ObjectiveTo explore the changes in volatile components， total polysaccharides， enzyme activity， and chromaticity value of Myristicae Semen Koji（MSK） during the fermentation process， and conduct correlation analysis. MethodsBased on gas chromatography-mass spectrometry（GC-MS）， the changes of volatile components in MSK at different fermentation times were identified. The phenol sulfuric acid method， dinitrosalicylic acid method（DNS）， and carboxymethyl cellulose sodium salt method（CMC-Na） were used to investigate the total polysaccharide content， amylase activity， and cellulase activity during the fermentation process. Visual analysis technology was used to explore the changes in chromaticity values， revealing the fermentation process of MSK and the dynamic changes of various measurement indicators， partial least squares-discriminant analysis（PLS-DA） was used to explore the differential compounds of MSK at different fermentation degrees， and Pearson correlation analysis was used to explore the correlation between volatile components of MSK and total polysaccharides， enzyme activity， and chromaticity values. ResultsA total of 60 volatile compounds were identified from MSK， the relative contents of components such as （+）-α-pinene， β-phellandrene， β-pinene， （+）-limonene， and p-cymene obviously increased， while the relative contents of components such as safrole， methyl isoeugenol， methyleugenol， myristicin， and elemicin significantly decreased. During the fermentation process， the total polysaccharide content showed an upward trend， while the activities of amylase and cellulase showed an initial increase followed by a decrease， and reached their maximum value at 40 h. the overall brightness（L^*） and total color difference（ΔE^*） gradually increased， while the changes in red-green value（a^*） and yellow-blue value（b^*） were not obvious. PLS-DA results showed that MSK could be clearly distinguished at different fermentation times， and 13 differential biomarkers were screened out. Pearson correlation analysis results showed that the contents of α-terpinene， β-phellandrene， methyleugenol， β-cubebene and myristic acid had an obvious correlation with chromaticity values. ConclusionAfter fermentation， the volatile components， total polysaccharides， amylase activity， and cellulase activity of MSK undergo significant changes， and there is a clear correlation between them and chromaticity values， which reveals the dynamic changes in the fermentation process and related indicators of MSK， laying a foundation for the quality control.

Objective:To investigate the effect of the antioxidant Tempol on the skin depigmentation of a vitiligo-like mouse model induced by the combination of the endogenous reactive oxygen species (ROS) producer AAPH and tyrosinase-related protein 2 (TRP2) -180 nonapeptide.Methods:A vitiligo-like skin depigmentation model was established by immunizing mice with injections of a TRP2-180 nonapeptide mixture into the foot pads twice and into the tails twice, with the injection interval being 1 week. After the first injection, 12 immune-induced mouse models of vitiligo were randomly divided into 4 groups (3 mice per group) : a model group, an AAPH group, a Tempol group, and a combined treatment group; additionally, 3 untreated mice injected with an ovalbumin (OVA) 257-264 peptide served as a sham control group. Mice in the AAPH group, the Tempol group, and the combined treatment group were subcutaneously injected with AAPH into the tails, intraperitoneally injected with Tempol, and received the above both treatments, respectively, while mice in the model group and the sham control group received phosphate-buffered saline injections into the tail and/or abdomen. Drug interventions were carried out 3 times per week for 3 consecutive weeks. Six weeks after the last immunization, mice were sacrificed. The area of depigmented macules on the tail was measured using a point-counting method, X-gal staining and double immunofluorescence staining were performed to assess the distribution and number of melanocytes, mast cells, and CD8 + T cells in depigmented macules on the tail. HaCaT cells were in vitro co-cultured with AAPH and/or Tempol, and a conventional culture group served as the control. Cellular ROS levels were measured by dichlorofluorescin diacetate labeling and flow cytometry; Western blot analysis was performed to determine the expression of matrix metalloproteinase 9 (MMP9) and stem cell factor (SCF) in cell lysates, and to detect soluble SCF levels in the culture supernatant. Comparisons among multiple groups were conducted using one-way analysis of variance, and multiple comparisons were performed using least significant difference- t test. Results:Depigmented macules were observed on the tails of mice in all groups except the sham control group. The area of depigmented macules was significantly larger in the AAPH group (7.27 ± 0.31 cm 2) than in the model group and combined treatment group (3.53 ± 0.21 cm 2, 4.07 ± 0.40 cm 2; t = 13.48, 11.56, respectively, both P < 0.001), while there was no significant difference between the Tempol group (3.30 ± 0.40 cm 2) and