[Huangpu Tongqiao Capsule improves cognitive impairment in rats with Wilson disease by inhibiting endoplasmic reticulum stress-mediated apoptosis pathway].

OBJECTIVE: To investigate the neuroprotective effect of Huangpu Tongqiao Capsule (HPTQ) in a rat model of Wilson disease (WD) and explore the underlying mechanisms. METHODS: SD rat models of WD were established by feeding of coppersupplemented chow diet and drinking water for 12 weeks, and starting from the 9th week, the rats were treated with low-, moderate- and high-dose HPTQ, penicillamine, or normal saline by gavage on a daily basis for 3 weeks. Copper levels in the liver and 24-h urine of the rats were detected, and their learning and memory abilities were evaluated using Morris water maze test. HE staining was used to observe morphological changes of CA1 region neurons in the hippocampus, and neuronal apoptosis was detected with TUNEL staining. Hippocampal expressions of endoplasmic reticulum stress (ERS)-mediated apoptosis pathway-related proteins GRP78, CHOP, caspase-12, cleaved caspase-9, and cleaved caspase-3 at both the mRNA and protein levels were detected using RT-qPCR, immunofluorescence assay or Western blotting. RESULTS: Compared with normal control rats, the rat models with copper overload-induced WD exhibited significantly increased copper levels in both the liver and 24-h urine, impaired learning and memory abilities, obvious hippocampal neuronal damage in the CA1 region and increased TUNEL-positive neurons (P<0.01), with also lowered mRNA and protein expressions of GRP78, CHOP, caspase-12, cleaved caspase-9, and cleaved caspase-3 in the hippocampus (all P<0.01). Treatments with HPTQ and penicillamine significantly lowered copper level in the liver but increased urinary copper level, improved learning and memory ability, alleviated neuronal damage and apoptosis in the hippocampus, and decreased hippocampal expressions of GRP78, CHOP, caspase-12, cleaved caspase-9, and cleaved caspase-3 in the rat models (P<0.01 or 0.05). CONCLUSION: HPTQ Capsule has neuroprotective effects in rat models of WD possibly by inhibiting ERS-mediated apoptosis pathway.

The active components and potential mechanisms of Wuji Wan in the treatment of ethanol-induced gastric ulcer: An integrated metabolomics, network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Wuji Wan (WJW) is a traditional Chinese medicine formula that can be found in the "Prescriptions of Taiping Benevolent Dispensary" that has been employed in treating gastric discomfort, burning epigastric pain, and gastric reflux for hundreds of years and has shown promise for treating gastric ulcers (GUs). However, the active components and mechanism of action against GUs remain unclear. AIM OF THE STUDY: The aim of this study was to explore the active components of WJW and elucidate the underlying mechanism involved in treating GUs. MATERIALS AND METHODS: Initially, cell viability was measured by a cell counting kit 8 (CCK-8) assay to evaluate the efficacy of WJW-containing serum in vitro. The gastric ulcer index, ulcer inhibition rate, hematoxylin and staining (H&E), and periodic acid-Schiff (PAS) staining were used to evaluate the therapeutic effect of WJW in vivo. Subsequently, the levels of inflammatory factors and oxidative stress factors were determined using an enzyme-linked immunosorbent assays (ELISA) on in vitro and in vivo samples. Additionally, UPLC-Q Exactive Plus Orbitrap HRMS was used to analyze the components that were absorbed into the blood of WJW and its metabolites. Network pharmacology and metabolomics were subsequently used to identify the targets and pathways. Real-time quantitative PCR (RT‒qPCR) and Western blotting were used to verify the mRNA and protein levels of the key targets and pathways. Finally, the active components were identified by molecular docking to verify the binding stability of the components and key targets. RESULTS: WJW-containing serum ameliorated ethanol-induced damage in GES-1 cells and promoted cell healing. WJW-containing serum reduced IL-6, TNF-α, MDA, and LDH levels while increasing IL-10, SOD, and T-AOC levels in the cells. Moreover, WJW treatment resulted in decreased IL-6, TNF-α, and MDA levels and increased IL-10, SOD, PGE2, and NO levels in GUs rats. In addition, eight components of WJW were absorbed into the blood. The network pharmacology results revealed 192 common targets for blood entry components and GUs, and KEGG analysis revealed that apoptosis signaling pathways were the main pathways involved in WJW activity against GUs. Metabolomic screening was used to identify 13 differential metabolites. There were 23 common targets for blood entry components, GUs, and differential metabolites, with the key targets TNF (TNF-α), AKT1, PTGS2 (COX2) and MAPK1. WJW significantly inhibited the expression of Bax, Caspase-9, Caspase-3, cleaved Caspase-9, cleaved Caspase-3, TNF-α, COX2, and p-p44/42 MAPK while promoting the expression of Bcl-2 and p-AKT1. Molecular docking revealed that the active components of WJW for the treatment of GUs are berberine, palmatine, coptisine, evodiamine, rutaecarpine, evocarpine, and paeoniflorin. CONCLUSIONS: WJW treatment reduces inflammation and oxidative stress injury and inhibits apoptosis signaling pathways. The main active components are berberine, palmatine, coptisine, evodiamine, rutaecarpine, evocarpine, and paeoniflorin. In this paper, we provide a new strategy for exploring the active components of traditional Chinese medicine formulas for the treatment of diseases based on target mechanisms.

