Objective: To examine the effect of intracellular vesicles (IVs) and extracellular vesicles (EVs) that originated from periodontal ligament stem cells (PDLSCs) on the osteogenic differentiation of PDLSCs within a lipopolysaccharide (LPS)-simulated inflammatory microenvironment, and to provide new insights for the application of IVs in the repair and regeneration of periodontal tissue in periodontitis. Methods: Ethical approval was obtained from the institution. Human-origin PDLSCs were extracted, and the IVs and EVs from PDLSCs at the 3rd-6th passages were gathered and identified using transmission electron microscopy, nano flow cytometry (Nano FCM) analysis, and Western Blot. The 3rd-6th generations of PDLSCs were categorized into the following groups: Control group, LPS group, LPS + 100 μg/mL EVs group (LPS+EVs group), and LPS + 100 μg/mL IVs group (LPS+IVs group). The effects of the IVs and EVs on the anti-inflammatory and osteogenic differentiation of PDLSCs in an inflammatory microenvironment were assessed by using a Cell Counting Kit-8 (CCK-8), enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), Western Blot, alkaline phosphatase (ALP) staining, and alizarin red staining (ARS). Results: Under transmission electron microscopy, the IVs and EVs derived from PDLSCs displayed a double-layer membrane structure. NanoFCM analysis revealed that the average diameters of the IVs and EVs were 79.6 nm and 82.1 nm, respectively. Western Blot analysis indicated that the surface proteins CD9, CD63, and CD81 of the IVs and EVs were positively expressed, while calnexin was negatively expressed, indicating that IVs and EVs were successfully obtained. Compared with the Control group, the proliferation of PDLSCs in the LPS group was reduced, while the levels of inflammatory cytokine interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the cell supernatant were increased, the mRNA expressions of osteogenic differentiation-related genes, including osteoblast-related genes runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN) of PDLSCs were reduced, the protein expressions of RUNX2 and osteopontin (OPN) were also decreased (P<0.05); compared with the LPS group, the proliferation of PDLSCs in the LPS+EVs group and LPS+IVs group were significantly increased, while the levels of IL-6, TNF-α were significantly reduced, and the mRNA expressions of RUNX2, ALP, OCN were significantly increased, the protein expressions of RUNX2 and OPN were also significantly increased (P<0.05). Further, in the inflammatory microenvironment, Compared with EVs, IVs more significantly promote the proliferation of PDLSCs, inhibit TNF-α expression, enhance the expression of RUNX2 mRNA, upregulate the expression of RUNX2 and OPN proteins, increase ALP activity, and promote the formation of mineralized nodules (P<0.05). Conclusion IVs and EVs derived from PDLSCs can boost the proliferation of PDLSCs in an inflammatory microenvironment, inhibit the expression of inflammatory factors, and advance the osteogenic differentiation of PDLSCs. The anti-inflammatory and osteogenic effects of IVs are superior to those of EVs.

Recent clinical studies have shown that mutation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene in cancer cells may be associated with immunosuppressive tumor microenvironment (TME) and poor response to immune checkpoint blockade (ICB) therapy. Therefore, efficiently restoring PTEN gene expression in cancer cells is critical to improving the responding rate to ICB therapy. Here, we screened an adeno-associated virus (AAV) capsid for efficient PTEN gene delivery into B16F10 tumor cells. We demonstrated that intratumorally injected AAV6-PTEN successfully restored the tumor cell PTEN gene expression and effectively inhibited tumor progression by inducing tumor cell immunogenic cell death (ICD) and increasing immune cell infiltration. Moreover, we developed an anti-PD-1 loaded phospholipid-based phase separation gel (PPSG), which formed an in situ depot and sustainably release anti-PD-1 drugs within 42 days in vivo. In order to effectively inhibit the recurrence of melanoma, we further applied a triple therapy based on AAV6-PTEN, PPSG^@anti-PD-1 and CpG, and showed that this triple therapy strategy enhanced the synergistic antitumor immune effect and also induced robust immune memory, which completely rejected tumor recurrence. We anticipate that this triple therapy could be used as a new tumor combination therapy with stronger immune activation capacity and tumor inhibition efficacy.

