Loss of Bladder Epithelium Induced by Cytolytic Mast Cell Granules.

Programmed death and shedding of epithelial cells is a powerful defense mechanism to reduce bacterial burden during infection but this activity cannot be indiscriminate because of the critical barrier function of the epithelium. We report that during cystitis, shedding of infected bladder epithelial cells (BECs) was preceded by the recruitment of mast cells (MCs) directly underneath the superficial epithelium where they docked and extruded their granules. MCs were responding to interleukin-1ß (IL-1ß) secreted by BECs after inflammasome and caspase-1 signaling. Upon uptake of granule-associated chymase (mouse MC protease 4 [mMCPT4]), BECs underwent caspase-1-associated cytolysis and exfoliation. Thus, infected epithelial cells require a specific cue for cytolysis from recruited sentinel inflammatory cells before shedding.

Oxybutynin ameliorates LPS-induced inflammatory response in human bladder epithelial cells.

Urinary tract infection (UTI) mainly results from bacterial infections in the urinary tract and markedly impacts the normal lives of millions of patients worldwide. The infection and damage to urethral epithelial cells is the first and key step of UTI development and is a critical target for treating clinical UTI. Oxybutynin, an agent for treating urinary incontinence, is recently claimed with protective effects on bladder ultrastructure. Our study will assess the impact of Oxybutynin on inflammation in lipopolysaccharide (LPS)-stimulated bladder epithelial cells. Bladder epithelial T24 cells were treated with 1 µg/mL LPS with or without 10 and 20 µM Oxybutynin for 24 h. Increased levels of oxidative stress (OS) biomarkers, such as reactive oxygen species, 8-hydroxy-2'-deoxyguanosine, malondialdehyde, as well as upregulated inducible nitric oxide synthase and promoted release of nitric oxide, were observed in LPS-managed T24 cells, all of which were signally suppressed by Oxybutynin. Furthermore, severe inflammatory responses, including enhanced release of cytokines, upregulated matrix metallopeptidase-2 (MMP-2) and MMP-9, and raised monocyte chemoattractant protein-1 level, were found in LPS-challenged T24 cells, which were markedly reversed by Oxybutynin. Moreover, the activated toll-1ike receptor 4/nuclear factor-κB pathway observed in LPS-managed T24 cells was repressed by Oxybutynin. Collectively, Oxybutynin mitigated LPS-induced inflammatory response in human bladder epithelial cells.

Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

PapG subtype-specific binding characteristics of Escherichia coli towards globo-series glycosphingolipids of human kidney and bladder uroepithelial cells.

Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections (UTIs) in humans. P-fimbriae are key players for bacterial adherence to the uroepithelium through the Galα1-4Gal-binding PapG adhesin. The three identified classes I, II and III of PapG are supposed to adhere differently to host cell glycosphingolipids (GSLs) of the uroepithelial tract harboring a distal or internal Galα1-4Gal sequence. In this study, GSL binding characteristics were obtained in a nonradioactive adhesion assay using biotinylated E. coli UTI and urine isolates combined with enzyme-linked NeutrAvidin for detection. Initial experiments with reference globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer), globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) and Forssman GSL (GalNAcα1-3GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) revealed balanced adhesion toward the three GSLs for PapG I-mediated attachment. In contrast, E. coli carrying PapG II or PapG III increasingly adhered to growing oligosaccharide chain lengths of Gb3Cer, Gb4Cer and Forssman GSL. Binding studies with GSLs from human A498 kidney and human T24 bladder epithelial cells, both being negative for the Forssman GSL, revealed the less abundant Gb4Cer vs. Gb3Cer as the prevalent receptor in A498 cells of E. coli expressing PapG II or PapG III. On the other hand, T24 cells exhibited a higher relative content of Gb4Cer vs. Gb3Cer alongside dominant binding of PapG II- or PapG III-harboring E. coli toward Gb4Cer and vastly lowered attachment to minor Gb3Cer. Further studies on PapG-mediated interaction with cell surface-exposed GSLs will improve our knowledge on the molecular mechanisms of P-fimbriae-mediated adhesion and may contribute to the development of antiadhesion therapeutics to combat UTIs.

