Exploring the effect of disc displacement on the risk and severity of condylar erosion in adult temporomandibular disorder patients: A CBCT and MRI study.

OBJECTIVE: The objective of the study was to investigate the relationship between types of disc displacement (DD) diagnosed by magnetic resonance imaging (MRI), and the risk (presence or absence) and severity of condylar erosion (CE) graded using cone-beam computed tomography (CBCT) in adult Temporomandibular disorders (TMD) patients. METHODS: A total of 353 TMD patients (283 females, 70 males) underwent MRI scans to categorise DD as normal (NA), anterior displacement with reduction (ADDR), or anterior displacement without reduction (ADDNR). CE severity was graded on a scale of 0-3 (absence, mild, moderate or severe) using CBCT. To establish the plausibility and cut-off points for CE diagnosis, the severity of CE was then further divided into three classifications: Grade 0 versus 1 + 2 + 3; Grades 0 + 1 versus 2 + 3; Grades 0 + 1 + 2 versus 3. Logistic regression analysis was performed, adjusting for age, gender and joint correlation. RESULTS: ADDNR significantly increased the risk of CE compared with NA (OR = 10.04, 95% CI: [6.41, 15.73]) and showed a significant increase in CE severity across all classifications (ORs = 10.04-18.95). The effects of ADDNR were significant in both genders (p < .001) and had a greater impact in females. ADDR was predominantly associated with mild CE. CONCLUSIONS: ADDNR significantly increased the risk and severity of CE independent of gender when compared to NA, whereas ADDR was mainly associated with mild CE. Slight cortical discontinuity may represent a subclinical diagnosis requiring further investigation.

The effect of root orientation on inferior alveolar nerve injury after extraction of impacted mandibular third molars based on propensity score-matched analysis: a retrospective cohort study.

BACKGROUND: The injury of the inferior alveolar nerve (IAN) is one of the most serious complications of impacted mandibular third molars (IMTMs) extraction. The influence of the root orientation of IMTMs on IAN injury is still controversial. A deeper understanding of the risk factors of IAN injury conduces to better prevention of IAN injury. This study aims to explore whether root orientation is an independent risk factor of IAN injury during IMTMs extraction using the statistical strategy of propensity score matching (PSM). METHODS: This retrospective cohort study included 379 patients with 539 cases of high-risk IMTMs screened by panoramic radiography and cone beam computed tomography. The IAN injury incidence after extraction of different groups of IMTMs was analyzed using the chi-square test or Fisher's exact test. The correlation between third molar root orientation and impaction depth/contact degree with IAN was evaluated by the Lambda coefficient. Based on PSM for balancing confounding factors including age, sex, impaction depth, and contact degree, the effect of root orientation on the incidence of IAN injury was further analyzed using Fisher's exact test. RESULTS: There were significant group differences in IAN injury incidence in impaction depth, root orientation, and contact degree of root-IAC before PSM. Root orientation was correlated with impaction depth and contact degree of root-IAC. After PSM, there were 9 cases with IAN injury and 257 cases without IAN injury. There were significant group differences between the buccal and non-buccal groups after PSM, and the risk of IAN injury was higher when the root was located on the buccal side of IAC (OR = 8.448, RR = 8). CONCLUSIONS: Root orientation is an independent risk factor of IAN injury, and the risk is higher when the root is located on the buccal side of IAC. These findings could help better evaluate the risk of inferior alveolar nerve injury before the extraction of IMTMs.

Wnt/ß-catenin signaling pathway is activated in the progress of mandibular condylar cartilage degeneration and subchondral bone loss induced by overloaded functional orthopedic force (OFOF).

Objective: To explore the role of Wnt/ß-catenin signaling pathway in the pathogenesis and progression of temporomandibular joint osteoarthritis (TMJ OA) caused by overloaded force. Materials and methods: We generated a rat model of forward mandibular extension device to induce TMJ OA by overloaded force. Condylar cartilage samples were collected at 2wk, 4wk, and 8wk after appliances were installed. Changes of the condylar cartilage and subchondral bone were evaluated by hematoxylin and eosin (HE), Safranin O and Fast Green staining (SO&FG), micro-CT, tartrate resistant acid phosphatase (TRAP) staining. The expression levels of ß-catenin, COL-2, MMP3 and sclerostin (SOST) were detected by immunohistochemistry (IHC) and PCR. Results: HE, SO&FG, micro-CT, OARSI and Mankin scores showed that the condyle cartilage layer was significantly thinner and proteoglycan loss in the overloded group. TRAP staining exhibited that the number of positive osteoclasts increased and OPG level decreased in the overload group. IHC, PCR showed that the expression of COL2 and SOST decreased, while MMP3 and ß-catenin increased in the overload group. Conclusion: Wnt/ß-catenin signaling pathway is activated in the progress of mandibular condylar cartilage degeneration and subchondral bone loss induced by overloaded functional orthopedic force (OFOF).

