ObjectiveTo investigate the role and mechanism of podoplanin （PDPN） in hepatic stellate cell （HSC） activation and liver fibrosis. MethodsLiver biopsy samples were collected from 75 patients with chronic hepatitis B who attended Department of Infectious Diseases， Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine， for the first time from September 2019 to June 2022， and RT-PCR and immunohistochemistry were used to measure the expression of PDPN in liver tissue of patients in different stages of liver fibrosis. A total of 12 male C57/BL6 mice were randomly divided into control group and model group. The mice in the model group were given intraperitoneal injection of 10% CCl₄， and those in the control group were injected with an equal volume of olive oil， for 6 weeks. HE staining and Sirius Red staining were used to observe liver histopathological changes； primary mouse liver cells were separated to measure the mRNA expression of PDPN in various types of cells； primary mouse HSCs were treated with PDPN protein， followed by treatment with the NF-‍κB inhibitor BAY11-708， to measure the expression of inflammatory factors in HSCs induced by PDPN. The independent-samples t test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. The Spearman correlation analysis was used to investigate data correlation. ResultsAs for the liver biopsy samples， there was a relatively low mRNA expression level of PDPN in normal liver， and there was a significant increase in the mRNA expression level of PDPN in liver tissue of stage S3 or S4 fibrosis （all P<0.001）. Immunohistochemical staining showed that PDPN was mainly expressed in the fibrous septum and the hepatic sinusoid， and the PDPN-positive area in S4 liver tissue was significantly higher than that in S0 liver tissue （t=8.892， P=0.001）. In normal mice， PDPN was mainly expressed in the hepatic sinusoid， and there was a significant increase in the expression of PDPN in CCl₄ model mice （t=0.95， P<0.001）， mainly in the fibrous septum. RT-PCR showed a significant increase in the mRNA expression of PDPN in the CCl₄ model mice （t=11.25， P=0.002）. Compared with hepatocytes， HSCs， Kupffer cells， and bile duct endothelial cells， hepatic sinusoidal endothelial cells showed a significantly high expression level of PDPN （F=20.56， P<0.001）. Compared with the control group， the primary mouse HSCs treated by PDPN protein for 15 minutes showed significant increases in the mRNA expression levels of the inflammation-related factors TNFα， CCL3， CXCL1， and CXCR1 （all P<0.05）， and there were significant reductions in the levels of these indicators after treatment with BAY11-7082 （all P<0.05）. ConclusionThere is an increase in the expression of PDPN mainly in hepatic sinusoidal endothelial cells during liver fibrosis， and PDPN regulates HSC activation and promotes the progression of liver fibrosis via the NF-κB signaling pathway.

ObjectiveTo investigate the mechanism of action of Xiayuxue decoction in inhibiting nonalcoholic fatty liver disease （NAFLD） induced by high-fat diet in mice by regulating nucleotide binding oligomerization domain like receptor containing pyrin domain protein 6 （NLRP6）. MethodsA total of 15 male C57BL/6 mice were randomly divided into low-fat diet （LFD） group， high-fat diet （HFD） group， and Xiayuxue decoction-HFD group （XYXD group）， with 5 mice in each group. Liver function parameters （alanine aminotransferase ［ALT］ and aspartate aminotransferase ［AST］） and blood lipid metabolic indicators （triglycerides ［TG］ and total cholesterol ［TC］） were measured； HE staining and oil red O staining were performed for liver tissue to observe histomorpholoty and lipid droplet deposition； quantitative real-time PCR was used to measure the expression levels of inflammatory factors （tumor necrosis factor-α ［TNF-α］， interleukin-1β ［IL-1β］， interleukin-18 ［IL-18］， and NLRP6） in liver tissue； Western blot was used to measure the protein expression levels of NLRP6， nuclear factor-kappa B （NF-κB）， and NF-κB p65； immunohistochemistry was used to measure the expression of NLRP6 and CD68. Mouse Raw264.7 cells were treated with palmitic acid （PA）， lipopolysaccharide， and serum containing Xiayuxue decoction to observe inflammation. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the LFD group， the HFD group had significant increases in the serum levels of ALT， AST， TC， and TG （all P<0.05）. Liver histopathological examination showed that the HFD group had marked hepatic steatosis and a signficant increase in NAS score （P<0.05）， and quantitative real-time PCR showed significant increases in the inflammatory factors such as IL1β and IL-18 and a significant reduction in the expression of NLRP6 （all P<0.05）. Immunohistochemistry showed that the expression of NLRP6 showed a similar trend as that of the macrophage marker CD68. Western blot showed that after the downregulation of NLRP6 expression， there was a significant increase in phosphorylated NF-κB p65 （P<0.05）. Compared with the HFD group， Xiayuxue decoction effectively improved liver inflammation， upregulated the expression of NLRP6， and downregulated phosphorylated NF-κB p65 in HFD mice （all P<0.05）. After Raw264.7 cells were treated with PA， NLRP6 was downregulated to promote the progression of inflammation （P<0.05）， and treatment with Xiayuxue decoction could upregulate NLRP6 and inhibit inflammation NF-κB （P<0.05）. ConclusionXiayuxue decoction can effectively improve hepatic steatosis and liver inflammation in a mouse model of NAFLD， possibly by regulating NLRP6/NF-κB to alleviate macrophage activation.