Neurotoxicity study of ethyl acetate extract of Zanthoxylum armatum DC. on SH-SY5Y based on ROS mediated mitochondrial apoptosis pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum armatum DC. (ZADC) is a traditional medicinal plant with various pharmacological activities and is widely used in China, Japan, India, and other regions. Previous studies have revealed that the methanol extract of ZADC can cause neurotoxicity symptoms in rats, such as drooling, decreased appetite, decreased movement, and increased respiratory rate. However, the basis of these toxic substances and the mechanism of neurotoxicity remain unclear. AIM OF THE STUDY: To evaluate the effects of ZADC on nerve cells and their damage mechanisms and discuss the possible toxic substance basis. MATERIALS AND METHODS: The ethyl acetate extract of ZADC is obtained by extracting the methanol extract of ZADC with ethyl acetate. The Q-Orbitrap LC-MS/MS method was employed to analyze the chemical composition of the EA extract of ZADC. SH-SY5Y cells were incubated with different concentrations of the ethyl acetate extract of ZADC. The cytotoxicity of the extract was evaluated using CCK-8, LDH, and ROS assays, and the oxidative stress status of cells was assessed using MDA, GSH, and SOD. Cell apoptosis was detected using flow cytometry. Damage to mitochondrial function was evaluated by labeling mitochondria, ATP, and MMP with fluorescence. Cyto-C, Caspase-3, Caspase-9, Apaf-1, Bax, and reduced Bcl2 expression were measured to evaluate the activation of the mitochondrial apoptosis pathway. Finally, NAC intervention was used to detect changes in the relevant indicators. The activation of mitochondrial apoptosis pathway was evaluated by measuring Cyto-C, Caspase-3, Caspase-9, Apaf-1, and Bax and Bcl2 expression. Finally, NAC intervention was utilized to detect changes in the relevant indicators. RESULTS: After treating SY-SY5Y cells with EA extract from ZADC, cell viability decreased significantly, and the intracellular ROS level increased in a dose-dependent manner. Meanwhile, ZADC can cause cellular oxidative stress and increase MDA and SOD concentrations while decreasing GSH concentrations. It can also shorten the mitochondrial cristae and decrease the number of mitochondria. In contrast, it can reduce ATP synthesis in the mitochondria and mitochondrial membrane potential (MMP). Furthermore, it increased the apoptosis rate and the expression of Cyto-C, Caspase-3, Caspase-9, Apaf-1, and Bax and reduced Bcl2 expression. NAC intervention alleviated the reduction in SH-SY5Y cell survival and the accumulation of reactive oxygen species induced by the EA extract in ZADC. It also inhibits signaling pathways dominated by proteins, such as Cyto-C, reducing cell apoptosis and cytotoxicity. A total of 46 compounds were identified in the extracts. CONCLUSIONS: The results suggest that EA extract of ZADC can induce the mitochondrial apoptotic pathway by accumulating ROS in cells, leading to apoptosis. Antioxidants had a good inhibitory and protective effect against cell damage caused by the EA extract of ZADC. The neurotoxic components of ZADC may be organic acids and compounds containing amino groups.

Periploca forrestii Schltr. ameliorate liver injury caused by fluorosis in rat.

To investigate the impact of the ethanoic fractions of Periploca forrestii Schltr. (P. forrestii) in ameliorating the liver injury caused by fluoride ingestion and to explore the potential mechanisms. Initially, an in vitro fluorosis cell model was constructed using the human normal liver cell line (L-02) induced by fluoride. Cell viability was assessed using the CCK-8 assay kit. The lactate dehydrogenase (LDH) assay kit was utilized to measure LDH content in the cell supernatant, while the malonic dialdehyde (MDA) assay kit was employed to determine MDA levels within the cells. Subsequently, a fluorosis rat model was established, and LDH content in the cell supernatant was measured using the LDH assay kit. Various parameters, including MDA, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and reactive oxygen species (ROS) content within the cells, were detected using appropriate assay kits. Additionally, cell apoptosis rate was determined using the Annexin V-FITC/PI cell apoptosis assay kit. The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Cleaved Caspase-3, Caspase-9, and Cleaved Caspase-9 were analyzed through Western blotting. Compared to the model group, the ethanolic fraction D of P.forrestii (Fr.D) increased cell viability (P < 0.01) and decreased LDH and MDA levels (P < 0.01). In the high-dose Fr.D treatment group of fluoride-poisoned rats, serum ALT, AST, LDH and MDA levels significantly decreased (P < 0.01). Results from rat primary cells exhibited that the Fr.D administration group exhibited significantly higher cell survival rates than the fluoride group (P < 0.01). Similarly, primary rat cells treated with Fr.D showed enhanced cell viability (P < 0.05) and reduced apoptosis rate, LDH, MDA, SOD, GSH-Px, CAT, and ROS levels (P < 0.05) compared to the model group. Western blot analysis indicated that the Fr.D treatment group elevated the Bcl-2/Bax protein expression ratio and reduced Caspase-3 and Caspase-9 activation levels (P < 0.01) compared to the model group. The results suggest that components within the Fr.D from Periploca forrestii may alleviate fluoride-induced liver injury by potentially counteracting oxidative stress and cell apoptosis.

[Mechanism of Gegen Qinlian Decoction in improving glucose metabolism in vitro and in vivo by alleviating hepatic endoplasmic reticulum stress].

This study investigated the mechanism of Gegen Qinlian Decoction(GQD) in improving glucose metabolism in vitro and in vivo by alleviating endoplasmic reticulum stress(ERS). Molecular docking was used to predict the binding affinity between the main effective plasma components of GQD and ERS-related targets. Liver tissue samples were obtained from normal rats, high-fat-induced diabetic rats, rats treated with metformin, and rats treated with GQD. RNA and protein were extracted. qPCR was used to measure the mRNA expression of ERS marker glucose-regulated protein 78(GRP78), and unfolded protein response(UPR) genes inositol requiring enzyme 1(Ire1), activating transcription factor 6(Atf6), Atf4, C/EBP-homologous protein(Chop), and caspase-12. Western blot was used to detect the protein expression of GRP78, IRE1, protein kinase R-like ER kinase(PERK), ATF6, X-box binding protein 1(XBP1), ATF4, CHOP, caspase-12, caspase-9, and caspase-3. The calcium ion content in liver tissues was determined by the colorimetric assay. The ERS-HepG2 cell model was established in vitro by inducing with tunicamycin for 6 hours, and 2.5%, 5%, and 10% GQD-containing serum were administered for 9 hours. The glucose oxidase method was used to measure extracellular glucose levels, flow cytometry to detect cell apoptosis, glycogen staining to measure cellular glycogen content, and immunofluorescence to detect the expression of GRP78. The intracellular calcium ion content was measured by the colorimetric assay. Whereas Western blot was used to detect GRP78 and ERS-induced IRE1, PERK, ATF6, and eukaryotic translation initiation factor 2α(eIF2α) phosphorylation. Additionally, the phosphorylation levels of insulin receptor substrate 1(IRS1), phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85), and protein kinase B(Akt), which were involved in the insulin signaling pathway, were also measured. In addition, the phosphorylation levels of c-Jun N-terminal kinases(JNKs), which were involved in both the ERS and insulin signaling pathways, were measured by Western blot. Molecular docking results showed that GRP78, IRE1, PERK, ATF4, and various compounds such as baicalein, berberine, daidzein, jateorhizine, liquiritin, palmatine, puerarin and wogonoside had strong binding affinities, indicating that GQD might interfere with ERS-induced UPR. In vivo results showed that GQD down-regulated the mRNA transcription of Ire1, Atf6, Atf4, Grp78, caspase-12, and Chop in diabetic rats, and down-regulated GRP78, IRE1, PERK, as well as ERS-induced apoptotic factors ATF4 and CHOP, caspase-12, caspase-9, and caspase-3, while up-regulating XBP1 to enhance adaptive UPR. In addition, GQD increased the calcium ion content in liver tissues, which facilitated correct protein folding. In vitro results showed that GQD increased glucose consumption in ERS-induced HepG2 cells without significantly affecting cell viability, increased liver glycogen synthesis, down-regulated ATF6 and p-eIF2α(Ser51), and down-regulated IRE1, PERK, and GRP78, as well as p-IRS1(Ser312) and p-JNKs(Thr183/Tyr185), while up-regulating p-PI3Kp85(Tyr607) and p-Akt(Ser473). These findings suggested that GQD alleviates excessive ERS in the liver, reduces insulin resistance, and improves hepatic glucose metabolism in vivo and in vitro.