Excessive mechanical stress induced temporomandibular joint osteoarthritis via osteoclasts-mediated osteogenic differentiation of BMSCs.

BACKGROUND: Bone homeostasis is a dynamic process maintained by osteoblasts and osteoclasts, which may be regulated by excessive mechanical stress (EMS). OBJECTIVES: Our study aimed to explore the relationship between osteogenic differentiation of BMSCs and EMS-activated osteoclast differentiation of RAW 264.7 cells in order to optimise orthodontic treatment. METHODS: We established the model of EMS in vivo and in vitro. In vivo, HE, Safranin-O staining, micro-CT, and immunofluorescence double-labelling were utilised to assess the changes in condylar, the distributions of osteoblasts, osteoclasts and MAPKs. In vitro, the effects of EMS-activated osteoclast differentiation exerting on osteogenic differentiation of BMSCs were observed by Western Blot, qRT-PCR and Alizarin Red staining. Furthermore, the role of MAPKs in this progress was explored by using inhibitors of MAPKs and co-culture supernatants. RESULTS: In vivo, EMS led to the degradation of condylar cartilage and destruction of subchondral bone, diagnosed as temporomandibular joint osteoarthritis (TMJ OA). Osteoclasts and osteoblasts were both enriched in subchondral bone, but osteoclast predominated. The expressions of p-JNK, p-ERK1/2, and p-p38 were all activated in vitro and in vivo, which were localised mainly in the Trap+ area in subchondral bone. Interestingly, only the inactivation of p-ERK1/2 in osteoclasts significantly inhibited the osteogenic differentiation of BMSCs in vitro. This revealed that p-ERK1/2 played a key role in the osteoclasts-induced osteogenic differentiation of BMSCs. CONCLUSION: Our results proved that EMS led to TMJ OA, in which upregulated p-ERK1/2 in osteoclasts was mechanosensitive and facilitated the osteogenic differentiation of BMSCs.

Senegenin alleviates Aß1-42 induced cell damage through triggering mitophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Senegenin (SEN), an active compound extracted from the traditional Chinese herb Polygala tenuifolia Willd. (a species in the genus Polygala, family Polygalaceae), could nourish neurons and resist neuronal damage in mouse models of Alzheimer's disease (AD). Amyloid-ß (Aß) depositions in neuronal cells may cause pathological changes such as oxidative stress which one return could cause severe damage to mitochondria in AD patients or animal models. Mitophagy is an important mechanism to selectively remove damaged mitochondria. In neurons, this process is mainly mediated by PTEN-induced putative kinase 1 (PINK1)/Parkin pathway. Previous studies have shown that SEN could reduce mitochondrial damage and inhibit apoptosis in neurons. Therefore, this study speculated that SEN might activate mitophagy to clear damaged mitochondria, thereby mitigating Aß-induced cell damage in neuronal cells. AIM OF THE STUDY: This study aimed to determine the effects of SEN on Aß-induced cell damage, and further to explore whether SEN could induce mitophagy. Moreover, the regulatory role of mitophagy in the neuroptrotective effect of SEN would be elucidated. MATERIALS AND METHODS: This study established an in vitro cell damage model using Aß1-42 to treat mouse hippocampal neuron HT22 cells. The effects of SEN on cell damage were determined by MTT assay and lactate dehydrogenase (LDH) release assay. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected by Cytation™5 cell imaging microplate detection system. The apoptotic rate was analyzed by flow cytometry. The effects of SEN on mitophagy were detected by transmission electron microscope, immunofluorescence and immunoblotting. RESULTS: Firstly, HT22 cells were treated with 30 µM Aß1-42 for 24 h to establish the damage model. It was found that 30 µM Aß1-42 caused neuronal damages as evidenced by reduced cell viability, increased LDH release and ROS, collapsed MMP and elevated apoptosis. Secondly, Aß1-42-incubated cells were treated with 10, 20, 40 and 60 µM SEN for 24 h. SEN significantly reduced the damage of Aß1-42-incubated cells as shown by recovered cell viability and MMP, reduced apoptosis and ROS. Notably, SEN induced the formation of mitophagosomes and mitolysosomes, and elevated the conversion of LC3 I to LC3 II. Moreover, SEN down-regulated the expression of p62, promoted the accumulation of full-length PINK1 and the translocation of Parkin to mitochondria, decreased the expression of mitochondrial matrix protein HSP60, thus activating the PINK1/Parkin-mediated mitophagy. However, when cells were pretreated with 5 µM CsA (Cyclosporine A, a mitophagy inhibitor) for 2 h and then co-treated with 20 and 40 µM SEN for 24 h, the protective effects of SEN were compromised. CONCLUSIONS: The present study demonstrated that SEN could alleviate Aß1-42-induced cell damage through PINK1/Parkin-mediated mitophagy. Our findings justify the traditional use of P. tenuifolia in China with anti-aging or anti-neurodegenerative effects.