Objective·To explore the role of advanced platelet-rich fibrin(A-PRF)in osteochondral regeneration.Methods·Bone-marrow mesenchymal stem cells(BMSCs)and knee joint chondrocytes were obtained from New Zealand rabbits.A-PRF was obtained by low-speed centrifugation of the heart blood of rabbits.The histological structure of A-PRF was observed by an optical microscope.The release of growth factors in A-PRF was detected by ELISA,including platelet-derived growth factor,transforming growth factor-β,insulin-like growth factor,vascular endothelial growth factor,epidermal growth factor and fibroblast growth factor.A-PRF's cytotoxicity and capability for promoting the proliferation of rabbit BMSCs were detected by live/dead double staining and MTT methods.The effect of A-PRF on the gene expression of type Ⅱ collagen,aggrecan,alkaline phosphatase(ALP)and osteocalcin(OCN)in rabbit BMSCs was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Transwell chambers were used to determine the effect of A-PRF on the migration ability of rabbit BMSCs and the chondrocytes.Rabbit knee osteochondral defect models were established,and 18 rabbits were randomly divided into 3 groups.The A-PRF group(n=6)was implanted with A-PRF in the defect,the A-PRF+BMSCs group(n=6)was implanted with rabbit BMSCs on A-PRF,and the control group(n=6)did not undergo implantation.The rabbits were sacrificed 12 weeks after surgery and the knee joint specimens were stained with hematoxylin-eosin(H-E),toluidine blue and safranin O/fast green.Based on the surface morphology and histology of the knee joints,the International Cartilage Repair Society(ICRS)scoring system was used for macroscopic and histological scoring.Results·A-PRF had a loose network structure and can slowly release growth factors.No cytotoxicity to rabbit BMSCs was observed after adding A-PRF,and the the capability for promoting the proliferation of rabbit BMSCs was significantly increased at 24,48 and 72 h after adding A-PRF(all P<0.05).Chondrogenesis-related gene Ⅱ collagen and aggrecan,as well as osteogenesis-related genes ALP and OCN were significantly up-regulated(all P<0.05).After adding A-PRF,the migration abilities of rabbit BMSCs and chondrocytes were significantly enhanced(both P<0.05),and the migration ability of rabbit BMSCs was significantly higher than that of chondrocytes(P=0.025).The joint surface morphology in the rabbit knee joint defect models was observed.It can be seen that the defects in the A-PRF group and the A-PRF+BMSCs group were basically restored,while the the defects in the control group were only covered by soft tissue.In the ICRS macroscopic score,there was no statistical difference between the A-PRF group and the A-PRF+BMSCs group,but the scores of the two groups were all significantly higher than those of the control group(all P<0.05).According to the histological results,both the A-PRF group and the A-PRF+BMSCs group formed osteochondral repair,but the cartilage in the A-PRF group was more mature,while the control group formed fibrous repair.In the ICRS histological score,there was no statistical difference between the A-PRF group and the A-PRF+BMSCs group,but the scores of both the groups were significantly higher than those of the control group(both P<0.05).Conclusion·Autologous A-PRF has good biocompatibility and the capability for promoting the proliferation of BMSCs.It can promote the repair of cartilage and subchondral bone both in vitro and in vivo.

