Elemene induces cell apoptosis via inhibiting glutathione synthesis in lung adenocarcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Curcuma wenyujin Y.H. Chen & C. Ling, also known as Wen-E-Zhu, has been used for cancer treatment since ancient times, with roots dating back to the Song Dynasty. Elemene (EE), a sesquiterpene extract with potent anticancer properties, is extracted from Wen-E-Zhu, with ß-elemene (BE) being its main active compound, along with trace amounts of ß-caryophyllene (BC), γ-elemene and δ-elemene isomers. EE has demonstrated broad-spectrum anti-cancer effects and is commonly used in clinical treatments for various types of malignant cancers, including lung cancer. Studies have shown that EE can arrest the cell cycle, inhibit cancer cell proliferation, and induce apoptosis and autophagy. However, the exact mechanism of its anti-lung cancer activity remains unclear and requires further research and investigation. AIM OF THE STUDY: In this study, the possible mechanism of EE and its main active components, BE and BC, against lung adenocarcinoma was investigated by using A549 and PC9 cell lines. MATERIALS AND METHODS: The subcutaneous tumor model of nude mice was constructed to evaluate the efficacy of EE in vivo, then the in vitro half-inhibitory concentration (IC50) of EE and its main active components, BE and BC, on A549 and PC9 cells at different concentrations were determined by CCK-8. Flow cytometry was used to detect the apoptosis and cycle of A549 and PC9 cells treated with different concentrations of BE and BC for 24 h. Non-targeted metabolomics analysis was performed on A549 cells to explore potential target pathways, which were subsequently verified through kit detection and western blot analysis. RESULTS: Injection of EE in A549 tumor-bearing mice effectively suppressed cancer growth in vivo. The IC50 of EE and its main active components, BE and BC, was around 60 µg/mL. Flow cytometry analysis showed that BE and BC blocked the G2/M and S phases of lung adenocarcinoma cells and induced apoptosis, leading to a significant reduction in mitochondrial membrane potential (MMP). Results from non-targeted metabolomics analysis indicated that the glutathione metabolism pathway in A549 cells was altered after treatment with the active components. Kit detection revealed a decrease in glutathione (GSH) levels and an increase in the levels of oxidized glutathione (GSSG) and reactive oxygen (ROS). Supplementation of GSH reduced the inhibitory activity of the active components on lung cancer and also decreased the ROS content of cells. Analysis of glutathione synthesis-related proteins showed a decrease in the expression of glutaminase, cystine/glutamate reverse transporter (SLC7A11), and glutathione synthase (GS), while the expression of glutamate cysteine ligase modified subunit (GCLM) was increased. In the apoptosis-related pathway, Bax protein and cleaved caspase-9/caspase-9 ratio were up-regulated and Bcl-2 protein was down-regulated. CONCLUSIONS: EE, BE, and BC showed significant inhibitory effects on the growth of lung adenocarcinoma cells, and the mechanism of action was linked to the glutathione system. By down-regulating the expression of proteins related to GSH synthesis, EE and its main active components BE and BC disrupted the cellular redox system and thereby promoted cell apoptosis.

Beneficial effects of non-herbal supplements on patients with diabetes.

BACKGROUND AND AIMS: Controlling glycemic levels is crucial for patients with diabetes mellitus to improve their disease management and health outcomes. Beyond lifestyle modification and pharmacotherapy, some supplements have been shown to lower blood glucose as well as mitigate diabetic complications. METHODS: Information was primarily gathered by employing various PubMed scholarly articles for real-world examples in addition to data extraction from supplementary manuscripts. Only original human trials were used, and those published within the past two decades were primarily chosen. However, background information may contains review articles. RESULTS: Some non-herbal supplements have been suggested to lower fasting blood glucose, postprandial glucose, glycated glucose (HbA1c), lipid profiles, oxidative stress, and inflammation, as well as improving body composition, insulin sensitivity, blood pressure, and nephropathy. CONCLUSION: This review discusses ten non-herbal supplements that have been reported to have beneficial effects among different types of patients with diabetes as well as potential future clinical application. However, more long-term studies with a larger amount and more diverse participants need to be conducted for a robust conclusion. Also, mechanisms of action of antidiabetic effects are poorly understood and need further research.

Oral short-chain fatty acids administration regulates innate anxiety in adult microbiome-depleted mice.