The anti-tussive, anti-inflammatory effects and sub-chronic toxicological evaluation of perilla seed oil.

BACKGROUND: Perilla seed oil (PSO) is the main constituent of perilla seeds currently being used in the food industry, however it also has great clinical potential in the regulation of lung function as a nutrition supplement because of the high content of α-linolenic acid (ALA). In this study, the pharmacological activities including anti-tussive, expectorant and anti-inflammatory effect of PSO were performed. Furthermore, the 90-day sub-chronic oral toxicity with a 30 day recovery period was evaluated in Wistar rats. RESULTS: The pharmacological studies demonstrated that PSO inhibited cough frequency induced by capsaicine in mice. PSO also inhibited the leukotriene B4 (LTB4) release from the calcium ionophore A23187-induced polymorphonuclear neutrophils (PMNs) to some extent. In this sub-chronic toxicity study, mortality, clinical signs, body weight, food consumption, hematology, serum biochemistry, urinalysis, organ weight, necropsy, and histopathology were used to evaluate the toxicity of PSO. Lower body weight and various negative impacts on liver related parameters without histopathological lesion were observed in the 16 g kg-1 groups. No clinically significant changes were discovered in the 4 g kg-1 group during the test period. CONCLUSION: In summary, PSO exhibited anti-tussive and anti-inflammatory activities in vivo and in vitro. These sub-chronic toxicity studies inferred that the 'no-observed adverse effect level' (NOAEL) of PSO in Wistar rats was determined to be 4 g kg-1 . These results may provide a safety profile and a valuable reference for the use of PSO. © 2020 Society of Chemical Industry.

Novel Insights Into the Role of N6-Methyladenosine RNA Modification in Bone Pathophysiology.

Thus far, there are more than known 150 modifications to RNA, in which common internal modifications of mRNA include N6-methyladenosine (m6A), N1-methyladenosine, and 5-methylcytosine. Among them, m6A RNA modification is one of the highest abundance modifications in eukaryotes, regulating mechanisms controlling gene expression at the post-transcription level. As an invertible and dynamic epigenetic marker, m6A base modification influences almost all vital biological processes, cellular components, and molecular functions. Once the m6A modification process is abnormal, a series of diseases-including cancer, neurological diseases, and growth disorders-will be caused. Besides, several base modification activities also have been created by noncoding RNAs (ncRNAs), for instance, microRNAs, and circular RNAs, long ncRNAs, which were dynamically regulated during bone and cartilage pathophysiology processes. Therefore, it has now been clear that dynamic modification on coding RNAs and ncRNAs represents a completely new way to modulate genetic information. In this review, we highlight up-to-date progress and applications of m6A RNA modification in bone and cartilage pathophysiology, and we discuss the pathological roles and underlying molecular mechanism of m6A modifications in osteoarthritis and osteoporosis and osteosarcoma pathogenesis.

Overloaded Orthopedic Force Induces Condylar Subchondral Bone Absorption by Stimulating Rat Mesenchymal Stem Cells Differentiating into Osteoclasts via mTOR-Regulated RANKL/OPG Secretion in Osteoblasts.