[Moxibustion with seed-size moxa cone enhances anti-tumor effect of cyclophosphamide on Hepa1-6 hepatoma-bearing mice by regulating apoptosis factor cysteine family].

OBJECTIVE: To observe the anti-tumor effect of moxibustion with seed-sized moxa cones on Hepa1-6 liver cancer bearing (HLCB) mice and its regulatory mechanism on cell apoptosis. METHODS: A total of 40 male C57BL/6 mice were randomly divided into control, moxibustion, cyclophosphamide (CTX) and moxibustion+CTX groups, with 10 mice in each group. The HLCB model was established by subcutaneous inoculation of Hepa1-6 cancer cells into the right armpit. Mice of the CTX and moxibustion+CTX groups were given intraperitoneal injection of CTX (30 mg/kg), once daily for 3 days. Moxibustion with seed-sized moxa cones were applied to "Dazhui" (GV14), bilateral"Zusanli" (ST36) and "Sanyinjiao" (SP6), with 5 moxa cones for each acupoint, once daily for 10 consecutive days. The survival status scores and body weight of HLCB mice were observed, and the tumor weight and tumor inhibition rate were detected. HE staining was used to observe the morphological changes of tumor tissue. ELISA was used to detect the levels of serum interleukin (IL)-2, IL-4 and tumor necrosis factor-α (TNF-α). Western blot and fluorescent quantitative real-time PCR were used to detect the protein and mRNA expressions of cysteine aspartate protease (Caspase) -3 and Caspase-9 in tumor tissues, separately. RESULTS: Compared with the control group, the survival status scores, body weight, serum IL-2 and TNF-α levels were significantly increased (P<0.05, P<0.01), the tumor weight and serum IL-4 levels were significantly decreased (P<0.05) in the moxibustion group；while the survival status, body weight, tumor weight, serum IL-2 and IL-4 levels were significantly decreased (P<0.01, P<0.05), the content of TNF-α was significantly increased (P<0.01) in the CTX group. The protein and mRNA expressions of Caspase-3 and Caspase-9 in the 3 trentment groups were significantly increased (P<0.05, P<0.01). In comparison with the moxibustion group, the survival status scores, body weight and tumor weight, serum content of IL-2 were significantly decreased (P<0.01, P<0.05). In contrast to the CTX group, the survival status scores, body weight, serum IL-2 and TNF-α content, and the expressions of Caspase-3 and Caspase-9 in tumor tissue were significantly increased (P<0.01, P<0.05), and the tumor weight and serum IL-4 content were significantly decreased (P<0.05) in the moxibustion+CTX group. Results of HE staining showed that the tumor cells in the control group had clear nuclear membranes and nucleoli, with more dividing cells； while less nuclear division and an increase in tumor necrosis areas were found in the 3 treatment groups. CONCLUSION: Moxibustion with seed-size moxa cone can enhance the anti-tumor effect of CTX and improve the quality of life of HLCB mice, which may be related with its effect in activating the expressions of Caspase-3 and Caspase-9 in tumor tissue.

Alpha-ketoglutaric acid mitigates the detrimental effects of soy antigenic protein on the intestinal health and growth performance of Mirror carp Cyprinus carpio.

The study investigated the alleviated effects of Alpha-ketoglutaric acid (AKG) on the intestinal health of mirror carp (Cyprinus carpio Songpu) caused by soy antigenic protein. The diets were formulated from fishmeal (CON), 50% soybean meal (SBM), the mixture of glycinin and ß-conglycinin (11 + 7S) and adding 1% AKG in the 11 + 7S (AKG). Carp (~ 4 g) in triplicate (30 fish per tank) was fed to apparent satiation thrice a day for six weeks. Compared with CON, SBM treatment resulted in significantly poor growth performance (P < 0.05), whereas 11 + 7S and AKG treatments were not significantly different from CON (P > 0.05). Gene expression of tumor necrosis factor (TNF-α) and interleukin-1 ß (IL-1ß) in proximal intestines (PI) and distal intestines (DI) were increased (P < 0.05), and transforming growth factor (TGF-ß) in PI and middle intestines (MI) was decreased (P < 0.05) in both SBM and 11 + 7S. The caspase-3 in DI increased in SBM (P < 0.05) and the caspase-3 and caspase-9 in DI increased in 11 + 7S (P < 0.05); conversely, TGF-ß in PI and MI was increased, TNF-α and IL-1ß in the MI, caspase-3, and caspase-9 in DI was decreased in AKG (P < 0.05). The TOR (target of rapamycin) in PI and MI, ACC in PI, MI and DI was decreased in SBM (P < 0.05), the AMPK in the PI and DI, TOR in PI, MI and DI, ACC in PI and DI, 4E-BP in DI was reduced in 11 + 7S (P < 0.05). AMPK in the PI and DI, ACC in the PI and MI, TOR in PI, MI, and DI, 4E-BP in PI and DI was recovered by AKG supplementation (P < 0.05). Lipids and lipid-like metabolism, organic acids and derivatives metabolism increased in AKG dietary treatment. In conclusion, AKG reduces the expression of intestinal inflammation and apoptosis pathway and changes glycerophospholipid metabolism and sphingolipid metabolism in the intestine of fish.