Anxiety is characterized by feelings of tension and worry even in the absence of threatening stimulus. Pathological condition of anxiety elicits defensive behavior and aversive reaction ultimately impacting individuals and society. The gut microbiota has been shown to contribute to the modulation of anxiety-like behavior in rodents through the gut-brain axis. Several studies observed that germ-free (GF) and the broad spectrum of antibiotic cocktail (ABX)-treated rodents display lowered anxiety-like behavior. We speculate that gut microbial short-chain fatty acids (SCFA) modulate the innate anxiety response. Herein, we administered SCFA in the drinking water in adult mice treated with ABX to deplete the microbiota and tested their anxiety-like behavior. To further augment the innate fear response, we enhanced the aversive stimulus of the anxiety-like behavior tests. Strikingly, we found that the anxiety-like behavior in ABX mice was not altered when enhanced aversive stimulus, while control and ABX mice supplemented with SCFA displayed increased anxiety-like behavior. Vagus nerve serves as a promising signaling pathway in the gut-brain axis. We determined the role of vagus nerve by subdiaphragmatic vagotomy (SDV) in ABX mice supplemented with SCFA. We found that the restored anxiety-like behavior in ABX mice by SCFA was unaffected by SDV. These findings suggest that gut microbiota can regulate anxiety-like behavior through their fermentation products SCFA.

[Effect of wheat-grain moxibustion on the expression of Beclin-1/GRP78 in spinal dorsal horn in rats with cervical spondylotic radiculopathy].

OBJECTIVE: To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14) on the expressions of Beclin-1 and GRP78 in spinal dorsal horn in rats with cervical spondylotic radiculopathy (CSR), and to explore the possible analgesic mechanism of wheat-grain moxibustion for CSR. METHODS: A total of 48 SD rats were randomly divided into a sham operation group, a model group, a wheat-grain moxibustion group and a wheat-grain moxibustion+3-MA group, 12 rats in each group. The CSR model was prepared by spinal cord insertion method. Three days after modeling, the rats in the model group were intraperitoneally injected with 1 mL of 0.9% sodium chloride solution; the rats in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14, 6 cones per time) on the basis of the model group; the rats in the wheat-grain moxibustion+3-MA group were intraperitoneally injected with 3-MA solution and wheat-grain moxibustion at "Dazhui" (GV 14, 6 cones per time). The three groups were intervened for 7 days, once a day. The gait score and mechanical pain threshold were observed before treatment and 7 days into treatment; after the treatment, the expressions of mRNA and protein of Beclin-1 in spinal dorsal horn were detected by real-time fluorescence quantitative PCR and immunohistochemistry; the expression of GRP78 protein in spinal dorsal horn was detected by Western blot method; the autophagosomes and ultrastructure in spinal dorsal horn neurons were observed by electron microscope. RESULTS: After the treatment, compared with the sham operation group, in the model group, the gait score was increased and the mechanical pain threshold was decreased (P<0.01), and the expression of GRP78 protein in spinal dorsal horn was increased (P<0.01). Compared with the model group and the wheat-grain moxibustion+3-MA group, in the wheat-grain moxibustion group, the gait score was decreased and mechanical pain threshold was increased (P<0.01), and the expression of GRP78 protein in spinal dorsal horn was decreased, and the expressions of mRNA and protein of Beclin-1 were increased (P<0.01). Under electron microscope, the ultrastructure of spinal dorsal horn neurons in the wheat-grain moxibustion group was not significantly damaged, and its structure was basically close to normal, and the number of autophagosomes was more than the other three groups. CONCLUSION: Wheat-grain moxibustion at "Dazhui" (GV 14) has analgesic effect on CSR rats. The mechanism may be related to moderately up-regulate the expression of Beclin-1, enhance autophagy and reduce endoplasmic reticulum stress.

[Effect of mild moxibustion on pain and expression of LC3 and Bax in spinal cord in cervical spondylotic radiculopathy rats].