Objective To investigate the effect of non-alcoholic fatty liver disease(NAFLD)induced by high-fat diet(HFD)on the kidneys of mice and the protective effect and mechanism of Xiayuxue Decoction.Methods A total of 25 healthy controls and 25 NAFLD patients who attended Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from September 2020 to September 2021 were enrolled,and the levels of total cholesterol(TC),triglyceride(TG),blood urea nitrogen(BUN),creatinine(Cr),and uric acid(UA)were measured.A total of 24 male C57BL/6 mice were randomly divided into low-fat diet(LFD)group,HFD group,and HFD+Xiayuxue Decoction group(XYXD group),with 8 mice in each group,and since week 13,XYXD was administered by gavage once a day for 6 weeks till the end of week 18.The level of TC,TG,BUN,and Cr were measured for each group.HE staining and oil red staining were used to observe the pathological changes of the liver and the kidneys;immunohistochemical double staining was used to measure the expression levels of CD68 and alpha-smooth muscle actin(α-SMA);quantitative real-time PCR was used to measure the expression levels of sterol regulatory element binding protein 1(SREBP1),fatty acid synthase(FASN),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),Desmin,and α-SMA in renal tissue;Western blot was used to measure the protein expression levels of SREBP1 and TNF-α.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for pairwise comparison;the independent-samples t-test was used for comparison between two groups.Results Compared with the healthy controls,NAFLD patients showed significant increases in the levels of TC,TG,BUN,Cr,and UA(all P<0.05).Compared with the LFD group,the HFD group had significant increases in body weight,TC,TG,BUN,and Cr(all P<0.001),and compared with the HFD group,the XYXD group showed significant inhibition of the expression of TC,TG,BUN,and Cr(all P<0.001).Liver pathological examination showed that compared with the LFD group,the HFD group showed significant increases in hepatic steatosis and inflammatory infiltration,while the XYXD group showed significant alleviation of lesions.Renal pathological examination showed that compared with the LFD group,the HFD group had significant inflammatory infiltration,steatosis,and collagen formation in renal tissue,and compared with the HFD group,XYXD significantly alleviated inflammatory infiltration and inhibited steatosis and collagen formation.Quantitative real-time PCR showed that compared with the LFD group,the HFD group had significant increases in the relative mRNA expression levels of SREBP1,FASN,IL-6,TNF-α,Desmin,and α-SMA in renal tissue(all P<0.001),and compared with the HFD group,the XYXD group had significant reductions in the relative expression levels of these indicators(all P<0.001).Western blot showed that compared with the LFD group,the HFD group had significant increases in the protein expression levels of SREBP1 and TNF-α(P<0.05),and compared with the HFD group,the XYXD group had significant reductions in the protein expression levels of SREBP1 and TNF-α(P<0.05).Immunohistochemical staining showed that compared with the LFD group,the HFD group had significant increases in the positive staining or the double positive staining of α-SMA and CD68(P<0.05),and compared with the HFD group,the XYXD group showed significant reductions(P<0.05).Conclusion HFD can induce renal steatosis,inflammatory infiltration,and collagen formation,and XYXD might exert a protective effect on the kidneys by inhibiting the expression of macrophages and myofibroblasts in renal tissue.

Objective To explore the characteristics of response to joint attention(RJA)under diverse guiding behaviors for pre-schoolers with moderate to severe autism spectrum disorder(ASD). Methods From March to May,2023,21 children with moderate to severe ASD and 16 children with developmental de-lays matched the physiological ages were selected from Jiaxing Sunlight Rehabilitation Kindergarten,and 16 typ-ical developmental children matched the physiological ages were selected from the kindergartens nearby.They accepted a behavioral experiment on RJA.The number of RJA,frequence of RJA and the coefficient of variation of guiding behaviors needed to RJA were compared among the three groups. Results About half of the ASD group responded after guiding of head-turning,and the others required higher levels of guiding.The frequence of RJA after guiding of head-turning was less in the ASD group than in the typical devel-opment group(χ2>6.170,P<0.05),and the coefficient of variation of guiding behaviors was more(d=4.039,P<0.001). Conclusion Preschoolers with moderate to severe ASD are able to respond to joint attention,and this ability is poorer than typically developing children.The guiding behavior of the evaluator should be considered in assessing and intervening RJA in preschoolers with ASD.