[Stellera chamaejasme extract against multidrug resistance of breast cancer cell line MCF-7].

This study explored the effect and underlying mechanism of Stellera chamaejasme extract(SCE) on multidrug resistance of breast cancer. The chemotherapy-sensitive breast cancer cell line MCF-7 and adriamycin(ADR)-resistant cell line MCF-7/ADR were used as experimental subjects. MTT assay was used to detect cell proliferation activity. Pi staining was used to detect the cell cycle. 4',6-Diamidino-2-phenylindole, dihydrochloride(DAPI) staining and flow cytometry were used to detect apoptosis. Dansylcadaverine(MDC) staining and GFP-LC3B-Mcherry adenovirus transfection were used to detect autophagy. The protein expression of Bcl-2, Bax, caspase-9, caspase-3, LC3B, p62, and Beclin-1 was detected by Western blot. The results showed that SCE could significantly inhibit the proliferation of both sensitive and resistant breast cancer cell lines. The drug resistance factor was 0.53, which was significantly lower than 59 of ADR. Meanwhile, the proportion of sensitive/resistant cells in the G_0/G_1 phase increased significantly after SCE treatment. In addition, DAPI staining showed that a series of apoptosis phenomena such as nuclear pyknosis, staining deepening, and nuclear fragmentation appeared in sensitive/resistant cell lines after SCE administration. Moreover, the results of flow cytometry double staining showed that the proportion of apoptotic cells in sensitive/resistant cell lines increased significantly after SCE administration. Besides, Western blot showed that the protein expression levels of caspase-3, caspase-9, and Bcl-2 significantly decreased and the expression level of Bax protein significantly increased in both breast cancer cell lines after SCE administration. Furthermore, SCE could also increase the positive fluorescent spots after MDC staining and yellow fluorescent spots after GFP-LC3B-mcherry transfection, and up-regulate the expression levels of autophagy-related proteins LC3B-Ⅱ, p62, and Beclin-1 in breast cancer cells. In summary, SCE may play the role of anti-multidrug resistance by blocking the cell cycle of breast cancer multidrug-resistant cells, blocking autophagy flow, and ultimately interfering with the apoptosis resistance of drug-resistant cells.

Porcine cardiac blood - Salvia miltiorrhiza root alleviates cerebral ischemia reperfusion injury by inhibiting oxidative stress induced apoptosis through PI3K/AKT/Bcl-2/Bax signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bge. mixed with porcine cardiac blood (PCB-DS) is mainly employed for the treatment of brain ischemia-induced mental disturbances, palpitations and phlegm confusion based on the traditional principle of Menghe medical sect. PCB is the guide to DS and enhances the effect of DS. However, the potential mechanism of PCB-DS preventing cerebral ischemia/reperfusion injury (CIRI) from the perspective of oxidative stress induced cell apoptosis remains unknown. AIM OF THE STUDY: To investigate the pharmacological activity and molecular mechanism of PCB-DS against CIRI. MATERIALS AND METHODS: DS samples processed with different methods were prepared and UPLC-Q-TOF-MS/MS was employed for qualitative analysis of the respective processing product. The middle cerebral artery occlusion reperfusion model was then established to investigate the pharmacological activities of PCB-DS. Pathological changes in the rat brain were observed by triphenyl tetrazolium chloride (TTC), hematoxylin-eosin, and TUNEL staining. The levels of IL-6, IL-1ß, and TNF-α were detected by ELISA to evaluate the inflammatory damage. Metabolomics of cerebrospinal fluid was further used to explore the potential mechanism of PCB-DS in preventing CIRI. Based on this, the levels of oxidative stress-related lactate dehydrogenase (LDH), reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were determined. The protein levels of PI3K, AKT, Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9 proteins of the cerebral infarct zone were finally measured by western blotting. RESULTS: Forty-seven components were identified in four processing products. Compared to DS, the content of total aqueous components in PCB-DS was significantly increased including salvianolic acid B isomer, salvianolic acid D, salvianolic acid F, and salvianolic acid H/I/J. Among the DS, DS processed with wine, DS processed with pig blood, and DS processed with porcine cardiac blood, PCB-DS best alleviated the CIRI through the neurological score, brain infarct volume, brain histopathology and the levels of inflammatory factors in the brain. Twenty-five significant metabolites in the cerebrospinal fluid were screened out between the sham and I/R groups. They were mainly involved in the beta-alanine metabolism, histidine metabolism, and lysine degradation, which indicated that PCB-DS may inhibit oxidative stress-induced apoptosis to achieve treating ischemic stroke. The results of biomedical examination showed that PCB-DS could alleviate oxidative damage, significantly downregulate the expression of Bax, cleaved caspase-3 and cleaved caspase-9, and upregulate the expression of p-PI3K, p-AKT, and Bcl-2. CONCLUSION: In summary, this study demonstrated that PCB-DS alleviated CIRI and the molecular mechanism may be related to inhibiting the oxidative stress induced apoptosis through PI3K/AKT/Bcl-2/Bax signaling pathway.

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.

Hawthorn fruit extract protect against MC-LR-induced hepatotoxicity by attenuating oxidative stress and apoptosis.

Microcystins (MCs) is a class of cyclic heptapeptide compounds with biological activity. There is no effective treatment for liver injury caused by MCs. Hawthorn is a medicinal and edible plant traditional Chinese medicine with hypolipidemic, reducing inflammation and oxidative stress in the liver. This study discussed the protective effect of hawthorn fruit extract (HFE) on liver damage caused by MC-LR and the underlying molecular mechanism. After MC-LR exposure, pathological changes were observed and hepatic activity of ALT, AST and ALP were increased obviously, but they were remarkably restored with HFE administration. In addition, MC-LR could significantly reduce SOD activity and increase MDA content. Importantly, MC-LR treatment resulted in mitochondrial membrane potential decreased, and Cytochrome C release, eventually leading to cell apoptosis rate increase. HFE pretreatment could significantly alleviate the above abnormal phenomena. To examine the mechanism of protection, the expression of critical molecules in the mitochondrial apoptosis pathway was examined. The levels of Bcl-2 was inhibited, and the levels of Bax, Caspase-9, Cleaved Caspase-9, and Cleaved caspase-3 were upregulated after MC-LR treatment. HFE reduced MC-LR-induced apoptosis via reversing the expression of key proteins and genes in the mitochondrial apoptotic pathway. Hence, HFE could alleviate MC-LR induced hepatotoxicity by reducing oxidative stress and apoptosis.