Quantitative Proteomic Analysis Reveals Caffeine-Perturbed Proteomic Profiles in Normal Bladder Epithelial Cells.

Lower urinary tract symptoms (LUTSs) are highly prevalent among the elderly and negatively impact quality of life. Since caffeinated beverages are enjoyed worldwide and the relationship between LUTS and caffeine is still not fully understood, it would be of particular interest to examine the underlying mechanisms that drive caffeine's influence on LUTS development and progression. The aim of this study is to characterize the effects of caffeine on hTert-immortalized normal bladder epithelial cells by investigating whether exposure to caffeine can cause potential changes in the bladder proteome and/or biological pathways. In labeled LC-MS/MS proteomic analysis, 57 proteins are found as being differentially expressed in caffeine-treated bladder epithelial cells, compared to controls; this included 32 upregulated and 25 downregulated proteins. Further functional gene enrichment analysis reveals that caffeine affects major biological pathways, including those for "muscle contraction" and "chromatin assembly." These findings provide new scientific insights that may be useful in future studies investigating the role of caffeine in bladder dysfunctions.

GM1 and GD1a gangliosides modulate toxic and inflammatory effects of E. coli lipopolysaccharide by preventing TLR4 translocation into lipid rafts.

Exogenous gangliosides are known to inhibit the effects of Escherichia coli lipopolysaccharide (LPS) in different cells exhibiting anti-inflammatory and immunosuppressive activities. The mechanisms underlying ganglioside action are not fully understood. Because LPS recognition and receptor complex formation occur in lipid rafts, and gangliosides play a key role in their maintenance, we hypothesize that protective effects of exogenous gangliosides would depend on inhibition of LPS signaling via prevention of TLR4 translocation into lipid rafts. The effect of GM1 and GD1a gangliosides on LPS-induced toxic and inflammatory reactions in PC12 cells, and in epithelial cells isolated from the frog urinary bladder, was studied. In PC12 cells, GD1a and GM1 significantly reduced the effect of LPS on the decrease of cell survival and on stimulation of reactive oxygen species production. In epithelial cells, gangliosides decreased LPS-stimulated iNOS expression, NO, and PGE2 production. Subcellular fractionation, in combination with immunoblotting, showed that pretreatment of cells with GM1, GD1a, or methyl-ß-cyclodextrin, completely eliminated the effect of LPS on translocation of TLR4 into lipid rafts. The results are consistent with the hypothesis that ganglioside-induced prevention of TLR4 translocation into lipid rafts could be a mechanism of protection against LPS in various cells.

Abnormal Akt signalling in bladder epithelial cell explants from patients with interstitial cystitis/bladder pain syndrome can be induced by antiproliferative factor treatment of normal bladder cells.