Objective:To explore the surgical method and clinical effect of bionic double eyelid operation with "8" suture and anchoring.Methods:A retrospective study was conducted on the received double eyelid operation with "8" -shaped suture and anchor in the Department of Plastic Surgery People’s Hospital of Jinhua City, Zhejiang Province (Jinhua Hospital Affiliated to Wenzhou Medical University) during November 2016 to October 2020. The upper eyelid layer is dissected, the normal double eyelid structure of oriental is simulate. Using "8" -shaped suture anchorage technique, through horizontal mattress "8" -shaped suture, the orbital septum-upper eyelid levator aponeurosis complex (A) and the orbicularis oculi muscle (M) of the lower lip of the incision were relocated and healed on the tarsal plate (including pretarsal fascia) (T) the dynamic structure of the oriental double eyelid was reconstructed. In follow up, the upper eyelid flatness, the upper eyesight extent, the distance between the eye and eyebrow, the forced sensation of opening the eyes, bilateral eyes’ symmetry, the curve and the stability of the double eyelid, the eyelash curl, incisional scar and complications were recorded and evaluated.Results:A total of 103 patients, 5 males and 98 females, with a mean age of 29.5 years (17-65 years), were included. After the postoperative follow-up of 6 to 24 months, the upper eyelid was smooth, plump and moderate, the upper vision increased, the distance between the eyebrow and eyes was relatively shorter than the preoperative, the eyes openings were widened, the two sides were basically symmetrical, the double eyelid line was natural and smooth, the crease was stable, the eyelashes were slightly curled, the incision scar was not obvious. complications, such as infection and ptosis did occur.Conclusion:The "8" -shaped suture anchoring bionic double eyelid operation introduces the concept of "breaking" then "building", the upper eyelid can be dissected clearly, the reduction is accurate, the fixation is accurate, and the mechanical structure is stable. Postopertively, the shape of the upper eyelid was natural and stable.

Objective:To explore the surgical method and clinical effect of bionic double eyelid operation with "8" suture and anchoring.Methods:A retrospective study was conducted on the received double eyelid operation with "8" -shaped suture and anchor in the Department of Plastic Surgery People’s Hospital of Jinhua City, Zhejiang Province (Jinhua Hospital Affiliated to Wenzhou Medical University) during November 2016 to October 2020. The upper eyelid layer is dissected, the normal double eyelid structure of oriental is simulate. Using "8" -shaped suture anchorage technique, through horizontal mattress "8" -shaped suture, the orbital septum-upper eyelid levator aponeurosis complex (A) and the orbicularis oculi muscle (M) of the lower lip of the incision were relocated and healed on the tarsal plate (including pretarsal fascia) (T) the dynamic structure of the oriental double eyelid was reconstructed. In follow up, the upper eyelid flatness, the upper eyesight extent, the distance between the eye and eyebrow, the forced sensation of opening the eyes, bilateral eyes’ symmetry, the curve and the stability of the double eyelid, the eyelash curl, incisional scar and complications were recorded and evaluated.Results:A total of 103 patients, 5 males and 98 females, with a mean age of 29.5 years (17-65 years), were included. After the postoperative follow-up of 6 to 24 months, the upper eyelid was smooth, plump and moderate, the upper vision increased, the distance between the eyebrow and eyes was relatively shorter than the preoperative, the eyes openings were widened, the two sides were basically symmetrical, the double eyelid line was natural and smooth, the crease was stable, the eyelashes were slightly curled, the incision scar was not obvious. complications, such as infection and ptosis did occur.Conclusion:The "8" -shaped suture anchoring bionic double eyelid operation introduces the concept of "breaking" then "building", the upper eyelid can be dissected clearly, the reduction is accurate, the fixation is accurate, and the mechanical structure is stable. Postopertively, the shape of the upper eyelid was natural and stable.