[Protective effect of moxibustion preconditioning in rats with premature ovarian insufficiency].

OBJECTIVE: To observe the effects of moxibustion preconditioning on ovarian function, fertility and ovarian granulosa cell apoptosis in rats with premature ovarian insufficiency (POI), so as to investigate its underlying mechanism in improving POI. METHODS: Forty-two female SD rats with two complete estrous cycles were randomly divided into control group, model group and pre-moxibustion group, with 14 rats in each group. The pre-moxibustion group was pretreated with mild moxibustion for 14 days before POI model establishment at 1) "Guanyuan" (CV4) and "Zhongwan" (CV12) and 2) bilateral "Shenshu" (BL23) as two sets of acupoints on alternate days, once each day, for 10 min each acupoint. After 14-day mild moxibustion intervention, 75 mg·kg-1·d-1 tripterygium glycoside tablet suspension was administered to rats in the pre-moxibustion group and the model group by gavage, for 14 consecutive days, while equivalent saline was given to rats in the control group in the same way. After modeling, the effect of moxibustion preconditioning on ovarian reserve function was evaluated by the estrous cycles, pregnancy rate and embryo number, morphological changes of ovaries, and serum sex hormone levels. TUNEL staining was used to detect the rate of granulosa cell apoptosis in ovaries. Immunohistochemistry and real time quantitative PCR were used to detect the relative expression of Caspase-3 and Caspase-9 proteins and mRNA levels in ovaries. RESULTS: Compared with the control group, the estrous cycles were disturbed; the pregnancy rate and number of embryos, the wet weight of ovary and ovarian index, the number of total follicles and different level of follicles, serum Estradiol (E2) and anti-mullerian hormone (AMH) levels were all significantly decreased (P<0.01,P<0.05), while the number of atretic follicles, serum follicule-stimulating hormone (FSH) and luteinizing hormone (LH) levels, the number of TUNEL-positive granulosa cells, the expression of ovarian Caspase-3 and Caspase-9 proteins and mRNAs were significantly increased (P<0.01) in the model group. Compared with the model group, the disordered estrous cycles were improved; the pregnancy rate, the embryo numbers, the wet weight of ovary, and the total follicle number and primary follicle number, serum AMH level were significantly increased (P<0.01，P<0.05), while the number of atretic follicles, serum FSH level, the number of TUNEL-positive granulosa cells, expression of ovarian Caspase-3 and Caspase-9 proteins and mRNAs were all significantly decreased (P<0.01,P<0.05) in the moxibustion group. CONCLUSION: Moxibustion preconditioning could improve ovarian function and improve fertility of POI rats, which may be associated with reducing the apoptosis of ovarian granulosa cells.

Curcumol, a major terpenoid from Curcumae Rhizoma, attenuates human uterine leiomyoma cell development via the p38MAPK/NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Uterine fibroids (UFs) are the most common benign tumors in women of reproductive age. Curcumae Rhizoma, the main essential oil component of which is curcumol, is widely used for the treatment of phymatosis in China due to its antitumor, anti-inflammatory, antithrombin, anti-tissue fibrosis and anti-oxygen pharmacological activities, but its potential for the treatment of UFs has not been evaluated. AIM OF THE STUDY: This study aimed to investigate the effects and mechanisms of curcumol intervention in human uterine leiomyoma cells (UMCs). MATERIALS AND METHODS: Putative targets of curcumol intervention in UFs were identified using network pharmacology strategies. Molecular docking was performed to assess the binding affinity of curcumol to core targets. A concentration gradient of curcumol (0, 50, 100, 200, 300, 400 and 500 µM) or RU-486 (mifepristone, 0, 10, 20, 40, 50, and 100 µM) was applied to UMCs, and cell viability was detected by the CCK-8 assay. Cell apoptosis and cell cycle were examined by flow cytometry, and cell migration was assessed by a wound-healing assay. Additionally, the mRNA and protein expression levels of critical pathway components were evaluated by RT‒PCR and western blotting. Finally, the actions of curcumol on different tumor cell lines were summarized. RESULTS: Network pharmacology predicted 62 genes with roles in the treatment of UFs with curcumol, and MAPK14 (p38MAPK) displayed a higher interaction degree. GO enrichment and KEGG analyses revealed that the core genes were abundantly enriched in the MAPK signaling pathway. The molecular binding of curcumol to core targets was relatively stable. In UMCs, 200, 300 and 400 µM curcumol treatment for 24 h decreased cell viability compared with that in the control group, and the greatest effect was detected at 48 h and maintained until 72 h. Curcumol arrested cells in the G0/G1 phase and subsequently suppressed mitosis, promoted early apoptosis and reduced the degree of wound healing in a concentration-dependent manner in UMCs. Furthermore, 200 µM curcumol decreased the mRNA and protein expression of p38MAPK, the mRNA expression of NF-κB, and the protein expression of Ki-67 and increased the mRNA and protein expression of Caspase 9. Curcumol (300 and 400 µM) decreased the mRNA and protein expression of p38MAPK, NF-κB, and Ki-67 and increased the protein expression of Caspase 9 in UMCs. Curcumol was demonstrated to treat tumor cell lines, including breast cancer, ovarian cancer, lung cancer, gastric cancer, liver cancer and nasopharyngeal carcinoma, but its effects on benign tumors have not yet been reported. CONCLUSION: Curcumol suppresses cell proliferation and cell migration while arresting the cell cycle in the G0/G1 phase and inducing cell apoptosis in UMCs via a mechanism related to p38MAPK/NF-κB pathway regulation. Curcumol may be a potential therapeutic and preventive agent in the treatment of benign tumors such as UFs.

Ginger polysaccharide promotes myeloid-derived suppressor cell apoptosis by regulating lipid metabolism.