OBJECTIVES: To determine whether protein kinase B (Akt) signalling and secretion of specific downstream effector proteins are abnormal in specific cell fractions of bladder epithelial cells from patients with interstitial cystitis/bladder pain syndrome (IC/BPS), as explanted bladder epithelial cells from patients with IC/BPS produce a frizzled 8-related glycopeptide antiproliferative factor (APF) that inhibits normal bladder epithelial cell proliferation and expression of several proteins known to be regulated by Akt signalling. A related secondary objective was to determine whether treatment of normal bladder epithelial cells with active synthetic asialo-antiproliferative factor (as-APF) induces similar changes in Akt signalling and specific downstream effector proteins/mRNAs. PATIENTS AND METHODS: Cell proteins were extracted into four subcellular fractions from primary bladder epithelial explants of six patients who fulfilled modified National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) criteria for IC/BPS and six age- and gender-matched controls. Total and/or phosphorylated cellular Akt, glycogen synthase kinase 3ß (GSK3ß), and ß-catenin; total cellular JunB; and secreted matrix metalloproteinase 2 (MMP2) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) levels were determined by Western blot. MMP2, JunB, p53, uroplakin 3 (UPK3), and ß-actin mRNAs were quantified by quantitative reverse transcriptase-polymerase chain reaction. Akt activity was determined by nonradioactive assay. RESULTS: IC/BPS cells had lower Akt activity, along with lower Akt ser473- and GSK3ß ser9-phosphorylation and higher ß-catenin ser33,37/thr41-phosphorylation in specific fractions as compared with matched control cells. IC/BPS explants also had evidence of additional downstream abnormalities compared with control cells, including lower nuclear JunB; lower secreted MMP2 and HB-EGF; plus lower MMP2, JunB, and UPK3 mRNAs but higher p53 mRNA relative to ß-actin. Each of these IC/BPS cell abnormalities was also induced in normal cells by as-APF. CONCLUSION: These findings indicate that IC/BPS cells have abnormal Akt activity with downstream protein expression abnormalities including decreased MMP2 and HB-EGF secretion. They also support the hypothesis that APF plays a role in the pathogenesis of IC/BPS via its effects on cell Akt signalling and HB-EGF production.

Human milk oligosaccharides protect bladder epithelial cells against uropathogenic Escherichia coli invasion and cytotoxicity.

The invasive pathogen uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). Recurrent infection that can progress to life-threatening renal failure has remained as a serious global health concern in infants. UPEC adheres to and invades bladder epithelial cells to establish infection. Studies have detected the presence of human milk oligosaccharides (HMOs) in urine of breast-fed, but not formula-fed, neonates. We investigated the mechanisms HMOs deploy to elicit protection in human bladder epithelial cells infected with UPEC CFT073, a prototypic urosepsis-associated strain. We found a significant reduction in UPEC internalization into HMO-pretreated epithelial cells without observing any significant effect in UPEC binding to these cells. This event coincides with a rapid decrease in host cell cytotoxicity, recognized by LIVE/DEAD staining and cell detachment, but independent of caspase-mediated or mitochondrial-mediated programmed cell death pathways. Further investigation revealed HMOs, and particularly the sialic acid-containing fraction, reduced UPEC-mediated MAPK and NF-κB activation. Collectively, our results indicate that HMOs can protect bladder epithelial cells from deleterious cytotoxic and proinflammatory effects of UPEC infection, and may be one contributing mechanism underlying the epidemiological evidence of reduced UTI incidence in breast-fed infants.

Mycobacterium bovis bacille Calmette-Guerin-derived extracellular vesicles as an alternative to live BCG immunotherapy.

For over 40 years, the gold standard treatment for non-muscular invasive bladder cancer (NMIBC) has been repeated administration of Mycobacterium bovis bacille Calmette-Guerin (BCG). Upon administration, BCG initiates a cascade of immunological events that lead to the recruitment of immune cells to the bladder that eliminates NMIBC cells in a multi-mechanistic, yet incompletely defined manner. Despite its effectiveness, live BCG immunotherapy is often impacted by limited supply and availability and can cause rare but serious side effects. Bacterial extracellular vesicles (EV) are nanoparticles secreted by live bacteria. EVs are composed of multiple surface proteins, sugars, and lipid that can elicit cellular responses and host recognition similar to live bacteria. In this study, we sought to evaluate the cellular responses of epithelial bladder cancer cells (BCC) to BCG EVs and live BCG. We compared the effect of each treatment on BCC cytokine production, cellular viability and apoptosis. Our data suggest that BCG EVs are as effective as live BCG in eliciting cytokine responses and halting cancer cell growth by, in part, inducing apoptosis. These results indicate that BCG EVs warrant investigation as an alternative to live BCG for NMIBC immunotherapy.

