OBJECTIVE: To compare the clinical efficacy between penetrating needling of three eye acupoints combined with sodium hyaluronate eye drops and sodium hyaluronate eye drops alone for the treatment of dry eye. METHODS: A total of 156 patients (312 eyes) with dry eye were randomly assigned to an observation group and a control group, with 78 patients (156 eyes) in each group. The control group was treated with sodium hyaluronate eye drops, one drop per eye, four times daily, for 4 weeks. In addition to the sodium hyaluronate treatment, the observation group received penetrating needling of three eye acupoints. Acupoints included bilateral Cuanzhu (BL2), Sizhukong (TE23), Sibai (ST2), and Jingming (BL1). Needling was performed once daily, four times a week, for 4 weeks. The subjective ocular symptom scores, neuropathic pain symptom inventory-eye (NPSI-Eye) scores, ocular surface disease index (OSDI) scores, corneal fluorescein staining (FL) scores, tear break-up time (BUT), SchirmerⅠtest (SⅠT), central tear meniscus height (TMH), and tear levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were evaluated before and after treatment in the two groups. Clinical efficacy was also compared between the two groups. RESULTS: After treatment, both groups showed significant improvements in subjective ocular symptom scores, NPSI-Eye scores, OSDI scores, FL scores, and reductions in tear IL-6 and TNF-α levels (P<0.01). Additionally, BUT, SⅠT, and TMH were increased significantly in both groups (P<0.01). After treatment, the subjective ocular symptom scores, NPSI-Eye score, OSDI score, FL score, and tear levels of IL-6 and TNF-α in the observation group were lower than those in the control group (P<0.01, P<0.05), while BUT, SⅠT, and TMH were significantly improved compared to the control group (P<0.01). The markedly effective rate and total effective rate in the observation group were 83.3% (65/78) and 100.0% (78/78), respectively, which were higher than 52.6% (41/78, P<0.01) and 92.3% (72/78, P<0.05) in the control group. CONCLUSION The penetrating needling of three eye acupoints combined with sodium hyaluronate eye drops can effectively alleviate symptoms of dry eye, reduce inflammatory response, and has superior efficacy to sodium hyaluronate eye drops alone.
Humans ; Hyaluronic Acid/administration & dosage* ; Male ; Female ; Dry Eye Syndromes/genetics* ; Middle Aged ; Acupuncture Points ; Ophthalmic Solutions/administration & dosage* ; Adult ; Aged ; Treatment Outcome ; Acupuncture Therapy ; Interleukin-6/genetics* ; Young Adult ; Tumor Necrosis Factor-alpha/metabolism*

BACKGROUND: Lung cancer currently ranks first globally in both incidence and mortality. Pemetrexed (PMX) serves as a first-line treatment for lung adenocarcinoma (LUAD), but the patients often develop drug resistance during therapy. Milk exosome (mEXO) have the advantages of low immunogenicity, high tissue affinity, and low cost, and mEXO itself has anti-tumor effects. Hyaluronan (HA) naturally bind to CD44, a receptor which is highly expressed in LUAD tissues. This study aims to construct hyaluronan-modified milk exosome (HA-mEXO) and preliminarily investigate their molecular mechanisms for reversing PMX resistance through cellular experiments. METHODS: Exosomes were extracted from milk using high-speed centrifugation, and HA-mEXO was constructed. PMX-resistant A549 and PC-9 cell lines were treated with mEXO and HA-mEXO, respectively. CCK-8 assays, colony formation assays, Transwell assays, and flow cytometry were performed to evaluate proliferation, colony formation, migration, invasion, and apoptosis phenotypes in the treated resistant cell lines. Finally, transcriptomic sequencing, analysis, and cellular functional recovery experiments were conducted to investigate the mechanism by which HA-mEXO reverses PMX resistance in LUAD cells. RESULTS: The expression of CD44 in A549 and PC-9 LUAD drug-resistant cell lines was significantly higher than that in parental cells, and the uptake rate of HA-mEXO by drug-resistant cell lines was significantly higher than that of mEXO. Compared to the mEXO group, HA-mEXO-treated A549 and PC-9 resistant cells exhibited significantly reduced half maximal inhibitory concentration (IC50) values for PMX, markedly diminished clonogenic, migratory, and invasive capabilities, and a significantly increased proportion of apoptotic cells. Western blot analysis revealed that, compared to parental cells, A549 and PC-9 drug-resistant cells exhibited downregulated ZNF516 expression and upregulated ABCC5 expression. Immunofluorescence analysis revealed that HA-mEXO treatment downregulated ABCC5 expression in A549 and PC-9 drug-resistant cells compared to the PBS group, whereas co-treatment with HA-mEXO and ZNF516 knockdown showed no significant change in ABCC5 expression. CONCLUSIONS HA-mEXO carrying ZNF516 suppress ABCC5 expression, thereby enhancing the sensitivity of A549 and PC-9 LAUD drug-resistant cells to PMX.
Humans ; Hyaluronic Acid/chemistry* ; Drug Resistance, Neoplasm/drug effects* ; Exosomes/chemistry* ; Adenocarcinoma of Lung/genetics* ; Pemetrexed/pharmacology* ; Animals ; Lung Neoplasms/pathology* ; Milk/chemistry* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Cell Line, Tumor ; Hyaluronan Receptors/metabolism*