the model group ( P = 0.424). X-gal staining and double immunofluorescence staining showed that the number of melanocytes in the normal skin around the depigmented macules was significantly lower in the AAPH group and the combined treatment group than in the model group ( t = 6.31, 5.16, respectively, both P < 0.001), and no significant difference was observed between the AAPH group and the combined treatment group ( P = 0.516). The numbers of CD8 + T cells and mast cells were significantly higher in the AAPH group than in the model group and the combined treatment group (all P < 0.001). The numbers of the 3 types of cells mentioned above in the Tempol group did not differ from those in the model group (all P > 0.05). The ROS levels in HaCaT cells in the AAPH group were the highest, and significantly higher than those in the control group and the combined treatment group (both P < 0.001). Western blot analysis showed that the MMP9 level in the cell lysates and soluble SCF level in the culture supernatant were significantly higher in the AAPH group than in the control group and the combined treatment group (all P < 0.05) ; no significant difference was observed in the membrane-bound SCF level in cell lysates among the groups ( F = 0.06, P = 0.977) . Conclusion:The antioxidant Tempol could inhibit the formation of skin depigmented macules in vitiligo-like mouse models under AAPH-induced oxidative stress.

Objective:To investigate effects of rutin on ultraviolet irradiation (UVR) -induced human dermal fibroblast (FB) senescence and melanogenesis in mouse ear skin.Methods:The third- to fifth-passage FBs were divided into 4 groups: a blank control group, a UVR group, a rutin group, and a combined treatment group. In the UVR group, FBs were irradiated using an ultraviolet irradiator at a single dose of 0.6 J/cm 2 UVA combined with 0.03 J/cm 2 UVB once daily for 5 consecutive days; FBs in the rutin group were cultured in Dulbecco's modified Eagle medium containing 50 μmol/L rutin for 5 days; the combined treatment group received both UVR and the treatment with 50 μmol/L rutin for 5 days; the blank control group underwent no treatment. β-Galactosidase staining was performed to assess the senescence of FBs, real-time quantitative PCR (qPCR) to measure the telomere length in FBs, and Western blot analysis to detect the expression levels of stem cell factor (SCF) in FB cell lysates and culture supernatants. FB culture supernatants were collected from each group, and mixed with M254 medium at a ratio of 3∶1 to prepare conditioned medium, which was then used to treat PIG1 melanocytes for 24 hours. Western blot analysis was conducted to determine the tyrosinase (TYR) expression in PIG1 melanocytes in each group, while the 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay was applied to assess the proliferative activity of PIG1 cells in each group. Ten Dct-LacZ transgenic mice were divided into a control group and a UVR group. For each mouse, 5% rutin-containing cream was topically applied to the right ear after UVR, while the left ear treated with the cream base alone served as a control. Skin biopsies were performed after 4 weeks, followed by X-gal staining and Avidin/fluorescein isothiocyanate (FITC) staining to count the numbers of melanocytes and mast cells in mouse ear skin. Results:In the UVR group, the number of senescent FBs (25.67 ± 2.89), relative protein expression levels of SCF (1.95 ± 0.22), and relative levels of SCF in the cell culture supernatant (1.52 ± 0.34) were all significantly higher than those in the blank control group (5.67 ± 1.56, 0.95 ± 0.11, 1.01 ± 0.31, respectively), while these indicators were significantly lower in the combined treatment group (12.00 ± 1.63, 1.32 ± 0.19, 1.15 ± 0.32, respectively) than in the UVR group (all P < 0.05). The relative telomere length in FBs was significantly shorter in the UVR group (0.49 ± 0.12) than in the blank control group (0.94 ± 0.11; LSD- t = 3.15, P = 0.021), but significantly longer in the combined treatment group (0.81 ± 0.13) than in the UVR group (LSD- t = 4.30, P = 0.034). After the treatment with FB conditioned medium, the relative expression level of TYR in PIG1 melanocytes and the number of EdU-positive cells were significantly higher in the UVR group (2.54 ± 0.21, 33.54 ± 3.21, respectively) than in the blank control group (0.97 ± 0.19, 21.45 ± 2.51, respectively; both P < 0.001), but significantly lower in the combined treatment group (1.63 ± 0.12, 18.54 ± 3.87, respectively) than in the UVR group (both P < 0.001). X-gal staining and Avidin/FITC staining showed that the numbers of melanocytes and mast cells in the mouse left ear skin in the UVR group (5.00 ± 1.22, 98.60 ± 8.47, respectively) were significantly higher than those in the mouse left ear skin in the control group (1.80 ± 0.45, 53.80 ± 5.76, respectively) and those in the mouse right ear skin treated with the rutin-containing cream in the UVR group (2.80 ± 0.45, 69.60 ± 8.89, respectively) (all P < 0.05) . Conclusion:Rutin may inhibit UVR-induced skin melanogenesis by suppressing the senescence of dermal FBs and paracrine secretion of SCF.