Appropriate orthopedic force led to bone remodeling of mandibular condyle, while overloaded orthopedic force (OOF) induced condylar bone absorption. Bone absorption is ascribed to the imbalanced activities between osteoclasts (OCs) and osteoblasts (OBs), mechanism of which remains unclear. This study aimed to observe the condylar changes induced by OOF by mandible advancement appliance and to further investigate the role of mammalian target of Rapamycin (mTOR) and RANKL/OPG in osteoclastic differentiation of stem cells in vivo and in vitro. In vivo, the results of micro-CT analysis indicated that condylar bone resorption was induced by OOF through mandibular advancement appliance for 2 weeks and worsened time dependently. Morphologically, cartilage thickness was reduced, subchondral cortical bone line appeared not continuous, and subchondral bone exhibited irregular-shaped and owned uneven surface. The bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were decreased accomplished with the increased trabecular separation (Tb.Sp) determined by micro-CT. In addition, based on immunofluorescent labeling, OOF activated both OCs and OBs, but osteoclastogenesis prevailed over osteogenesis. The mTOR activation and ratio of RANKL/OPG in OBs were elevated by OOF. In vitro, the results of western blot and polymerase chain reaction (PCR) consistently suggested that the mTOR and RANKL/OPG ratio were upregulated by overloaded mechanical stretch. Pretreatment with mTOR inhibitor, rapamycin, could attenuate the activation of mTOR and the secretion of RANKL in OBs. Interestingly, based on the Trap staining, the supernatant of OBs exposed to OOF could promote osteoclastic differentiation of mesenchymal stem cells (MSCs), while its role was weakened by inhibition of mTOR in OBs. Collectively, OOF induced condylar bone absorption; in the process, osteoclastogenesis was prominent than osteogenesis. The activation of mTOR and secretion of RANKL/OPG were enhanced by OOF and were involved in promoting MSCs differentiating into OCs.

The role of endoplasmic reticulum stress in lead (Pb)-induced mitophagy of HEK293 cells.

It is well-documented that lead (Pb) toxicity can affect almost all systems in living organisms. It can induce selective autophagy of mitochondria (mitophagy) by triggering reactive oxygen species production. Emerging evidence has suggested that Pb-induced autophagy can also be activated by the endoplasmic reticulum (ER) stress pathway. However, the interplay between ER stress and mitophagy remains to be elucidated. In this study, human embryonic kidney HEK293 cells were employed to investigate the role of ER stress in Pb-induced mitophagy. The results showed that the cell viability was decreased and cell damage was induced after exposure to Pb (0, 0.5, 1, 2, and 4 mM) for 24 h in a dose-dependent manner. Moreover, the expression of LC3-Ⅱ was significantly increased, and the expression of HSP60 was dramatically decreased after exposure to 1 mM and 2 mM Pb, indicating the induction of mitophagy following Pb exposure. Meanwhile, the expressions of activating transcription factor 6, inositol-requiring protein-1α, CCAAT/enhancer binding protein homologous protein, and glucose-regulated protein 78 were dramatically increased after Pb treatment, signifying the initiation of ER stress. Notably, the mitophagic effect was significantly compromised when ER stress was inhibited by 0.5 mM 4-phenylbutyrate, which was evidenced by lesser decreases in HSP60 expression and level of LC3-Ⅱ, suggesting Pb-induced mitophagy may be activated by the ER stress. Taken together, these findings provide a better understanding of Pb toxicity and suggest that Pb-induced ER stress may play a regulatory role in the upstream of mitophagy.

[Autophagy induced by cyclic mechanical stretch inhibited caspase-dependent apoptosis of myoblasts].

PURPOSE: The aim of this study was to investigate the molecular mechanism of autophagy and apoptosis induced by cyclic mechanical stretch and the potential role of autophagy in stretch-induced apoptosis of myoblasts. METHODS: Loading model of L6 myoblasts was established in vitro. The cells were then subjected to cyclic mechanical stretch involving 3 s of 15% stretch alternating with 3 s of relaxation. The cells were collected after mechanical stretch for 6 h, 12 h and 24 h, respectively. Control cells were cultured on the same plates without mechanical strain. Apoptosis of myoblasts was assessed by Hoechst 33258 staining and Annexin V binding and propidium iodide staining. Autophagy was determined by MDC staining and transmission electron microscopy(TEM). The level of proteins associated with apoptosis and autophagy was detected by Western blot. The data were analyzed with SPSS 17.0 software package. RESULTS: The results of Hoechst 33258 staining and Annexin V binding and propidium iodide staining indicated that mechanical stretch notably induced apoptosis of myoblasts. Caspase inhibitor z-VAD-fmk effectively abrogated apoptosis of myoblasts, indicating mechanical stretch induced caspase-dependent apoptosis. In addition, the results of TEM, MDC staining and Western blot proved that mechanical stretch elicited autophagy of myoblasts. Inhibition of autophagy using 3-MA enhanced caspase-dependent apoptosis induced by mechanical stretch. CONCLUSIONS: Cyclic mechanical stretch induced apoptosis and autophagy of myoblasts time-dependently. Protective autophagy, acting as the compensatory mechanism, inhibited caspase-dependent apoptosis induced by mechanical stretch.