OBJECTIVE: To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation. METHODS: Firstly, hyaluronic acid was oxidized with NaIO ₄ and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young's modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR). RESULTS: FTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young's modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point ( P<0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group ( P<0.05). CONCLUSION The MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.
Hydrogels ; Hyaluronic Acid/pharmacology* ; Vascular Endothelial Growth Factor A/metabolism* ; Tissue Engineering/methods* ; Cell Differentiation ; Myoblasts/metabolism* ; Cell Proliferation

The high- and the low-molecular weight hyaluronic acids (HMW-HA and LMW-HA, respectively) showed different biological activities in inflammation. However, the role of LMW-HA in inflammatory response is controversial. In this study, we aimed to investigate the effect of bioactive hyaluronan (B-HA) on lipopolysaccharide (LPS)-induced inflammatory responses in human macrophages and mice. B-HA was produced from HA treated with glycosylated recombinant human hyaluronidase PH20. Human THP-1 cells were induced to differentiate into macrophages. THP-1-derived macrophages were treated with B-HA, LPS, or B-HA + LPS. The mRNA expression and the production of inflammatory cytokines were determined using quantitative real-time PCR and enzyme-linked immunosorbent assay. The phosphorylation levels of proteins in the nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and IRF-3 signaling pathways were measured using Western blot. The in vivo efficacy of B-HA was assessed in a mouse model of LPS-induced inflammation. Results showed that B-HA inhibited the expression of TNF-α, IL-6, IL-1, and IFN-β, and enhanced the expression of the antiinflammatory cytokine IL-10 in LPS-induced inflammatory responses in THP-1-derived macrophages and in vivo. B-HA significantly suppressed the phosphorylation of the TLR4 signaling pathway proteins p65, IKKα/β, IκBα, JNK1/2, ERK1/2, p38, and IRF-3. In conclusion, our results demonstrated that the B-HA attenuated the LPS-stimulated inflammatory response by inhibiting the activation of the TLR4 signaling pathway. B-HA could be a potential anti-inflammatory drug in the treatment of inflammatory disease.
Animals ; Cytokines ; Hyaluronic Acid ; Lipopolysaccharides ; Mice ; NF-kappa B/metabolism* ; Signal Transduction ; Toll-Like Receptor 4

Hyaluronic acid (HA) is widely used in many fields, such as medicine, cosmetics and food. The bioactivity of HA depends on its molecular weight (Mw). Owing to the important physiological activities and special physiological functions, HA oligosaccharides have important application prospects in medicine fields. Streptococcus zooepidemicus has wide applications in commercial production of HA, due to its short fermentation cycle and strong production intensity. In order to efficiently synthesize HA oligosaccharides and solve the dissolved oxygen in the fermentation process, in this study, we overexpressed HA synthase (HasA) and introduced and optimized the leech hyaluronidase LHAase in Streptococcus zooepidemicus WSH-24. As a result, HA oligosaccharides were efficiently produced with improved dissolved oxygen. After 24 h, HA oligosaccharides production intensity reached to 294.2 mg/(L·h), and the concentration accumulated to 0.97 g/L in flask cultures, which was 1.82 times of the wild strain. Impressively, HA oligosaccharides were increased to 7.06 g/L in 3 L fermentor. The constructed Streptococcus zooepidemicus strain for producing HA oligosaccharides would have broad application prospects.
Bioreactors ; Fermentation ; Hyaluronan Synthases ; genetics ; metabolism ; Hyaluronic Acid ; genetics ; metabolism ; Industrial Microbiology ; Oligosaccharides ; genetics ; metabolism ; Streptococcus equi ; genetics ; metabolism