Targeting Host Tyrosine Kinase Receptor EPHA2 Signaling Affects Uropathogen Infection in Human Bladder Epithelial Cells.

Urinary tract infections (UTIs) affect a major proportion of the world population but have limited non-antibiotic-based therapeutic and preventative strategies against UTIs. Facultative intracellular uropathogens such as strains of uropathogenic E. coli, K. pneumoniae, E. faecalis, E. cloacae are well-known uropathogens causing UTIs. These pathogens manipulate several host-signaling pathways during infection, which contributes to recurrent UTIs and inappropriate antibiotic application. Since host cell receptor tyrosine kinases (RTKs) are critical for the entry, survival and replication of intracellular pathogens, we investigated whether different uropathogens require host EPHA2 receptors for their intracellular survival using a cell culture model of intracellular infection in human bladder epithelial cells (BECs). Infection of BECs with seven different uropathogens enhanced the expression levels and activation of EPHA2. The significance of EPHA2 signaling for uropathogen infection was investigated by silencing EPHA2 expression using RNA interference or by inhibiting the kinase activity of EPHA2 using small-molecule compounds such as dasatinib or ALW-II-41-27. Both preventive and therapeutic tyrosine kinase inhibition significantly reduced the intracellular bacterial load. Thus, our results demonstrate the involvement of host cell EPHA2 receptor during intracellular uropathogen infection of BECs, and targeting RTK activity is a viable non-antibiotic therapeutic strategy for managing recurrent UTIs.

Estrogen receptors in human bladder cells regulate innate cytokine responses to differentially modulate uropathogenic E. coli colonization.

The bladder epithelial cells elicit robust innate immune responses against urinary tract infections (UTIs) for preventing the bacterial colonization. Physiological fluctuations in circulating estrogen levels in women increase the susceptibility to UTI pathogenesis, often resulting in adverse health outcomes. Dr adhesin bearing Escherichia coli (Dr E. coli) cause recurrent UTIs in menopausal women and acute pyelonephritis in pregnant women. Dr E. coli bind to epithelial cells via host innate immune receptor CD55, under hormonal influence. The role of estrogens or estrogen receptors (ERs) in regulating the innate immune responses in the bladder are poorly understood. In the current study, we investigated the role of ERα, ERß and GPR30 in modulating the innate immune responses against Dr E. coli induced UTI using human bladder epithelial carcinoma 5637 cells (HBEC). Both ERα and ERß agonist treatment in bladder cells induced a protection against Dr E. coli invasion via upregulation of TNFα and downregulation of CD55 and IL10, and these effects were reversed by action of ERα and ERß antagoinsts. In contrast, the agonist-mediated activation of GPR30 led to an increased bacterial colonization due to suppression of innate immune factors in the bladder cells, and these effects were reversed by the antagonist-mediated suppression of GPR30. Further, siRNA-mediated ERα knockdown in the bladder cells reversed the protection against bacterial invasion observed in the ERα positive bladder cells, by modulating the gene expression of TNFα, CD55 and IL10, thus confirming the protective role of ERα. We demonstrate for the first time a protective role of nuclear ERs, ERα and ERß but not of membrane ER, GPR30 against Dr E. coli invasion in HBEC 5637 cells. These findings have many clinical implications and suggest that ERs may serve as potential drug targets towards developing novel therapeutics for regulating local innate immunity and treating UTIs.

Speciation of arsenic in exfoliated urinary bladder epithelial cells from individuals exposed to arsenic in drinking water.