Activation of the heart normally begins in the sinoatrial node (SAN). Electrical impulses spontaneously released by SAN pacemaker cells (SANPCs) trigger the contraction of the heart. However, the cellular nature of SANPCs remains controversial. Here, we report that SANPCs exhibit glutamatergic neuron-like properties. By comparing the single-cell transcriptome of SANPCs with that of cells from primary visual cortex in mouse, we found that SANPCs co-clustered with cortical neurons. Tissue and cellular imaging confirmed that SANPCs contained key elements of glutamatergic neurotransmitter system, expressing genes encoding glutamate synthesis pathway (Gls), ionotropic and metabotropic glutamate receptors (Grina, Gria3, Grm1 and Grm5), and glutamate transporters (Slc17a7). SANPCs highly expressed cell markers of glutamatergic neurons (Snap25 and Slc17a7), whereas Gad1, a marker of GABAergic neurons, was negative. Functional studies revealed that inhibition of glutamate receptors or transporters reduced spontaneous pacing frequency of isolated SAN tissues and spontaneous Ca

Objective:To analyse the clinical presentation and pathogenic gene mutations of a family diagnosed with primary familial brain calcification (PFBC).Methods:A pedigree with primary familial brain calcification was recruited. The clinical data of the proband who was admitted to the Affiliated Hospital of Guizhou Medical University in March 2020 and the family members were collected. The DNA sequence of myogenesis regulating glycosidase (MYORG) gene was detected by Sanger sequencing in the proband and some available family members.Results:The proband is a male, 30 years old. There was only one patient of PFBC in this family. The first symptom of the proband was vagueness of speech, and gradually extrapyramidal symptoms such as slow and flexible movement and advanced cognitive impairment appeared. The brain CT of the proband and his second brother showed extensive symmetrical calcifications, mainly located in the bilateral cerebellar hemispheres, basal ganglia and thalamus. A homozygous mutation in the exon 2 of the MYORG gene [c.1967T>C(p.I656T)] was identified in the proband and an asymptomatic patient. The heterozygous mutation of MYORG gene was also detected in four healthy family members.Conclusions:All patients with homozygous mutations of MYORG gene showed calcification in CT scan, and most of the lesions were located in basal ganglia, cerebellum, subcortical white matter and thalamus. Compared with the patients with autosomal dominant gene mutation, the patients with MYORG gene mutation had more extensive intracranial calcification lesions, and the pontocerebellar lesions were more common. The most common symptoms of MYORG gene mutation patients were dyskinesia, mainly tremor paralysis and unclear speech.

Objective To investigate the therapeutic effect of Taohong Siwu decoction on a mouse model of carbon tetrachloride (CCl 4 )-induced liver fibrosis and its mechanism of action. Methods A total of 24 male C57BL/6 mice were randomly divided into normal group, model group, and Taohong Siwu decoction group, with 8 mice in each group. The mice in the model group and the Taohong Siwu decoction group were given intraperitoneal injection of 10% CCl 4 , and Taohong Siwu decoction was given by gavage since week 3 for 4 consecutive weeks. Liver function [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] was measured, and liver pathomorphology was observed. Real-time PCR was used to measure the mRNA expression of α-smooth muscle actin (α-SMA), hyaluronic acid synthase-2 (HAS-2), and collagen type Ⅰ(Col1), and Western blotting was used to measure the protein expression of α-SMA, Col1, and HAS-2. Primary hepatic stellate cells (HSCs) were isolated, and HAS-2 was silenced by siRNA to observe its influence on HSC activation. The t -test was used for comparison of continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the SNK or least significant difference t -test was used for further comparison between two groups. Results Compared with the normal group, the model group had significant increases in serum liver function parameters (ALT, AST) and the Taohong Siwu decoction group had significant reductions in the serum levels of ALT and AST (all P < 0.01). Pathological staining showed that the model group had marked inflammatory cell infiltration and formation of fibrous septa by proliferated collagen fibers, and the Taohong Siwu decoction group had loose fibrous septa and alleviated inflammatory cell infiltration. Compared with the model group, the Taohong Siwu decoction group had significant reductions in the mRNA and protein expression of α-SMA and Col1(all P < 0.001). Compared with the normal group, the model group had a significant increase in the mRNA expression level of HAS-2 in liver tissue ( t =6.14, P < 0.05), and compared with the model group, the Taohong Siwu decoction group had a significant reduction in the protein expression level of HAS-2 (0.29±0.10 vs 1.00±0.12, t =70.73, P < 0.001). After HAS-2 was silenced by siRNA, the Si HAS-2+transforming growth factor β (TGFβ) group (treated with TGFβ) had significant reductions in the mRNA expression levels of α-SMA and Col-Ⅰ compared with the NC+TGFβ group ( P < 0.01). Conclusion Taohong Siwu decoction exerts a marked therapeutic effect on CCl 4 -induced liver fibrosis in mice by inhibiting HAS-2.