Objective: To elucidate the possible role of human lysozyme-like protein 4 (LYZL4) in fertilization and characterize its enzymatic properties. METHODS: The localization of LYZL4 in human spermatozoa was investigated by immunofluorescence staining, the sources of LYZL4 on the sperm surface examined by RT-PCR, and the role of LYZL4 in fertilization assessed by the zona-free hamster egg penetration test. The recombinant plasmid pPIC9K-LYZL4 was constructed and its expression induced with methanol after transformed into competent Pichia pastoris GS115. The recombinant LYZL4 protein (rLYZL4) was purified from the fermentation supernatant and subsequently identified by Western blot. The hyaluronan binding ability of rLYZL4 was determined by ELISA and the muramidase activity, hyaluronidase activity, and free radical scavenging ability examined by spectrophotometric methods. RESULTS: Immunodetection with a specific antiserum localized LYZL4 on the acrosomal membrane of mature spermatozoa, which was exclusively secreted from the testis and epididymis as shown by RT-PCR. Immunoneutralization of LYZL4 significantly decreased the number of human spermatozoa bound to zona-free hamster eggs in a dose-dependent manner in vitro. The recombinant protein was expressed successfully by the P. pastoris strain GS115. Purified rLYZL4 exhibited a potent hyaluronan binding ability and a strong free radical scavenging ability but no muramidase or hyaluronidase activity. CONCLUSIONS LYZL4 secreted from the testis and epididymis is localized on the acrosomal membrane of mature spermatozoa and plays a role in sperm-egg binding as well as in binding hyaluronan and scavenging free radicals, which suggests that it might be a multi-functional molecule contributive to sperm protection and sperm-egg binding.
Acrosome ; enzymology ; Animals ; Blotting, Western ; Cricetinae ; Enzyme-Linked Immunosorbent Assay ; Epididymis ; Female ; Fertilization ; physiology ; Free Radical Scavengers ; metabolism ; Humans ; Hyaluronic Acid ; metabolism ; Male ; Muramidase ; analysis ; physiology ; Pichia ; Plasmids ; metabolism ; Recombinant Proteins ; analysis ; metabolism ; Sperm-Ovum Interactions ; physiology ; Spermatozoa ; enzymology ; Testis

OBJECTIVE: To construct a novel non-viral vector loaded with growth and differentiation factor-5 (GDF-5) plasmid using chitosan, hyaluronic acid, and chondroitin sulfate for osteoarthritis (OA) gene therapy. METHODS: Nano-microspheres (NMPs) were prepared by mixing chitosan, hyaluronic acid, and chondroitin sulfate. GDF-5 plasmid was encapsulated in the NMPs through electrostatic adsorption. The basic characteristics of the NMPs were observed, and then they were co-cultured with chondrocytes to observe their effects on extracellular matrix (ECM) protein expression. Finally, NMPs loaded with GDF-5 were injected into the articular cavities of rabbits to observe their therapeutic effects on OA in vivo. RESULTS: NMPs exhibited good physicochemical properties and low cytotoxicity. Their average diameter was (0.61±0.20) μm, and encapsulation efficiency was (38.19±0.36)%. According to Cell Counting Kit-8 (CCK-8) assay, relative cell viability was 75%-99% when the total weight of NMPs was less than 560 μg. Transfection efficiency was (62.0±2.1)% in a liposome group, and (60.0±1.8)% in the NMP group. There was no significant difference between the two groups (P>0.05). Immunohistochemical staining results suggested that NMPs can successfully transfect chondrocytes and stimulate ECM protein expression in vitro. Compared with the control groups, the NMP group significantly promoted the expression of chondrocyte ECM in vivo (P<0.05), as shown by analysis of the biochemical composition of chondrocyte ECM. When NMPs were injected into OA model rabbits, the expression of ECM proteins in chondrocytes was significantly promoted and the progression of OA was slowed down. CONCLUSIONS Based on these data, we think that these NMPs with excellent physicochemical and biological properties could be promising non-viral vectors for OA gene therapy.
Animals ; Cell Differentiation ; Cell Survival/drug effects* ; Chitosan/chemistry* ; Chondrocytes/cytology* ; Chondroitin Sulfates/chemistry* ; Drug Carriers ; Extracellular Matrix/metabolism* ; Genetic Therapy/methods* ; Growth Differentiation Factor 5/genetics* ; Hyaluronic Acid/chemistry* ; Microspheres ; Nanomedicine ; Osteoarthritis/therapy* ; Plasmids/metabolism* ; Rabbits

Hyaluronic acid (HA) is an important biomaterial as the extracellular matrix in human body. We produced HA by fermentation of Streptococcus iniae (Strep.). Production of HA by Strep. was evaluated and further improved by strain mutation by ultraviolet. One strain with higher HA yield and lower content of protein was obtained. Its HA yield increased from (82.3±3.3) mg/L to (120±10.6) mg/L, and protein decreased from (0.178±0.011) mg/L to (0.032±0.017) mg/L. The molecular weight (MW) of HA yield from Strep. is about 3.0×10⁵ Da. Using the method of freezing and thawing, HA aqueous solution was transferred into hydrogel. This HA hydrogel, casted on sterilized non-woven fabric, was applied to repair rabbit skin with full-thickness defect. The preliminary results of the animal tests displayed that HA hydrogel obviously reduced the inflammation around the wound and promoted the skin regeneration comparing with the control tests.
Animals ; Biocompatible Materials ; Fermentation ; Hyaluronic Acid ; biosynthesis ; Hydrogels ; Molecular Weight ; Rabbits ; Regeneration ; Skin ; drug effects ; growth & development ; Streptococcus iniae ; metabolism ; Wound Healing