Recently, targeting myeloid-derived suppressor cells (MDSCs) which mainly play an immunosuppressive role in tumor microenvironment has become a hot spot in tumor immunotherapy. This study focuses on biological effect of ginger polysaccharide extracted from natural plants on promoting apoptosis of MDSCs by regulating lipid metabolism. An MTT assay was used to detect the inhibitory effect of ginger polysaccharide on the growth of an MDSC-like cell line (MSC-2). The apoptosis-promoting effect of ginger polysaccharide on MSC-2 cells was detected by flow cytometry. Expression levels of apoptosis proteins (caspase 9 and Bcl-2) and lipid metabolism enzymes (fatty acid synthase (FASN) and diacylglycerol acyltransferase 2) in MSC-2 cells treated with different concentrations of ginger polysaccharide were detected by western blot assay. Nile red staining was used to quantitatively detect the effect of ginger polysaccharide on lipid droplet synthesis. Ginger polysaccharide inhibited proliferation of MSC-2 cells and promoted their apoptosis by upregulating pro-apoptotic caspase 9 protein, downregulating anti-apoptotic Bcl-2 protein, inhibiting expression of FASN and diacylglycerol acyltransferase 2 (key enzymes in fatty acid synthesis and lipid droplet formation, respectively). Ginger polysaccharide promoted apoptosis of MDSCs by regulating key lipid metabolism enzymes, inhibiting fatty acid synthesis and lipid droplet accumulation, and reducing the energy supply of cells.

A network pharmacology approach and experimental validation to investigate the anticancer mechanism and potential active targets of ethanol extract of Wei-Tong-Xin against colorectal cancer through induction of apoptosis via PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Wei-Tong-Xin (WTX), derives from the Chinese herbal decoction (CHD) of Wan-Ying-Yuan in ancient China, has been shown to be effective therapeutic herbal decoction for treating gastrointestinal diseases. Present studies have demonstrated that WTX had potential to alleviate the symptoms of gastrointestinal inflammation, gastric ulcer and improve gastric motility. AIM OF THE STUDY: The study primarily focused on exploring the therapeutic effect and possible pharmacological mechanism of WTX on colorectal cancer (CRC) based on network pharmacology, in vitro and in vivo experiments. MATERIALS AND METHODS: Firstly, colorectal cancer and WTX associated with targets were searched from GeneCards database and TCM Systems Pharmacology Database and Analysis Platform (TCMSP) respectively. The protein-protein interaction (PPI) network also was constructed to screening key targets. In addition, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to predict the underlying biological function and mechanism involving in the anti-colorectal cancer effect of WTX. Next, CCK-8, colony formation and transwell assays were performed to verify the influence of proliferation and metastasizing ability of HCT116 cells after treated with WTX. Cell cycle, apoptosis and reactive oxygen species (ROS) were analysis by flow cytometry. Hoechst 33258 staining was conducted to observe nuclear morphology changes. Protein expression of apoptosis and PI3K/AKT signaling as well as mRNA expression of ferroptosis and apoptosis were determined by Western Blotting and RT-qPCR. The effects of WTX and LY294002 combination on the PI3K/Akt/mTOR signaling pathway were measured by Western Blotting. Finally, the xenograft tumor mouse model was established by subcutaneous injection of CT26 cells to measure tumors volume and weight. Hematoxylin and eosin (HE) staining and immunohistochemical analysis were used to observe the pathological changes and the protein expression in tumor tissues. RESULTS: There were 286 potential treatment targets from 130 bioactive compounds in WTX, 1349 CRC-related targets were identified. Eleven core targets (TP53, AKT1, STAT3, JUN, TNF, HSP90AA1, IL-6, MAPK3, CASP3, EGFR, MYC) were found by PPI network analysis constructed of 142 common targets. The results of KEGG enrichment displayed PI3K/AKT signaling pathway as core pathway. After the treatment of WTX, the inhibitory of viability, metastases and cell cycle arrest at G2/M phase were observed in HCT116 cells. Moreover, WTX induced an increase in the expression of apoptosis proteins (Bak, cytochrome c, cleaved caspase-9/caspase-9 and cleaved caspase-3/caspase-3) and the levels of ROS and MDA, a decrease in the expression of PI3K/AKT signaling related proteins (PI3K, p-PI3K, p-AKT/AKT and p-mTOR/mTOR) and the level of SOD. WTX treatment significantly reduced the tumor weight, increased cleaved caspase-3 positive area and decreased that of ki67 in xenograft mouse model. CONCLUSION: Through a network pharmacology approach and in vitro experiments, we predicted and verified the effect of WTX on colorectal cancer cells mainly depended on the regulation of intrinsic apoptosis via PI3K/AKT signaling pathway, and further animal experiments proved that WTX has a good anti-colon cancer effect in vivo.

GPR78 Regulates Autophagy and Drug Resistance in Non-small Cell Lung Cancer.

Context: Lung cancer is one of the most common forms of cancer. Autophagy and apoptosis play an important role in the development of lung cancer. Researchers have found upregulation of GRP78 expression in cancer cells of various types. Objective: The study intended to explore the mechanism of G protein-coupled receptor 78(GPR78) in regulating autophagy and drug resistance in non-small cell lung cancer (NSCLC). Design: The research team performed a laboratory study. Setting: The study took place in the Department of Thoracic Surgery at Hainan General Hospital of the Hainan Affiliated Hospital of Hainan Medical University in Haikou, Hainan, China. Intervention: The research team cultured immortalized, normal, human bronchial epithelial cells C3 (HBEC3) lines and HBEC4 lines in a serum medium without keratinocytes and infected the expression of GPR78 in knockdown A549 cells using lentiviral agents. The team divided the cells into a control group and a shRNA-GPR78 group, the intervention group. The lentiviral silencing vector expressing shRNA targets human GPR78#1 and GPR78 #2aadam10. Outcome Measures: The research team analyzed the mRNA expression of GPR78 in the NSCLC cell lines H1975, H1299, and A549 and in HBEC3 and HBEC4 using a real time-polymerase chain reaction (RT-PCR) and measured the proliferation of A549 cells at 0h, 24h, 48h, 72h, and 96h using yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The team also analyzed the migration and invasion ability of cells using wound healing and Transwell tests as well as measured the protein expression of the autophagy-related factors Beclin-1, microtubule-associated protein light chain 3-I/II (LC3-I/LC3-II), ubiquitin-binding protein p62 and c-Jun N-terminal kinase (JNK) using a Western blot test. The team also analyzed the protein expressions of caspase-9, caspase-3, and caspase-12 related to apoptosis using a Western blot. To detect the cell viability induced by cisplatin, the team used a Cell Counting Kit 8 (CCK-8) at the concentrations of 1µM, 3µM and 10µM. Results: The mRNA expression of GPR78 in the H1975, H1299, and A549 cell lines was significantly higher than that in the HBEC3 and HBEC4 cell lines (P < .05). At 48h, 72h, and 96h, the A549 cell proliferation in the shRNA-GPR78 group was significantly lower than that of the control group (P < .05). The cell migration and invasion of cells in the shRNA-GPR78 group was significantly lower than that in the control group (P < .05), and the cell viability of the shRNA-GPR78 group was significantly lower than that of control group (P < .05). The expression of Beclin-1 and JNK protein in shRNA-GPR78 group was significantly higher than that in the control group (P < .05), and the expression of LC3-I/LC3-II and p62 protein in shRNA-GPR78 group was significantly lower than that in the control group (P < .05). The protein expressions of caspase-9, caspase-3, and caspase-12 in the shRNA-GPR78 group were significantly higher than those of the control group (P < .05), and the protein activities of RhoA and Rac1 in the shRNA-GPR78 group were significantly lower than those in the control group (P < .05). Conclusion: NSCLC upregulated GPR78. The knockdown of GPR78 can attenuate the proliferation, migration, and invasion of NSCLC cells and increase the apoptosis and autophagy of NSCLC cells that cisplatin has induced. Therefore, targeting GPR78 may be a promising treatment strategy for NSCLC patients.