Objective:To evaluate the functional outcomes and postoperative complications associated with modified Toupet-like (mToupet-like) anastomosis following proximal gastrectomy for patients with gastric tumors.Methods:After proximal gastrectomy, barbed sutures (2-3 stitches) in the seromuscular layer were used to secure the anterior wall of the stomach at a distance of 1-2 cm from the closure line and the posterior wall of the esophagus at a distance of 5.0 cm from the closure line. The remnant stomach was then positioned posterior to the esophagus on the greater curvature side. Esophagogastric anterior wall anastomosis (manual or circular stapling) was performed at the greater curvature of the remnant stomach, 3 cm distal to the gastroesophageal fixation point. A Toupet-like folding procedure was conducted by folding the reconstructed gastric fundus and wall anteriorly from behind the esophagus and embedding the esophagus within a 270° wrap at the site of stomach-esophagus fixation.Results:Twelve patients with gastric tumors underwent proximal partial gastrectomy with mToupet-like anastomosis in the Department of Gastric Surgery at Zhejiang Cancer Hospital from January to March 2024. Among them, 10 diagnosed as upper gastric adenocarcinoma, and 2 diagnosed as gastric gastrointestinal stromal tumors. The cohort included nine male patients and three female patients, aged 46 to 77 years old, with a body mass index (BMI) ranging from 19.7 to 27.3 kg/m2. The maximum tumor diameter was less than 4 cm, and the predicted residual gastric volume exceeded one-half. Laparoscopic surgery was performed in 11 patients, while only 1 patient underwent open surgery. The mean duration of mToupet-like anastomosis was 48.3±8.7 minutes with an estimated intraoperative blood loss was 53.0±11.2 ml. All the 12 patients successfully achieved R0 resection. Among these patietns, the median postoperative hospital stay was 8.5 (7.0, 11.0) days, and the average hospitalization cost was 5.0±0.2 ten thousand yuan. No Clavien-Dindo grade II or higher complications were observed during the perioperative period. Patients were followed up for 6 to 8 months after operation, and no cases of reflux esophagitis were detected by gastroscopy, and no patient required long-term oral proton pump inhibitors.Conclusions:mToupet-like anastomosis for digestive tract reconstruction after proximal gastrectomy is a safe and feasible technique, demonstrating favorable preliminary efficacy.