Periodontitis is a chronic inflammatory and immune reactive disease induced by the subgingival biofilm.The therapeutic effect for susceptible patients is often unsatisfactory due to excessive inflammatory response and oxidative stress.Sinensetin(Sin)is a nature polymethoxylated flavonoid with anti-inflammatory and antioxidant activities.Our study aimed to explore the beneficial effect of Sin on periodontitis and the specific molecular mechanisms.We found that Sin attenuated oxidative stress and inflammatory levels of periodontal ligament cells(PDLCs)under inflammatory conditions.Administered Sin to rats with ligation-induced periodontitis models exhibited a protective effect against periodontitis in vivo.By molecular docking,we identified Bach1 as a strong binding target of Sin,and this binding was further verified by cellular thermal displacement assay and immunofluorescence assays.Chromatin immunoprecipitation-quantitative polymerase chain reaction results also revealed that Sin obstructed the binding of Bach1 to the HMOX1 promoter,subsequently upregulating the expression of the key antioxidant factor HO-1.Further functional experiments with Bach1 knocked down and overexpressed verified Bach1 as a key target for Sin to exert its antioxidant effects.Additionally,we demonstrated that Sin prompted the reduction of Bach1 by potentiating the ubiquitination degradation of Bach1,thereby inducing HO-1 expression and inhibiting oxidative stress.Overall,Sin could be a promising drug candidate for the treatment of periodontitis by targeting binding to Bach1.

Objective The objective of this study was to provide methodological references for the inheritance of the experience of well-known Chinese medicine doctors in the treatment of kidney disease.Methods The study collected medical case data for IgA nephropathy,diagnosed and treated by Professor Yang Nizhi's outpatient department at Guangdong Provincial Hospital of Traditional Chinese Medicine from 2010 to 2020.The data was standardized and divided into three groups:urine and blood,urine turbidity,and renal failure groups.The study utilized the FangNet platform to apply the PageRank algorithm and calculate the THScore of different subgroups of core herbs for IgA nephropathy.The distribution pattern of syndrome differentiation and corresponding herb use regulations were visualized through Python(SciPy package,Clusterheatmap package),and the study explored and verified the drug prescription through exploratory and confirmatory factor analysis based on Pearson correlation coefficient.The weighted least squares estimation mean and variance adjusted(WLSMV)and the oblique rotated GEOMIN method were used with the Mplus software.Results The study included a total of 548 treatments for 145 patients with IgA nephropathy,with heamturia group(54 cases),urine turbidity group(51 cases),and renal failure group(40 cases).Results showed 9 basic syndromes such as Qi deficiency syndrome(91.79%),blood stasis syndrome(77.01%),damp-heat syndrome(66.06%),and Yin deficiency syndrome(38.69%).There are 24 core drugs in total,23 in the urine and blood group,21 in the urine turbidity group,and 16 in the renal failure group.These drugs mainly include qi-tonifying and yang-invigorating drugs,nourishing yin and blood drugs,promoting blood circulation and removing blood stasis drugs,and clearing heat and cooling blood drugs.The regulations for the differentiation and medication of IgA nephropathy(Z-Score>0.5 and P<0.05)were as follows:Huangqi,Shan Zhu Yu,and Tusizi were commonly used in Qi deficiency syndrome;Danshen,Ze Lan,and Shan Zhu Yu were commonly used in blood stasis syndrome;Pu Gong Ying,Shi Wei,Tao Ren,and Tu Fu Ling were commonly used in damp-heat syndrome;and Mo Han Lian,Tai Zi Shen,and Nv Zhen Zi were commonly used in Yin deficiency syndrome.Through exploratory and confirmatory factor analysis,the core drug combination factors for the treatment of IgA nephropathy by Professor Yang Nizhi were obtained as follows:F1(Tusizi,Shan Zhu Yu,Huangqi);F2(White Mao Gen,Xiao Ji,Qian Cao);F3(Nv Zhen Zi,Mo Han Lian,Tai Zi Shen);and F4(Ze Lan,Tao Ren).Conclusion This study analyzed the diagnosis and treatment experience of Professor Yang Nizhi in the treatment of IgA nephropathy by grouping,defining the core syndrome of"Qi deficiency and blood stasis,damp-heat and Yin deficiency",and the core treatment methods of"tonifying Qi,promoting blood circulation,clearing heat,and nourishing Yin"using the PageRank algorithm and Mplus factor analysis.The study provided methodological references for the inheritance of the experience of famous Chinese medicine doctors and promoted the development and utilization of traditional Chinese medicine.