[Ethoxysanguinarine directly targets CIP2A to inhibit proliferation and induce autophagy of SGC7901/DDP cells].

This study aims to investigate the effect of ethoxysanguinarine(Eth) on cisplatin(DDP)-resistant human gastric cancer cells and decipher the underlying mechanism. The human gastric cancer cell line SGC7901 and the DDP-resistant cell line SGC7901/DDP were used as the cell models. Western blot was employed to determine the expression levels of multidrug resistance-related proteins, and methyl thiazolyl tetrazolium(MTT) assay to detect the proliferation of SGC7901 and SGC7901/DDP cells exposed to DDP. After treatment with different concentrations of Eth, the proliferation of SGC7901 and SGC7901/DDP cells was detected by MTT assay, trypan blue exclusion assay, colony formation assay, and high-content imaging and analysis system. The apoptosis of SGC7901/DDP cells was detected by flow cytometry with Annexin V-FITC/PI staining. GFP-LC3 transfection was carried out to detect the effect of Eth on the autophagy of SGC7901/DDP cells. The expression levels of the multidrug resistance-related protein P-glycoprotein(P-gp), the apoptosis-related proteins [caspase-9, caspase-3, and poly(ADP-ribose) polymerase(PARP)], the autophagy-related protein light chain 3-Ⅱ(LC3-Ⅱ), the key effectors [mammalian target of rapamycin(mTOR), 70 kDa ribosomal protein S6 kinase(P70 S6 K), and 4 E binding protein 1(4 E-BP1)] of the mammalian target of rapamycin complex 1(mTORC1) signaling pathway, cancerous inhibitor of protein phosphatase 2A(CIP2A), and protein kinase B(Akt) were measured by Western blot. The mRNA level of CIP2A in the SGC7901/DDP cells exposed to Eth for 24 h was analyzed by RT-qPCR. After SGC7901/DDP cells were transfected with CIP2A expression vector pcDNA3.1-HA-CIP2A and treated with different concentrations of Eth, MTT assay was used to determine the prolife-ration of SGC7901/DDP cells and Western blot to detect the expression levels of related proteins. The interaction sites of Eth and CIP2A were predicted by molecular docking. The affinity between Eth and CIP2A was determined by drug affinity responsive target stability(DARTS) assay. The pharmacokinetic properties and drug-like activity of Eth were predicted by SwissADME. The results indicated that SGC7901/DDP cells were more sensitive to Eth than SGC7901 cells. Eth significantly inhibited proliferation and colony formation and changed the morphology, roundness, and area of SGC7901/DDP cells. Eth treatment caused the nucleus shrinking and significantly increased the apoptosis rate of the cells. Furthermore, Eth down-regulated the expression of caspase-9 and caspase-3 precursors and promoted the cleavage of PARP, which suggested that Eth induced the apoptosis of SGC7901/DDP cells. The GFP-LC3 in Eth-treated cells showed speckled aggregation. The up-regulated expression of LC3-Ⅱ by Eth indicated that Eth activated the autophagy of SGC7901/DDP cells. Eth down-regulated the expression of P-gp, the phosphorylation of mTOR, P70 S6K, and 4E-BP1, the expression of CIP2A, and the phosphorylation of Akt. Additionally, it increased the activity of PP2A, and had no significant effect on the expression of CIP2A in SGC7901/DDP cells. CIP2A overexpression antagonized the inhibition of cell proliferation and the activation of autophagy by Eth. Molecular docking suggested that Eth bound to CIP2A. The results of DARTS assay further proved the above binding effect. Eth has potential drug-like activity. The above results demonstrated that Eth inhibited the proliferation, induced the apoptosis, and activated the autophagy of SGC7901/DDP cells by targeting CIP2A and then down-regulating PP2A/mTORC1 signaling pathway. This study provided a new target for the treatment of cisplatin-resistant gastric cancer.

Abietic acid ameliorates nephropathy progression via mitigating renal oxidative stress, inflammation, fibrosis and apoptosis in high fat diet and low dose streptozotocin-induced diabetic rats.

BACKGROUND: Abietic acid (AA) has been reported to exhibit anti-inflammatory activity, however its protective effect against inflammation and its trigger factor i.e., oxidative stress and the related sequelae i.e., apoptosis and fibrosis in the kidney in diabetes mellitus (DM) is unknown. PURPOSE: To identify the ability of AA to mitigate the inflammatory and inflammation-related insults to the kidney in DM. METHODS & STUDY DESIGN: Adult male rats were induced type-2 DM by feeding with a high-fat diet for twelve weeks followed by injection with a single dose of streptozotocin (STZ) (30 mg/kg/bw) intraperitoneally at twelve weeks. Following DM confirmation, AA (10 and 20 mg/kg/day) was given orally for another four weeks. Then the fasting blood glucose (FBG) and renal profile were determined and oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) tests were performed. A day after the last treatment, rats were sacrificed and kidneys were harvested and subjected for histopathological and molecular biological analysis. RESULTS: AA treatment was found to reduce the FBG, serum urea and creatinine levels (p < 0.05) while improving the OGTT and ITT (p < 0.05) in diabetic rats. Besides, AA treatment also mitigated kidney histopathological changes, reduces kidney oxidative stress as reflected by reduced levels of RAGE and Keap1 but increased levels of kidney antioxidants Nrf2, SOD, CAT, GPX, HO-1 & NQO-1 (p < 0.05). Additionally, AA treatment also decreases kidney inflammation (NF-kB p65, IL-1ß, IL-6, TNF-α and iNOS) and fibrosis (TGF-ß1 and GSK-3ß) (p < 0/05). Kidney apoptosis decreased as reflected by decreased levels of Bax, caspase-3 and caspase-9 while its anti-apoptosis Bcl-2 protein levels increased (p < 0.05). CONCLUSION: AA helps to mitigate nephropathy development in DM via counteracting oxidative stress, inflammation and apoptosis.