PURPOSE: To investigate the therapeutic effects of mineral oil (MO) and hyaluronic acid (HA) mixture eye drops on the tear film and ocular surface in a mouse model of experimental dry eye (EDE). METHODS: Eye drops consisting of 0.1% HA alone or mixed with 0.1%, 0.5%, or 5.0% MO were applied to desiccating stress-induced murine dry eyes. Tear volume, corneal irregularity score, tear film break-up time (TBUT), and corneal fluorescein staining scores were measured at 5 and 10 days after treatment. Ten days after treatment, goblet cells in the conjunctiva were counted after Periodic acid-Schiff staining. RESULTS: There was no significant difference in the tear volume between desiccating stress-induced groups. The corneal irregularity score was lower in the 0.5% MO group compared with the EDE and HA groups. The 0.5% and 5.0% MO groups showed a significant improvement in TBUT compared with the EDE group. Mice treated with 0.1% and 0.5% MO mixture eye drops showed a significant improvement in fluorescein staining scores compared with the EDE group and the HA group. The conjunctival goblet cell count was higher in the 0.5% MO group compared with the EDE group and HA group. CONCLUSIONS: The MO and HA mixture eye drops had a beneficial effect on the tear films and ocular surface of murine dry eye. The application of 0.5% MO and 0.1% HA mixture eye drops could improve corneal irregularity, the corneal fluorescein staining score, and conjunctival goblet cell count compared with 0.1% HA eye drops in the treatment of EDE.
Animals ; Conjunctiva/*drug effects/pathology ; Cornea/metabolism ; Disease Models, Animal ; Drug Combinations ; Dry Eye Syndromes/*drug therapy/metabolism ; Emollients/administration & dosage ; Female ; Goblet Cells/drug effects/metabolism/pathology ; Hyaluronic Acid/*administration & dosage ; Mice ; Mice, Inbred C57BL ; Mineral Oil/*administration & dosage ; Ophthalmic Solutions ; Tears/*metabolism ; Viscosupplements/administration & dosage

OBJECTIVETo study the inhibitory effect of flavonoids from Glycyrrhiza uralensis on thioacetamide-induced chonic hepatic fibrosis in rats and the effect on the protein expressions of transforming growth factor-β1 (TGF-β1) and Caspase-3 in livers.

METHODMale Sprague-Dawley rats were randomly divided into totally seven groups: the normal control group, the model group, LF groups s (400, 200, 100, 50 mg · kg(-1) · d(-1)) and the silymarin positive control group (30 mg · kg(-1) · d(-1)). The hepatic fibrosis model was induced in the rats through intraperitoneal injection with 3% thioacetamide (TAA) at a dose of 150 mg · kg(-1) body weight twice a week for 12 weeks. During the course, the control group and the model group were orally administered with saline (1 mL · kg(-1) · d(-1)). After the modeling and drug intervention, the pathologic changes and fibrosis in liver tissues were observed by HE staining and Masson's Trichrome staining. The serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and liver hydroxyproline (HYP) contents were assayed by biochemical process. The serum hyaluronic acid (HA) was assessed by radioimmunoassay. In addition, the protein expressions of liver TGF-β1 and Caspase-3 were examined by immunohistochemical method. The mRNA expression of TGF-β1 in hepatic tissues was examined by quantitative Real-time PCR analysis.

RESULTCompared with the model group, flavonoids can protect the integrity of the structure of liver tissues, significantly reduce the hepatic cell degeneration and necrosis and the proliferation of fibrous tissues, notably reduce the serum AST, ALT, ALP and HA and HYP in hepatic tissues and down-regulate the protein expressions of liver TGF-β1 and Caspase-3 and the mRNA expression of TGF-β1 in hepatic tissues.

CONCLUSIONThe licorice flavonoids can resist the thioacetamide-induced hepatic fibrosis in rats. Its mechanism may be related to the down-regulation of the protein expressions of TGF-β1 and Caspase-3.

Animals ; Caspase 3 ; analysis ; Flavonoids ; pharmacology ; Glycyrrhiza uralensis ; chemistry ; Hyaluronic Acid ; blood ; Liver ; pathology ; Liver Cirrhosis, Experimental ; chemically induced ; metabolism ; prevention & control ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Thioacetamide ; Transforming Growth Factor beta1 ; analysis ; genetics