This paper compared the differences between two kinds of Bufonis Venenum produced by Bufo gargarizans gargarizans and B. gararizans andrewsi, and verified the rationality of the market value orientation of Bufonis Venenum based on the zebrafish mo-del. Twenty batches of Bufonis Venenum from Jiangsu province, Hebei province, Liaoning province, Jilin province, and Liangshan, Sichuan province, including B. gargarizans gargarizans and B. gararizans andrewsi, were collected. The UHPLC-LTQ-Orbitrap-MS combined with principal component analysis was used to compare the differences between two kinds of Bufonis Venenum. According to the limiting conditions of VIP>1, FC<0.5 or FC>2.0, and peak total area ratio>1%, 9 differential markers were determined, which were cinobufagin, cinobufotalin, arenobufagin, resibufogenin, scillaredin A, resibufagin, 3-(N-suberoylargininyl)-arenobufagin, 3-(N-suberoylargininyl)-marinobufagin, and 3-(N-suberoylargininyl)-resibufogenin. The content of 20 batches of Bufonis Venenum was determined according to the Chinese Pharmacopoeia(2020 edition) by high-performance liquid chromatography, and the 2 batches of Bufonis Venenum, CS7(8.99% of total content) and CS9(5.03% of total content), with the largest difference in the total content of the three quality control indexes of the Chinese Pharmacopoeia(bufalin, cinobufagin, and resibufogenin) were selected to evaluate their anti-liver tumor activity based on the zebrafish model. The tumor inhibition rates of the 2 batches were 38.06% and 45.29%, respectively, proving that only using the quality control indexes of the Chinese Pharmacopoeia as the value orientation of Bufonis Venenum market circulation was unreasonable. This research provides data support for the effective utilization of Bufonis Venenum resources and the establishment of a rational quality evaluation system of Bufonis Venenum.
Animals ; Zebrafish ; Bufanolides/analysis* ; Bufonidae ; Chromatography, High Pressure Liquid ; Quality Control ; Cell Line, Tumor

