential therapeutic target for multiple myeloma-related myocardial damage.

INTRODUCTION: Myocardial damage is a mostly incurable complication of multiple myeloma (MM) that seriously affects the treatment outcome and quality of life of patients. Exosomal circular RNAs (exo-circRNAs) play an important role in tumor occurrence and development and are considered key factors in MM pathogenesis. However, the role and mechanism of action of exo-circRNAs in MM-related myocardial damage are still unclear. This study aimed to investigate correlations between exo-circRNAs and MM and to preliminarily explore the role of exo-circRNAs in MM-related myocardial damage. METHODS: Six MM patients and five healthy controls (HCs) were included in the study. High-throughput sequencing and qRT-PCR verification were used to obtain a profile of abnormally expressed exo-circRNAs. GO, KEGG, miRanda, TargetScan and Metascape were used for bioinformatics analyses. H9C2 cells treated with exosomes from U266 cells were used in cell experiments. CCK-8, PCR, immunofluorescence and western blotting assays were used to detect cell proliferation and expression of autophagy-related indicators. Electron microscopy was used to observe the number of autophagic vesicles. RESULTS: Bioinformatics analysis showed that circRNAs with upregulated expression had the potential to promote MM-related myocardial damage. In addition, PCR results confirmed that circ-G042080 was abundantly expressed in the serum exosomes of 20 MM patients. Correlation analysis showed that the expression level of circ-G042080 was positively correlated with the clinical level of MM and MM-related myocardial damage and that circ-G042080 might interfere with MM-related myocardial damage through a downstream miRNA/TLR4 axis. Cell experiments demonstrated that the circ-G042080/hsa-miR-4268/TLR4 axis might exist in H9C2 cells incubated with exosomes and cause abnormal autophagy. CONCLUSION: Abnormal expression of serum exo-circRNAs was found to be associated with MM-related myocardial damage, suggesting that exo-circRNAs might become a new diagnostic marker of MM-related myocardial damage and a therapeutic target.

[Moxibustion at Gaohuang (BL 43) for chronic fatigue syndrome: a randomized controlled trial].

OBJECTIVE: To observe the clinical effect of chronic fatigue syndrome (CFS) treated with moxibustion at Gaohuang (BL 43). METHODS: With stratified block randomization, 72 patients accorded with inclusive criteria were divided into a moxibustion at Gaohuang (BL 43) group (moxibustion group) and an acupuncture group, 36 cases in each one. In the moxibustion group, Gaohuang (BL 43) was treated with big moxa cones as the main acupoint, 10 cones a time; Qihai (CV 6) and Zusanli (ST 36) were added with big moxa cones, 7 cones a time. In the acupuncture group, acupoints were the same as those in the moxibustion group, and twirling reinforcing method was used after qi arriving, 60 times one minute and 360° with range. In the two groups, 10-day treatment was made into one course and there were two days between courses. The treatment was given once a day for 3 courses. Changes of fatigue assessment index (FAI) before and after treatment and clinical effects were observed. RESULTS: The total effective rate was 88.9% (32/36) in the moxibustion group, which was better than 72.2% (26/36) in the acupuncture group apparently (P < 0.05). After treatment in the two groups, FAI scores were obviously declined compared with those before treatment (both P < 0.01) and FAI score in the moxibustion group was apparently lower than that in the acupuncture group (P < 0.05). CONCLUSION: Moxibustion at Gaohuang (BL 43) can improve the FAI score of patients with CFS and the clinical efficacy is definite.