Objective:To investigate the application value of gastric suspension method in supra-pancreatic lymph node dissection of laparoscopic radical gastrectomy of gastric cancer.Method:The retrospective cohort study was conducted. The clinicopathological data of 84 patients who under-went laparoscopic radical gastrectomy of gastric cancer at Zhejiang Cancer Hospital from August 2023 to July 2024 were collected. There were 61 males and 23 females, aged (64±11)years. Of the 84 patients, 42 patients undergoing supra-pancreatic lymph node dissection during laparoscopic radical gastrectomy of gastric cancer with traditional method for surgical field exposure were divided into the control group, and 42 patients undergoing supra-pancreatic lymph node dissection during laparoscopic radical gastrectomy of gastric cancer with gastric suspension method for surgical field exposure were divided into the suspension group. Observation indicators: (1) surgical conditions; (2) postoperative conditions. Comparison of measurement data with normal distribution between groups was conducted using the independent sample t test. Comparison of measurement data with skewed distribution between groups was conducted using the Mann-Whitney U test. Comparison of count data between groups was conducted using the chi-square test. Results:(1) Surgical condi-tions. The time for supra-pancreatic lymph node dissection of the control group was (78±14)minutes. Number of grasping operations was 116±34, number of bleeding sites caused by grasping operations was 7.8±2.7, and operation time was (3.9±0.8)hours. The above indicators of the suspension group were (59±12)minutes, 68±19, 2.1±1.5, and (3.3±0.7)hours, respectively. There were significant diffe-rences in the above indicators between the two groups ( t=5.42, 8.10, 8.31, 3.14, P<0.05). (2) Post-operative conditions. The tumor diameter was 2.5(2.0,3.5)cm for patients of the control group, versus 3.0(2.4, 4.4)cm for patients of the suspension group, showing a significant difference between the two groups ( Z=-1.98, P<0.05). Conclusion:Compared with the traditional non-suspension method, the gastric suspension method in laparoscopic radical gastrectomy of gastric cancer for supra-pancreatic lymph node dissection is associated with shorter operation time and less trauma.

Objective:To investigate the effect of the antioxidant Tempol on the skin depigmentation of a vitiligo-like mouse model induced by the combination of the endogenous reactive oxygen species (ROS) producer AAPH and tyrosinase-related protein 2 (TRP2) -180 nonapeptide.Methods:A vitiligo-like skin depigmentation model was established by immunizing mice with injections of a TRP2-180 nonapeptide mixture into the foot pads twice and into the tails twice, with the injection interval being 1 week. After the first injection, 12 immune-induced mouse models of vitiligo were randomly divided into 4 groups (3 mice per group) : a model group, an AAPH group, a Tempol group, and a combined treatment group; additionally, 3 untreated mice injected with an ovalbumin (OVA) 257-264 peptide served as a sham control group. Mice in the AAPH group, the Tempol group, and the combined treatment group were subcutaneously injected with AAPH into the tails, intraperitoneally injected with Tempol, and received the above both treatments, respectively, while mice in the model group and the sham control group received phosphate-buffered saline injections into the tail and/or abdomen. Drug interventions were carried out 3 times per week for 3 consecutive weeks. Six weeks after the last immunization, mice were sacrificed. The area of depigmented macules on the tail was measured using a point-counting method, X-gal staining and double immunofluorescence staining were performed to assess the distribution and number of melanocytes, mast cells, and CD8 + T cells in depigmented macules on the tail. HaCaT cells were in vitro co-cultured with AAPH and/or Tempol, and a conventional culture group served as the control. Cellular ROS levels were measured by dichlorofluorescin diacetate labeling and flow cytometry; Western blot analysis was performed to determine the expression of matrix metalloproteinase 9 (MMP9) and stem cell factor (SCF) in cell lysates, and to detect soluble SCF levels in the culture supernatant. Comparisons among multiple groups were conducted using one-way analysis of variance, and multiple comparisons were performed using least significant difference- t test. Results:Depigmented macules were observed on the tails of mice in all groups except the sham control group. The area of depigmented macules was significantly larger in the AAPH group (7.27 ± 0.31 cm 2) than in the model group and combined treatment group (3.53 ± 0.21 cm 2, 4.07 ± 0.40 cm 2; t = 13.48, 11.56, respectively, both P < 0.001), while there was no significant difference between the Tempol group (3.30 ± 0.40 cm 2) and the model group ( P = 0.424). X-gal staining and double immunofluorescence staining showed that the number of melanocytes in the normal skin around the depigmented macules was significantly lower in the AAPH group and the combined treatment group than in the model group ( t = 6.31, 5.16, respectively, both P < 0.001), and no significant difference was observed between the AAPH group and the combined treatment group ( P = 0.516). The numbers of CD8 + T cells and mast cells were significantly higher in the AAPH group than in the model group and the combined treatment group (all P < 0.001). The numbers of the 3 types of cells mentioned above in the Tempol group did not differ from those in the model group (all P > 0.05). The ROS levels in HaCaT cells in the AAPH group were the highest, and significantly higher than those in the control group and the combined treatment group (both P < 0.001). Western blot analysis showed that the MMP9 level in the cell lysates and soluble SCF level in the culture supernatant were significantly higher in the AAPH group than in the control group and the combined treatment group (all P < 0.05) ; no significant difference was observed in the membrane-bound SCF level in cell lysates among the groups ( F = 0.06, P = 0.977) . Conclusion:The antioxidant Tempol could inhibit the formation of skin depigmented macules in vitiligo-like mouse models under AAPH-induced oxidative stress.