OBJECTIVE: To observe the effect of mild moxibustion (Moxi) at "Dazhui" (GV14) on neuropathic pain, expression of autophagy and apoptosis factor LC3 and Bax proteins and mRNAs in the spinal cord tissue in rats with cervical spondylotic radiculopathy (CSR), so as to explore its underlying mechanism underlying relief of CSR-induced pain. METHODS: Forty rats (half male half female) were randomly divided into blank control, model, Moxi, Moxi+autophagy inhibitor 3-methyladenine (3-MA, Moxi+3-MA) groups, with 10 rats in each group. The CSR model was established by loose ligature of the local cervical nerve roots. Three days after modeling, mild Moxi was applied to GV14 for 10 min, once daily for 7 days. Rats of the Moxi+3-MA group received intraperitoneal injection of 3-MA(1 mL, 15 mg/kg+ saline) before Moxi, once daily for 7 consecutive days. Rats of the model and Moxi groups were also given normal saline (i.p., 1 mL), once daily for 7 days. The gait behavior score (1-3 points) was scaled according to the rats' pain reaction and foot paw contracture produced walking disorder and the mechanical pain threshold (MPT) was detected before and after the treatment. The expression of spinal cord LC3 and Bax proteins and mRNAs were detected by immunohistochemistry and quantitative RT-PCR, respectively. RESULTS: Compared with the blank control group, the gait disorder score, and percentage of Bax positive cells and expression of Bax mRNA were significantly increased (P<0.01, P<0.05), and MPT was markedly decreased in the model group (P<0.01). After the treatment, the gait disorder score, percentage of Bax positive cells and Bax mRNA expression were significantly down-regulated (P<0.01, P<0.05), while the MPT and percentage of LC3 positive cells and LC3 mRNA expression were considerably increased (P<0.01, P<0.05) in both Moxi and Moxi+3-MA groups. The therapeutic effects of mild Moxi were remarkably superior to those of Moxi+3-MA in downregulating gait disorder score, Bax positive cell percentage and Bax mRNA expression, and in up-regulating MPT, LC3 positive cell percentage and LC3 mRNA expression (P<0.05), suggesting a reduction of the function of mild Moxi after administration of 3-MA. CONCLUSION: Mild Moxi at GV14 can relieve neuropathic pain in CSR rats, which may be related to its functions in up-regulating LC3 autophagy, thereby inhibiting the expression of Bax pro-apoptotic protein in spinal cord to reduce apoptosis and to repair nerve injury.

Direct moxibustion exerts an analgesic effect on cervical spondylotic radiculopathy by increasing autophagy via the Act A/Smads signaling pathway.

BACKGROUND: Direct moxibustion (DM) is reported to be useful for cervical spondylotic radiculopathy (CSR), but the analgesic mechanism remains unknown. Autophagy plays a protective role in neuronal apoptosis, Act A/Smads signaling pathway has been confirmed to be associated with the activation of autophagy. The study aimed to explore the effect of DM on autophagy in rats with CSR and the involvement of Act A/Smads signaling pathway. METHODS: Rats were randomly divided into Sham, CSR, CSR + DM, CSR + DM + 3-MA (PI3K inhibitor), and CSR + DM + SB (Act A inhibitor) group. Three days after establishment of CSR model with a fish line inserted under the axilla of the nerve roots, DM at Dazhui (GV14) was performed six times once for seven consecutive days. Western blot and immunofluorescence staining were used to observe the expression of the neuronal autophagy molecule LC3II/I, Atg7, and Act A/Smads signaling molecule Act A, p-Smad2, and p-Smad3. Bcl-2/Bax mRNA expression was measured by real time PCR. RESULTS: DM improved the pain threshold and motor function of CSR rats and promoted the expression of Act A, p-Smad2, p-Smad3, LC3II/I, and Atg7 in the entrapped-nerve root spinal dorsal horn. DM reduced the expression of Bax mRNA and decreased the number of apoptotic neurons. 3-MA and Act A inhibitor SB suppressed the expression of above-mentioned proteins and reduced the protective effect of DM on apoptotic neurons. CONCLUSION: DM exerts analgesic effects by regulating the autophagy to reduce cell apoptosis and repair nerve injury, and this feature may be related to the Act A/Smads signaling pathway.

[Observation on clinical effect of acupuncture combined with wheat-grain moxibustion for mild to moderate postpartum depression].

OBJECTIVE: To compare the clinical effect of acupuncture combined with wheat-grain moxibustion and oral sertraline hydrochloride dispersible tablets in the treatment of mild to moderate postpartum depression. METHODS: Sixty patients with mild to moderate postpartum depression were randomly divided into an observation group and a control group, 30 cases in each group. Both groups were treated with psychotherapy. The control group was treated with oral sertraline hydrochloride dispersible tablets, 50 mg each time, once a day; the observation group was treated with acupuncture at Qihai (CV 6), Zusanli (ST 36), Xuehai (SP 10), Hegu (LI 4), Sanyinjiao (SP 6), Taixi (KI 3), etc. combined with wheat-grain moxibustion at Xinshu (BL 15), Pishu (BL 20), Ganshu (BL 18) and Shenshu (BL 23), once every other day, 3 times a week. Both groups were treated for 4 weeks as a course, with 2 consecutive courses of treatment. Before and after treatment and follow-up of 3 months after the end of treatment, the Hamilton depression scale (HAMD), Edinburgh postnatal depression scale (EPDS) and World Health Organization quality of life-BREF (WHOQOL-BREF) score of the two groups were compared, and the clinical effect was assessed. RESULTS: After treatment and during follow-up, the HAMD and EPDS scores of the two groups were lower than before treatment (P<0.05), and the WHOQOL-BREF scores of the two groups were higher than before treatment (P<0.05). In the control group, the scores of HAMD and EPDS during follow-up were higher than after treatment (P<0.05), and the score of WHOQOL-BREF during follow-up was lower than after treatment (P<0.05). After treatment and during follow-up, the HAMD and EPDS scores of the observation group were lower than those of the control group (P<0.05), and the WHOQOL-BREF score of the observation group was higher than that of the control group (P<0.05). The total effective rate of the observation group was 93.3% (28/30), which was higher than 86.7% (26/30) of the control group (P<0.05). CONCLUSION: Acupuncture combined with wheat-grain moxibustion can improve the depressive symptoms of patients with mild to moderate postpartum depression and improve their quality of life, and the clinical effect is more lasting and stable than oral sertraline hydrochloride dispersible tablets.