Objective:To analyze the burden and changing trends of periodontal disease in adults of the mainland of China from 1990 to 2019, and to predict the incidence trends of periodontal disease in the next 25 years, with a goal to provide a basis for reducing the burden of periodontal disease and formulating relevant prevention and treatment measures.Methods:Data on the incidence, prevalence, and disability adjusted life years (DALY) rate of periodontal disease among adults in the mainland of China from 1990 to 2019 were extracted from the global burden of disease study 2019 (GBD 2019) database. The estimated annual percent change (EAPC) was used to estimate the temporal trend of periodontal disease, and the age-period-cohort model (APC) was used to predict the age-standardized incidence of periodontal disease in Chinese adults from 2020 to 2044.Results:From 1990 to 2019, the incidence, prevalence, and DALY rate of adult periodontal disease in the mainland of China showed an increasing trend, with EAPCs of 0.3 (95% CI: 0.1-0.6), 0.5 (95% CI: 0.1-0.8), and 0.5 (95% CI: 0.1-0.8), respectively. The incidence and prevalence of periodontitis among the population aged 35-39 years old and 40-44 years old increased the most significantly, with EAPCs of 0.8 and 0.7, respectively, whereas the change in periodontal disease prevalence tended to be stable and the increase trend in prevalence was lower in the elderly group (EAPC=0.4). The incidence (EAPC=2.1), prevalence (EAPC=2.6) and DALY rate (EAPC=2.6) of periodontal disease in females increased more than those in males (EAPC=1.9, 2.4, and 2.4, respectively), of which the prevalence had exceeded that of males in 2019. The APC model predicted that the prevalence of periodontal disease in the period of 2020-2044 in China would still be on an upward trend, and the increase rate would be higher in females than in males. Conclusions:The burden of periodontal disease among adults in China had been increasing over the past 30 years, especially among young and middle-aged adults as well as females, and the incidence of periodontal disease will continue to increase over the next 25 years.

Indolealkylamines(IAAs) are the main hydrophilic substances in toad skin, mainly including free N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine, dehydrobufotenine, and binding bufothionine. In this study, the LPS-activated neutrophils were used to investigate the structure-activity relationship and anti-inflammatory mechanism of the above-mentioned five monomers from the toad skin in vitro. The neutrophils were divided into the control group, model group(1 μg·mL~(-1) LPS), positive drug group(100 μg·mL~(-1) indometacin), as well as the low-(50 μg·mL~(-1)), medium-(100 μg·mL~(-1)) and high-dose(200 μg·mL~(-1)) free N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine, dehydrobufotenine, and binding bufothionine groups. The levels of IL-6, TNF-α and IL-1β in the neutrophil supernatant of each group was measured by enzyme-linked immunosorbent assay(ELISA) after LPS stimulation, followed by the detection of apoptosis in each group after Annexin V/PI staining. The protein expression levels of caspase-3, Bax, Bcl-2, beclin1, LC3-I, and LC3-Ⅱ were assayed by Western blot. The results showed that IAAs reduced the excessive secretion of inflammatory cytokines caused by LPS compared with the model group. Besides, the activity of each free IAAs(N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine and dehydrobufotenine), especially bufotenine, was stronger than that of the binding bufothionine. As revealed by Annexin V/PI staining, LPS delayed the early apoptosis of neutrophils compared with the control group, while bufotenine promoted the apoptosis of neutrophils in a dose-dependent manner, which might be related to the elevated expression of apoptosis-related protein Bax/Bcl-2. In addition, LPS activated the autophagy pathways in neutrophils. This study confirmed the efficacy of IAAs in reducing the secretion of inflammatory cytokines in neutrophils induced by LPS for the first time. For instance, bufotenine exerts the anti-inflammatory effect possibly by inducing the apoptosis of neutrophils.
Animals ; Anti-Inflammatory Agents/pharmacology* ; Apoptosis ; Bufonidae ; Lipopolysaccharides/toxicity* ; Neutrophils ; Skin

Objective To determine the solubility of rhein in different vehicles and its partition coefficients in the n-octanol-water system for designing new formulations. Methods High performance liquid chromatography(HPLC) method was established to determine the concentration of rhein in water,different pH solutions and different solvents;the partition coefficients for the n-octanol- buffer solution systems were determined by shaking flask method. Results The equilibrium solubility of Rhein was 3.89 μg·mL-1and the lgP was 2.79 in water at 37 ℃.The solubility of rhein was increased with the raise of pH, which could reach 362.20 and 431.65 μg·mL-1in phosphate buffer solution at pH 6.8 and 7.4,respectively.Solubility of rhein in the semi polar solvent was relatively good,which can up to 2 971.74 μg·mL-1in PEG400.The oil-water partition coefficients of rhein were decreased with the increase of pH,and were 0.83 and 0.54 in phosphate buffer solution at pH 6.8 and 7.4,respectively. Conclusion Rhein is almost not soluble in water.With the raise of pH,the solubility is increased but the oil-water partition coefficient is decreased.