Objective:To investigate effects of rutin on ultraviolet irradiation (UVR) -induced human dermal fibroblast (FB) senescence and melanogenesis in mouse ear skin.Methods:The third- to fifth-passage FBs were divided into 4 groups: a blank control group, a UVR group, a rutin group, and a combined treatment group. In the UVR group, FBs were irradiated using an ultraviolet irradiator at a single dose of 0.6 J/cm 2 UVA combined with 0.03 J/cm 2 UVB once daily for 5 consecutive days; FBs in the rutin group were cultured in Dulbecco's modified Eagle medium containing 50 μmol/L rutin for 5 days; the combined treatment group received both UVR and the treatment with 50 μmol/L rutin for 5 days; the blank control group underwent no treatment. β-Galactosidase staining was performed to assess the senescence of FBs, real-time quantitative PCR (qPCR) to measure the telomere length in FBs, and Western blot analysis to detect the expression levels of stem cell factor (SCF) in FB cell lysates and culture supernatants. FB culture supernatants were collected from each group, and mixed with M254 medium at a ratio of 3∶1 to prepare conditioned medium, which was then used to treat PIG1 melanocytes for 24 hours. Western blot analysis was conducted to determine the tyrosinase (TYR) expression in PIG1 melanocytes in each group, while the 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay was applied to assess the proliferative activity of PIG1 cells in each group. Ten Dct-LacZ transgenic mice were divided into a control group and a UVR group. For each mouse, 5% rutin-containing cream was topically applied to the right ear after UVR, while the left ear treated with the cream base alone served as a control. Skin biopsies were performed after 4 weeks, followed by X-gal staining and Avidin/fluorescein isothiocyanate (FITC) staining to count the numbers of melanocytes and mast cells in mouse ear skin. Results:In the UVR group, the number of senescent FBs (25.67 ± 2.89), relative protein expression levels of SCF (1.95 ± 0.22), and relative levels of SCF in the cell culture supernatant (1.52 ± 0.34) were all significantly higher than those in the blank control group (5.67 ± 1.56, 0.95 ± 0.11, 1.01 ± 0.31, respectively), while these indicators were significantly lower in the combined treatment group (12.00 ± 1.63, 1.32 ± 0.19, 1.15 ± 0.32, respectively) than in the UVR group (all P < 0.05). The relative telomere length in FBs was significantly shorter in the UVR group (0.49 ± 0.12) than in the blank control group (0.94 ± 0.11; LSD- t = 3.15, P = 0.021), but significantly longer in the combined treatment group (0.81 ± 0.13) than in the UVR group (LSD- t = 4.30, P = 0.034). After the treatment with FB conditioned medium, the relative expression level of TYR in PIG1 melanocytes and the number of EdU-positive cells were significantly higher in the UVR group (2.54 ± 0.21, 33.54 ± 3.21, respectively) than in the blank control group (0.97 ± 0.19, 21.45 ± 2.51, respectively; both P < 0.001), but significantly lower in the combined treatment group (1.63 ± 0.12, 18.54 ± 3.87, respectively) than in the UVR group (both P < 0.001). X-gal staining and Avidin/FITC staining showed that the numbers of melanocytes and mast cells in the mouse left ear skin in the UVR group (5.00 ± 1.22, 98.60 ± 8.47, respectively) were significantly higher than those in the mouse left ear skin in the control group (1.80 ± 0.45, 53.80 ± 5.76, respectively) and those in the mouse right ear skin treated with the rutin-containing cream in the UVR group (2.80 ± 0.45, 69.60 ± 8.89, respectively) (all P < 0.05) . Conclusion:Rutin may inhibit UVR-induced skin melanogenesis by suppressing the senescence of dermal FBs and paracrine secretion of SCF.