[Effect of administration of acupuncture stimulation combined with Gushen Zhuyu Decoction on lumbar function in patients with disc herniation].

OBJECTIVE: To investigate the therapeutic effect of acupuncture stimulation combined with administration of"Gushen Zhuyu Tang"(decoction for consolidating kidney to eliminate blood stasis, DCKEBS) in the treatment of lumbar disc herniation (LDH) patients. METHODS: A total of 147 patients with LDH were randomly divided into DCKEBS, acupuncture and DCKEBS+acupuncture groups (n= 49 cases in each group). The patients of the acupuncture group received a) acupuncture stimulation of Dazhui (GV14), Ganshu (BL18), Shenshu (BL23), Tianshu (ST25), Yanglingquan (GB34), etc., b) fire needle pricking of the topical tendons, cord-like points, tender-points, c) row-needles stimulation of the attachment sites of muscles of the sacroiliac joint or crista iliaca, and d) acupotomy-debonding of the topical high-tension muscles, twice a week for 4 weeks. Those patients of the DCKEBS group were ordered to take DCKEBS [containing fried Yiyiren (Semen Coicis), Shanzhuyu (Fructus Corni), fried Baizhu (Rhizoma Astractylodis), Sangjisheng (Ramulus Loranthi), Duzhong (Cortex Eucommiae), Buguzhi (Fructus Psoraieae), etc.] 150 mL, twice daily, continuously for 4 weeks, and those of the DCKEBS+acupuncture group received the combined treatment mentioned above in the acupuncture and DCKEBS groups. The pain severity was assessed by using visual ana-logue scale (VAS, 0-10 points) and the modified Japan Orthopaedic Association questionnaire (M-JOA) score (0-30 points), separately, and the lumbar range of motion (ROM) and lumbar muscle strength were tested to evaluate the lumbar motor function. The levels of serum tumor necrosis factor α(TNF-α), matrix metalloproteinase-2 (MMP-2), and apoptosis related factors Caspase-3 and Caspase-9 were assayed using ELISA. The total effective rates of the three groups were compared. RESULTS: After the treatment, the VAS and M-JOA scores, contents of serum TNF-α, MMP-2, Caspase-3 and Caspase-9 were significantly decreased (P<0.01), and the myodynamia of lumbar muscular flexor and extensor was considerably increased (P<0.01) in the three groups, and the ROM angles of lumbar extending and buckling were increased (P<0.01) in the DCKEBS+acupuncture group compared with pretreatment. Comparison among the 3 groups showed that the VAS and M-JOA scores, and serum TNF-α, MMP-2, Caspase-3 and Caspase-9 contents of the DCKEBS+acupuncture group were significantly lower than those of both DCKEBS and acupuncture groups (P<0.01), while the ROM angles of lumbar extending and buckling, and the myodynamia of lumbar muscular flexor and extensor were obviously higher in the DCKEBS+acupuncture group than those of the DCKEBS and acupuncture groups (P<0.01). The total effective rate was 93.88%(46/49) in the DCKEBS+acupuncture group, higher than 75.51%(37/49) in the DCKEBS group and 71.43%(35/49) in the acupuncture group (P<0.05). CONCLUSION: Acupuncture combined with DCKEBS can relieve pain, improve lumbar muscle strength and lumbar movement function, and reduce serum TNF-α, MMP-2, Caspase-3 and Caspase-9 levels in LDH patients.

Apoptotic and Anti-metastatic Effects of Atractylodes lancea (Thunb.) DC. in a Hamster Model of Cholangiocarcinoma.

OBJECTIVES: Cholangiocarcinoma (CCA) is a highly aggressive tumor with a greater risk of distant metastasis. The promising anti-CCA activity and safety profile of Atractylodes lancea (AL) have previously been reported in a series of in vitro, in vivo and clinical studies. The present study investigated the effect of AL extract on apoptosis and metastasis signaling pathways in the Opisthorchis viverrini/dimethylnitrosamine (OV/DMN)-induced CCA hamster model. MATERIALS AND METHODS: Hamster liver tissues were obtained from the four groups (n = 5 per group), i.e., (i) 5-FU treated CCA (40 µg/mL); (ii) CCA; (iii) AL-treated CCA (5,000 mg/kg), and (iv) normal hamsters. Total RNA was isolated, and the expression levels of apoptosis-related and metastasis-related genes were determined by qRT-PCR analysis. RESULTS: The expression levels of p16, caspase-3, caspase-8, caspase-9, Apaf-1, p53 and Eef1a1 were downregulated, while that of the remaining genes were upregulated in CCA hamsters compared with normal hamsters. AL treatment increased the expression of p16, caspase-9, caspase-3, Apaf-1, p53 and E-cadherin and decreased the expression of cyclin D1, cdk4, Bax, Akt/PKB, Bcl-2, Mfge-8, Lass4, S100A6, TGF-ß, Smad-2, Smad-3, Smad-4, MMP-9, and N-cadherin. The expression of Eef1a1 was unchanged. CONCLUSION: The anti-CCA activity of AL in OV/DMN-induced CCA hamsters could be due to the induction of cell cycle arrest at the G1 phase and activation of the apoptosis pathway, resulting in cancer cell death. The activation of the apoptosis pathway mainly involved the intrinsic pathway (activation of caspase-3 and caspase-9 through p53 and Mfge-8 modulation and downregulation of anti-apoptotic genes Akt and Bcl-2). In addition, AL could also inhibit the canonical TGF-ß signaling pathway, MMP-9 and N-cadherin to suppress tumor metastasis.