A TAT peptide-based ratiometric two-photon fluorescent probe for detecting biothiols and sequentially distinguishing GSH in mitochondria.

Although biothiols, including cysteine (Cys), glutathione (GSH), and homocysteine (Hcy) can be used to diagnose many diseases and research physiological metabolism in many physiological processes, in situ real-time detection and differentiation of biothiols is still challenging because their similar chemical properties and molecular structures. Herein, we utilized the native chemical ligation (NCL) reaction mechanism to develop a Förster resonance energy transfer (FRET) strategy for designing a cell penetration peptide TAT-modified ratiometric two-photon biothiols probe (TAT-probe). The TAT-probe can not only rapidly enter into mitochondria assisted by TAT peptide, but also simultaneously detect biothiols and sequentially distinguish GSH. When the TAT-probe was excited with 404/820 nm wavelength light, it showed a change in the ratio of fluorescence after adding biothiols, including a quenched red fluorescence intensity (λem = 585 nm) and an enhanced signal in green fluorescence intensity (λem = 520 nm). Excitingly, the TAT-probe excited at 545 nm could display a red fluorescence (λem = 585 nm) towards GSH and a quenched signal towards Hcy or Cys. This specific fluorescence response indicated the TAT-probe could effectively detect biothiols and differentiate GSH from Cys/Hcy in mitochondria. This work pioneered a new approach to design and synthesize biothiol-probes based on peptides and NCL reaction mechanism.

Phosphorylated GSK­3ß protects stress­induced apoptosis of myoblasts via the PI3K/Akt signaling pathway.

Facial jaw muscle is involved in the occurrence, development, treatment and maintenance of maxillofacial deformities. The structure and function of this tissue can be altered by changes in external stimuli, and orthodontists can regulate its reconstruction using orthopedic forces. The PI3K/Akt signaling pathway is most well­known for its biological functions in cell proliferation, survival and apoptosis. In the present study, the effects of the PI3K/Akt signaling pathway in cyclic stretch­induced myoblast apoptosis were investigated. For this purpose, L6 rat myoblasts were cultured under mechanical stimulation and treated with the PI3K kinase inhibitor, LY294002, to elucidate the role of the PI3K/Akt signaling pathway. Cells were stained with Hoechst 33258 to visualize morphological changes and apoptosis of myoblasts, and western blotting was performed to detect expression of Akt, phosphorylated (p)­Akt (Ser473), glycogen synthase kinase 3ß (GSK­3ß) and p­GSK­3ß (Ser9). After addition of PI3K inhibitor, the expression of total Akt and GSK­3ß did not significantly differ among groups; however, the levels of p­Akt and p­GSK­3ß were lower in inhibitor­treated groups than in those treated with loading stress alone. In addition, the rate of apoptosis in myoblasts subjected to cyclic stretch increased in a time­dependent manner, peaking at 24 h. Collectively, it was also demonstrated that the PI3K/Akt/GSK­3ß pathway plays an important role in stretch­induced myoblast apoptosis.

NIX compensates lost role of parkin in cd-induced mitophagy in HeLa cells through phosphorylation.