Objective:To evaluate the functional outcomes and postoperative complications associated with modified Toupet-like (mToupet-like) anastomosis following proximal gastrectomy for patients with gastric tumors.Methods:After proximal gastrectomy, barbed sutures (2-3 stitches) in the seromuscular layer were used to secure the anterior wall of the stomach at a distance of 1-2 cm from the closure line and the posterior wall of the esophagus at a distance of 5.0 cm from the closure line. The remnant stomach was then positioned posterior to the esophagus on the greater curvature side. Esophagogastric anterior wall anastomosis (manual or circular stapling) was performed at the greater curvature of the remnant stomach, 3 cm distal to the gastroesophageal fixation point. A Toupet-like folding procedure was conducted by folding the reconstructed gastric fundus and wall anteriorly from behind the esophagus and embedding the esophagus within a 270° wrap at the site of stomach-esophagus fixation.Results:Twelve patients with gastric tumors underwent proximal partial gastrectomy with mToupet-like anastomosis in the Department of Gastric Surgery at Zhejiang Cancer Hospital from January to March 2024. Among them, 10 diagnosed as upper gastric adenocarcinoma, and 2 diagnosed as gastric gastrointestinal stromal tumors. The cohort included nine male patients and three female patients, aged 46 to 77 years old, with a body mass index (BMI) ranging from 19.7 to 27.3 kg/m2. The maximum tumor diameter was less than 4 cm, and the predicted residual gastric volume exceeded one-half. Laparoscopic surgery was performed in 11 patients, while only 1 patient underwent open surgery. The mean duration of mToupet-like anastomosis was 48.3±8.7 minutes with an estimated intraoperative blood loss was 53.0±11.2 ml. All the 12 patients successfully achieved R0 resection. Among these patietns, the median postoperative hospital stay was 8.5 (7.0, 11.0) days, and the average hospitalization cost was 5.0±0.2 ten thousand yuan. No Clavien-Dindo grade II or higher complications were observed during the perioperative period. Patients were followed up for 6 to 8 months after operation, and no cases of reflux esophagitis were detected by gastroscopy, and no patient required long-term oral proton pump inhibitors.Conclusions:mToupet-like anastomosis for digestive tract reconstruction after proximal gastrectomy is a safe and feasible technique, demonstrating favorable preliminary efficacy.

Objective:To investigate the application value of gastric suspension method in supra-pancreatic lymph node dissection of laparoscopic radical gastrectomy of gastric cancer.Method:The retrospective cohort study was conducted. The clinicopathological data of 84 patients who under-went laparoscopic radical gastrectomy of gastric cancer at Zhejiang Cancer Hospital from August 2023 to July 2024 were collected. There were 61 males and 23 females, aged (64±11)years. Of the 84 patients, 42 patients undergoing supra-pancreatic lymph node dissection during laparoscopic radical gastrectomy of gastric cancer with traditional method for surgical field exposure were divided into the control group, and 42 patients undergoing supra-pancreatic lymph node dissection during laparoscopic radical gastrectomy of gastric cancer with gastric suspension method for surgical field exposure were divided into the suspension group. Observation indicators: (1) surgical conditions; (2) postoperative conditions. Comparison of measurement data with normal distribution between groups was conducted using the independent sample t test. Comparison of measurement data with skewed distribution between groups was conducted using the Mann-Whitney U test. Comparison of count data between groups was conducted using the chi-square test. Results:(1) Surgical condi-tions. The time for supra-pancreatic lymph node dissection of the control group was (78±14)minutes. Number of grasping operations was 116±34, number of bleeding sites caused by grasping operations was 7.8±2.7, and operation time was (3.9±0.8)hours. The above indicators of the suspension group were (59±12)minutes, 68±19, 2.1±1.5, and (3.3±0.7)hours, respectively. There were significant diffe-rences in the above indicators between the two groups ( t=5.42, 8.10, 8.31, 3.14, P<0.05). (2) Post-operative conditions. The tumor diameter was 2.5(2.0,3.5)cm for patients of the control group, versus 3.0(2.4, 4.4)cm for patients of the suspension group, showing a significant difference between the two groups ( Z=-1.98, P<0.05). Conclusion:Compared with the traditional non-suspension method, the gastric suspension method in laparoscopic radical gastrectomy of gastric cancer for supra-pancreatic lymph node dissection is associated with shorter operation time and less trauma.