Microbiota regulate social behaviour via stress response neurons in the brain.

Social interactions among animals mediate essential behaviours, including mating, nurturing, and defence1,2. The gut microbiota contribute to social activity in mice3,4, but the gut-brain connections that regulate this complex behaviour and its underlying neural basis are unclear5,6. Here we show that the microbiome modulates neuronal activity in specific brain regions of male mice to regulate canonical stress responses and social behaviours. Social deviation in germ-free and antibiotic-treated mice is associated with elevated levels of the stress hormone corticosterone, which is primarily produced by activation of the hypothalamus-pituitary-adrenal (HPA) axis. Adrenalectomy, antagonism of glucocorticoid receptors, or pharmacological inhibition of corticosterone synthesis effectively corrects social deficits following microbiome depletion. Genetic ablation of glucocorticoid receptors in specific brain regions or chemogenetic inactivation of neurons in the paraventricular nucleus of the hypothalamus that produce corticotrophin-releasing hormone (CRH) reverse social impairments in antibiotic-treated mice. Conversely, specific activation of CRH-expressing neurons in the paraventricular nucleus induces social deficits in mice with a normal microbiome. Via microbiome profiling and in vivo selection, we identify a bacterial species, Enterococcus faecalis, that promotes social activity and reduces corticosterone levels in mice following social stress. These studies suggest that specific gut bacteria can restrain the activation of the HPA axis, and show that the microbiome can affect social behaviours through discrete neuronal circuits that mediate stress responses in the brain.

Plasma pharmacokinetics and lung distribution of tetrahydropalmatine after topical application of cold asthma recipe extract: Feishu (BL 13) versus Non-Feishu acupoint.

ETHNOPHARMACOLOGICAL RELEVANCE: Acupoint application of cold asthma recipe (CAR) was a traditional Chinese medicine (TCM) method, widely used as an alternative medicine for clinical prevention of the common winter diseases of asthma and bronchitis. Tetrahydropalmatine (THP) was a main active ingredient of CAR extract. The aim of this study is to compare plasma pharmacokinetics and lung distribution of THP between Feishu (FS) acupoint (BL 13) and Non-Feishu (NFS) acupoint application of CAR extract by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). MATERIALS AND METHODS: The extract of CAR was topically administrated in FS and NFS acupoint of rats for plasma pharmacokinetics, and topically administrated in FS and NFS acupoint of mice for lung distribution. The plasma and lung homogenates were pretreated by protein precipitation with acetonitrile. Chromatographic separation was performed on an ACQUITY UPLC BEH C18 column with a mobile phase consisted of 0.1% formic acid in acetonitrile and 0.1% formic acid in water. The detection was accomplished by multiple-reaction monitoring (MRM) scanning in the positive electrospray ionization (ESI(+)) mode. All pharmacokinetic parameters were estimated by non-compartmental analysis. RESULTS: A sensitive, accurate and precise UPLC-MS/MS method was successfully established for determination of THP in 100 µL plasma and lung homogenate. The lower limit of quantification (LLOQ) of THP was 0.05 ng/mL and 0.072 ng/mL, respectively. The pharmacokinetic results manifested that THP was absorbed and eliminated slowly in plasma. Additionally, it was found that there was significantly higher amount of THP absorbed into blood and lung after FS acupoint application compared to NFS acupoint application. CONCLUSIONS: Both of the rat plasma pharmacokinetics and mice lung distribution of THP could support that FS acupoint application of CAR extract has greater advantages of absorption into the blood circulation and distribution in target tissue over NFS acupoint application. The results might be helpful in providing a rational explanation for why the TCM chose the acupoint application and elucidating the underlying mechanism of this treatment.