Our previous study demonstrated that cadmium (Cd) is an effective inducer of mitophagy, which is mainly mediated by PINK1/Parkin pathway. However, the role of other mitophagy pathways in Cd-induced mitophagy remains elusive. The present study employed HeLa cells, lacking fully functional Parkin, as a cell model to study Parkin-independent mitophagy pathway induced by Cd. Our results showed that BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (Bnip3L/NIX), an outer mitochondrial membrane mitophagy receptor, could provide an alternate pathway for Cd-induced mitophagy in HeLa cells. Specifically, 10 µM Cd for 12 h induced mitophagy in GM00637 and HeLa cells which was assessed by mitochondrial fusion to lysosomes and decreased expression of mitochondrial markers such as COX-IV and HSP60. Notably, in GM00637 cells, Cd-induced mitophagy was predominantly mediated by PINK1/Parkin pathway as evinced by translocation of Parkin to mitochondria. Interestingly, in HeLa cells, significant increase in NIX expression was occurred and mitophagy was induced under Cd exposure, suggesting NIX compensates lost role of Parkin in Cd-induced mitophagy in HeLa cells. These results were verified by knocking down NIX using siRNA in HeLa cells, which lead to abolished mitophagy process. Moreover, NIX phosphorylation at serine-81 significantly increased in cells treated with Cd implying that phosphorylation of NIX plays an important role in NIX-mediated mitophagy. These findings reveal a novel mechanism of Cd toxicity and suggest a compensatory role of NIX in Cd-induced mitophagy.

[The role and mechanism of endoplasmic reticulum stress key factor PREK in myoblast apoptosis under stress loading].

PURPOSE: This study was aimed to figure out the way that cyclic-stretch influenced the apoptosis of myoblasts and evaluate the importance of PERK and its possible mechanism involved. METHODS: L6 rat myoblasts were cultured in vitro and mechanical stimulation model was constructed successfully. The myoblasts were imposed tension for 0, 2, 6, 12 and 24 hours respectively by multi-channel cell stress loading system. The force value was 15% cell deformation and the frequency was 10 cycles/min. Each cycle was consisted of stretch for 3 seconds and relaxation for 3 seconds, and the group without tension was used as the control group. The apoptotic myoblasts were dyed by DAPI and observed through fluorescence microscopy to detect the apoptosis rate; the mRNA levels of PERK and CHOP in different groups were detected by real-time PCR and protein levels of PERK and p-PERK in different groups were detected by Western blot. PERK inhibitor was used to clear the role of PERK in apoptosis induced by cyclic-stretch and clarify the relationship between the endoplasmic reticulum stress and apoptosis induced by cyclic-stretch. SPSS 17.0 software package was used to analyze the data statistically. RESULTS: DAPI nuclear stain showed that cyclical tensile stress can induce apoptosis in vitro cultured myoblast. Apoptosis rate showed a trend of rising gradually over time, peaked at 24 h. After dealt with the inhibitor of PERK, the apoptosis rate of the 24 h group under the cyclic stretch showed no difference compared with the control. The results of real- time PCR showed that the mRNA of CHOP was increased with the extension loading time, while the mRNA of PERK showed no difference compared with the control. Western blot results showed that the protein level of p-PERK was increased with the extension of loading time, while the expression of PERK showed no difference compared with the control group. When PERK inhibitor added, the mRNA level of CHOP along with the protein expression level of p-PERK showed no significant difference compared to the control. CONCLUSIONS: PERK signaling pathway is involved in the apoptosis of myoblasts induced by cyclic stretch, and the possible mechanism may be closely related to the phosphorylation of PERK.

Acute and sub-chronic 90-day oral toxicity study of Perilla seed oil in rodents and Beagle dogs.

Perilla seeds are used as food and traditional medicine in China. This study aimed to investigate the toxicity profile of Perilla seed oil (PSO), which is the main constituent of Perilla seeds in rodents and Beagle dogs. No significant treatment-associated toxicity or mortality was observed at PSO dosages of up to 50 g/kg and 20 g/kg in KM mice and Wistar rats, respectively, suggesting that PSO was well tolerated by the experimental rodents. Sub-chronic oral toxicity of PSO was studied in dogs at doses of 3, 6 and 12 g/kg/d for 90 days followed by a 30 day recovery period. The results indicated that the body weight increased in all-dose groups more than control group, typical of animals on diets rich in fatty acids. Treatment-related side effects, including changes in hematology and serum biochemistry parameters, histopathology of liver and lymph glands, were observed in the high and moderate-dose dogs. However, these changes disappeared after the doses were withdrawn during the recovery period, except for alteration of liver in the high-dose group. In conclusion, the "no observed adverse effect level" (NOAEL) of oral administration of PSO for 90 days in Beagle dogs was considered to be 3 g/kg/d.

Characterization of Microbial Communities in Chinese Rice Wine Collected at Yichang City and Suzhou City in China.