Objective To observe changes in relative pituitary hormones after severe traumatic brain injury (STBI) in children and to investigate the short-and long-term effects of mild hypothermia on pituitary function.Methods Included for this study were 81 children who had been admitted to the Brain Hospital of Liaocheng People's Hospital from June 2011 to January 2015 for STBI and abnormal secretion of pituitary-related hormones.They were divided by the wishes of patients and their families into 2 groups,subjected to mild hypothermia treatment (n=42) or conventional treatment (n=41).The levels were examined of growth hormone (GH),prolactin (PRL),adrenocorticotropic hormone (ACTH),free triiodothyronine (FT3),free thyroxine (FT4) and thyroid stimulating hormone (TSH) for both groups unpon admission and in the moming of 1,3,5,7 and 14 days after injury.At 6 months after injury the patients were followed up for Glasgow Outcome Score (GOS) and levels of pituitary-related hormones.Results Of the 83 patients,disturbance of one single hormone was found in 31 (30.10％) and disturbance of 2 or more hormones in 52 (50.48％).The top 3 hormones disturbed were PRL,ACTH and GH.On days 3,5 and 7 after injury,the levels of PRL and ACTH in the mild hypothermia treatment group were significantly lower than in the conventional treatment group (P＜0.05).On day 3 after injury,the level of FT3 in the mild hypothermia treatment group was significantly lower than in the conventional treatment group (P＜0.05).At 6 months after injury,the GOS score for the conventional treatment group was 3.04±0.38,significantly lower than that for the mild hypothermia treatment group (4.56±0.62) (P＜0.05).The incidence of hypopituitarism in the hypothermia treatment group (16.67％) was significantly lower than in the conventional treatment group (46.88％) (P＜0.05).Conclusions Incidence of pituitary dysfunction is high in children following severe traumatic brain injury.Mild hypothermia treatment can effectively alleviate early-stage parasecretion of pituitary hormones,and decrease the incidence of later pituitary dysfunction.

Objective To evaluate the clinical outcomes of minimally invasive internal fixation for femoral intertrochanteric fracture in osteoporotic elderly patients. Methods A retrospective study was conducted in 112 patients using interan nail (IN) or trochanteric antegrade nail (TAN) for the management of intertrochanteric femoral fracture from January 2009 to September 2015 in our hospital. According to AO classification, there were 34 cases of type 31-A.1, and 61 cases of type 31-A.2, 17 cases of type 31-A.3. Clinical and radiological follow-up were available. Surgical and fluoroscopic time, length of hospital stay, blood loss, complications and hip functions were compared between two groups. Results A total of 78 patients meeting the criteria were evaluated at a mean follow-up of 15 months (range, 3-27 months). The IN was used in 41 patients and the TAN in 37 patients. Operative time, fluoroscopy time and blood loss showed significant difference between the IN group and TAN group (respectively, 58.9±6.9 vs. 75.6±5.9min; 2.70±0.47 vs. 4.40±0.47min; 107.6±6.7 vs. 127.8±6.8ml, P<0.05), suggesting that patients treated with the IN experienced shorter operative and fluoroscopy times, less blood loss and better hip function (73%) than those with TAN (65%, P<0.05). Rate of postoperative complications was lower in the IN group (4.9%) than in the TAN group (10.8%, P<0.05). There was no difference in hospital stay and fracture healing time between the two groups (P>0.05). Conclusions For minimally invasive treatment of unstable femoral intertrochanteric fractures, use of either the IN or TAN is clinically effective. However, IN presents more advantages (e.g., easy operation, reliable fixation, less bleeding, better clinical outcomes, and less complications). The use of IN is a suitable option for the treatment of unstable intertrochanteric fractures in osteoporotic elderly patients.