BACKGROUND: The concentration of arsenic in urine has been used as a marker of exposure to inorganic As (iAs). Relative proportions of urinary metabolites of iAs have been identified as potential biomarkers of susceptibility to iAs toxicity. However, the adverse effects of iAs exposure are ultimately determined by the concentrations of iAs metabolites in target tissues. OBJECTIVE: In this study we examined the feasibility of analyzing As species in cells that originate in the urinary bladder, a target organ for As-induced cancer in humans. METHODS: Exfoliated bladder epithelial cells (BECs) were collected from urine of 21 residents of Zimapan, Mexico, who were exposed to iAs in drinking water. We determined concentrations of iAs, methyl-As (MAs), and dimethyl-As (DMAs) in urine using conventional hydride generation-cryotrapping-atomic absorption spectrometry (HG-CT-AAS). We used an optimized HG-CT-AAS technique with detection limits of 12-17 pg As for analysis of As species in BECs. RESULTS: All urine samples and 20 of 21 BEC samples contained detectable concentrations of iAs, MAs, and DMAs. Sums of concentrations of these As species in BECs ranged from 0.18 to 11.4 ng As/mg protein and in urine from 4.8 to 1,947 ng As/mL. We found no correlations between the concentrations or ratios of As species in BECs and in urine. CONCLUSION: These results suggest that urinary levels of iAs metabolites do not necessarily reflect levels of these metabolites in the bladder epithelium. Thus, analysis of As species in BECs may provide a more effective tool for risk assessment of bladder cancer and other urothelial diseases associated with exposures to iAs.

The multiple antibacterial activities of the bladder epithelium.

The urinary tract is subject to frequent challenges from the gut microflora. Indeed, up to 40% of women will experience at least one urinary tract infection (UTI) during their lifetime. Uropathogenic Escherichia coli (UPEC) contribute to an overwhelming majority of these cases and they typically initiate UTIs by invading the superficial epithelium that lines the bladder lumen. In addition to serving as an effective barrier to noxious agents found in urine, bladder epithelial cells (BECs) play a key physiological role in regulating bladder volume to accommodate urine flow. UPEC appear to coopt this latter property to circumvent this normally impregnable epithelial barrier. However, in spite of this shortcoming, recent studies suggest that BECs possess several immune mechanisms to combat bacterial invasion including expulsion of invading bacteria back into the bladder lumen following infection. These antibacterial activities of BECs are triggered and coordinated by sensory molecules located on the epithelial cell membrane and within the cells. Although, they are the primary targets of microbial attack, BECs appear to be equipped with a diverse repertoire of defense schemes to fend off many of these microbial challenges.

Role of uropathogenic Escherichia coli outer membrane protein T in pathogenesis of urinary tract infection.

OmpT is one of the members of the outer membrane protein family that has been identified as a virulence factor in most of the uropathogenic Escherichia coli (UPEC). However, the exact role of OmpT in the urinary tract infections (UTIs) remains unclear. To determine the role of OmpT in the pathogenesis of UPEC, an isogenic deletion mutant of ompT (COTD) was constructed by the λ Red recombination. Human bladder epithelial cell line 5637(HBEC 5637) was used to evaluate the ability of bacterial adhesion/invasion. A murine model of UTI was established to study the formation of intracellular bacterial communities (IBCs) in the process of UTIs. The cytokines were also examined during the pathogenesis. The results showed that the COTD strain was deficient in bacterial adhesion and invasion as well as in IBC formation compare to the parent strain. ELISA quantification analysis of cytokines showed that the levels of TNF-α, IL-6 and IL-8 in the serum, bladder and kidney tissues of the mice infected with COTD were lower than that of the CFT073 group. In summary, these results suggest that OmpT plays a multifaceted role in pathogenesis of UTI, including increased bacterial adhesiveness/invasiveness, formation of IBCs and upregulated proinflammatory cytokines.