Two typical microbial communities from Chinese rice wine fermentation collected in Yichang city and Suzhou city in China were investigated. Both communities could ferment glutinous rice to rice wine in 2 days. The sugar and ethanol contents were 198.67 and 14.47 mg/g, respectively, for rice wine from Yichang city, and 292.50 and 12.31 mg/g, respectively, for rice wine from Suzhou city. Acetic acid and lactic acid were the most abundant organic acids. Abundant fungi and bacteria were detected in both communities by high-throughput sequencing. Saccharomycopsis fibuligera and Rhizopus oryzae were the dominant fungi in rice wine from Suzhou city, compared with R. oryzae, Wickerhamomyces anomalus, Saccharomyces cerevisiae, Mucor indicus, and Rhizopus microsporus in rice wine from Yichang city. Bacterial diversity was greater than fungal diversity in both communities. Citrobacter was the most abundant genus. Furthermore, Exiguobacterium, Aeromonas, Acinetobacter, Pseudomonas, Enterobacter, Bacillus, and Lactococcus were highly abundant in both communities.

ATM mediates spermidine-induced mitophagy via PINK1 and Parkin regulation in human fibroblasts.

The ATM (ataxia telangiectasia mutated) protein has recently been proposed to play critical roles in the response to mitochondrial dysfunction by initiating mitophagy. Here, we have used ATM-proficient GM00637 cells and ATM-deficient GM05849 cells to investigate the mitophagic effect of spermidine and to elucidate the role of ATM in spermdine-induced mitophagy. Our results indicate that spermidine induces mitophagy by eliciting mitochondrial depolarization, which triggers the formation of mitophagosomes and mitolysosomes, thereby promoting the accumulation of PINK1 and translocation of Parkin to damaged mitochondria, finally leading to the decreased mitochondrial mass in GM00637 cells. However, in GM05849 cells or GM00637 cells pretreated with the ATM kinase inhibitor KU55933, the expression of full-length PINK1 and the translocation of Parkin are blocked, and the colocalization of Parkin with either LC3 or PINK1 is disrupted. These results suggest that ATM drives the initiation of the mitophagic cascade. Our study demonstrates that spermidine induces mitophagy through ATM-dependent activation of the PINK1/Parkin pathway. These findings underscore the importance of a mitophagy regulatory network of ATM and PINK1/Parkin and elucidate a novel mechanism by which ATM influences spermidine-induced mitophagy.

EGCG inhibits Cd(2+)-induced apoptosis through scavenging ROS rather than chelating Cd(2+) in HL-7702 cells.

CONTEXT AND OBJECTIVE: Epigallocatechin-3-gallat (EGCG), the major catechin in green tea, shows a potential protective effect against heavy metal toxicity to humans. Apoptosis is one of the key events in cadmium (Cd(2+))-induced cytotoxicity. Nevertheless, the study of EGCG on Cd(2+)-induced apoptosis is rarely reported. The objective of this study was to clarify the effect and detailed mechanism of EGCG on Cd(2+)-induced apoptosis. METHODS: Normal human liver cells (HL-7702) were treated with Cd(2+) for 21 h, and then co-treated with EGCG for 3 h. Cell viability, apoptosis, intracellular reactive oxygen species (ROS), malondialdehyde (MDA), mitochondrial membrane potential (MMP) and caspase-3 activity were detected. On the other hand, the chelation of Cd(2+) with EGCG was tested by UV-Vis spectroscopy analysis and Nuclear Magnetic Resonance ((1)H NMR) spectroscopy under neutral condition (pH 7.2). RESULTS AND CONCLUSION: Cd(2+) significantly decreased the cell viability and induced apoptosis in HL-7702 cells. Conversely, EGCG co-treatment resulted in significant inhibition of Cd(2+)-induced reduction of cell viability and apoptosis, implying a rescue effect of EGCG against Cd(2+) poisoning. The protective effect most likely arises from scavenging ROS and maintaining redox homeostasis, as the generation of intracellular ROS and MDA is significantly reduced by EGCG, which further prevents MMP collapse and suppresses caspase-3 activity. However, no evidence is observed for the chelation of EGCG with Cd(2+) under neutral condition. Therefore, a clear conclusion from this work can be made that EGCG could inhibit Cd(2+)-induced apoptosis by acting as a ROS scavenger rather than a metal chelating agent.