Niaogan Prescription protects bladder epithelial cells against uropathogenic Escherichia coli invasion / 中草药

Objective To observe the role of Niaogan Prescription against uropathogenic Escheriehia coli (UPEC) in invading bladder epithelial cells, and to discuss its mechanism of action. Methods After HTB-9 infected with UPEC, the cell invasion model was used to observe the protective effects of drug urine in human bladder cancer cell 5637 (HTB-9) against UPEC invasion. Effects of Niaogan Prescription on the main steps of signaling pathway: Toll-like receptor 4 (TLR4), adenylate cyclase 3 (AC3), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and Rac-1 was investigated by molecular biology techniques. Results Compared with the blank urine high dose group (10% volume fraction of blank urine), treatment with the drug-containing urine high dose group of Niaogan Prescription (10% volume fraction of drug urine) resulted in a significant decrease in UPEC invasion, the bacterial invasion rate of the rat blank urine group had no significant difference compared with the model group. Compared with the blank urine high dose group, the drug-containing urine high dose group of Niaogan Prescription improved TLR4 and AC3 protein expression, increased intracellular cAMP content, promoted PKA activation, and inhibited Rac1 activity. Conclusion Niaogan Prescription have the certain capacity against UPEC invasion of bladder epithelial cells, which mechanism is related to TLR4/cAMP signaling pathway. Niaogan Prescription; uropathogenic Escheriehia coli; invasion; human bladder epithelial cells; TLR4/cAMP signal pathway

Chitosan promotes proliferation of rabbit bladder epithelial cells in vitro / 第二军医大学学报

Objective: To study the influence of chitosan on proliferation of bladder epithelial cells, so as to discuss its feasibility in treatment of interstitial cystitis. Methods: Bladder epithelial cells were harvested by enzymatic digestion of the epithelium of New-Zealand rabbit bladder. The cells were cultured in different concentrations of chitosan(0.3, 0.6, 1.2, 2.4 and 4.8 g/L) for 72 h; untreated cells served as control. The growth and proliferation of cells were observed under microscope. The effects of chitosan on proliferation of cells were studied by NAG assay and cell counting. Results: Immunohistochemistry staining revealed that the cultured cells were epithelial cells. Chitosan (>0.3 g/L) promoted the growth of epithelial cells, and the promoting effect was significantly when the concentration of chitosan was 1.2 g/L (P<0.01). The promoting effects were decreased when the concentrations of chitosan were 2.4 and 4.8 g/L, but were still higher than that of the control group (P<0.01). Conclusion: Chitosan can promote the growth of the bladder epithelial cell in vitro, which might contribute to the treatment of interstitial cystitis.

Experimental study of chitosan on proliferation of bladder epithelial cells in vitro in rabbit ZHENG / 中华泌尿外科杂志

Objective To study chitosan's improving proliferation effect to the bladder epithelial cells,thus providing experlimental foundation for the treatment of interstitial cystitis.Methods Bladder epithelial cells were harested by the enzymatic digestion of the epithelium exposed by the eversion of reseeted New-Zealand hare's bladder.The cells were cultured in different concentrations(0.3、0.6、1.2、2.4、4.8 g/L)of chitosan group and control group,after 72 h,observing their growth and proliferation with optical microscopy;The effects of chitosan on proliferation of rabbit bladder epithelial cells were studied by NAG assay.EGFR mRNA was measured by PT-PCR.Results The growth of cells in the sample added chitosan is much better than that of in the control group.Chitosan could promote the proliferation of bladder epithelial cells at higher than 0.3 g/L of concentration in a dose dependent way.The optimum concentration to increase proliferation of eonjunctival epithelial cells was 1.2 g/L.The proliferative effect of EGFR mRNA increased with the elevated chitosan concentration after 72 h.Conclusions Chitosan can promote the growth of the bladder epithelial eell,which can provides a valuable evidence for further studies of interstitial cystitis's treatment.This proliferation effect is perhaps related to chitosan's promoting EGFR combinating specificity with EGFR.

Chitosan promotes proliferation of rabbit bladder epithelial cells in vitro / 第二军医大学学报

0.3 g/L)promoted the growth of epithelial cells,and the promoting effect was significantly when the concentration of chitosan was 1.2 g/L(P