Neutral Polysaccharides From Ophiocordyceps Lanpingensis Alleviate Cisplatin-induced Nephrotoxicity

Cisplatin(cis-diamminedichloroplatinum,DDP)is one of the most effective chemotherapy drugs for the treatment of solid tumors.However,the application of DDP in chemotherapy is limited due to its tissue toxicity,especially nephrotoxicity.Acute kidney injury(AKI)caused by cisplatin seriously affects the patient’s chemotherapy effect and quality of life.At present,angiotensin-converting enzyme inhibitors and diuretics are mainly used in clinic to alleviate DDP nephrotoxicity,but these treatment measures will produce other side effects.while natural drugs have shown unique safety and effectiveness in the prevention and treatment of DDP nephrotoxicity.Ophiocordyceps lanpingensis is a medicinal and edible fungus distributed in the Hengduan Mountains in northwestern Yunnan Province.For a long time,local ethnic minorities use O.lanpingensis for alleviation and health care of urinary system diseases,and no obvious side effects have been found,and its safety and effectiveness have been recognized.However,the pharmacological mechanism of O.lanpingensis is still unclear.Our previous studies have found that polysaccharides might be important biologically active components of O.lanpingensis.This study aims to explore the role and mechanism of neutral polysaccharides of O.lanpingensis(ONP)in alleviating acute kidney injury induced by DDP.This research is mainly divided into the following three aspects:1.Preparation and component analysis of neutral polysaccharides from O.lanpingensisThe crude polysaccharides solution was obtained by water extraction and alcohol precipitation method.The protein was removed by Seavge method,and the distilled water eluent was collected by gradient elution on DEAE-anion exchange column.ONP was obtained by freeze-drying.The molecular weight,monosaccharide composition and functional group structure of ONP were further analyzed.The results showed that the average molecular weight of ONP is about 6.1×10~4 Da.ONP is a heteropolysaccharide mainly composed of mannose,glucose,galactose and arabinose.The molar ratio of monosaccharides is 21.9:27.5:19.5:31.1.2.Attenuation of DDP-induced AKI in mice by neutral polysaccharides from O.lanpingensis.Ninety C57BL/6 male mice were randomly divided into five groups(18 mice in each group)including control group,model group(DDP group),O.lanpingensis neutral polysaccharides group(ONP group),O.lanpingensis neutral polysaccharide low and high dose groups(DDP-ONP-L and DDP-ONP-H groups).The control group was given 0.9%normal saline by gavage or intraperitoneal injection every day;the DDP group was given cisplatin solution(5.0 mg/kg)by intraperitoneal every other day;the ONP group was given 800 mg/kg of ONP by gavage every day;DDP-ONP-L group and DDP-ONP-H group was given 200 mg/kg and 800 mg/kg of ONP every day,except DDP was given every other day as the model,respectively.The experiment period was14 days.The changes in diet,drinking water and body weight of the mice were recorded.H&E staining was performed on kidney tissue and the contents of BUN,CRE,Ca and P in the serum of mice were detected by the kit.The results showed that compared with the control group,the weight of the mice in the DDP group was decreased significantly,and the weight in the ONP group was not changed much.The weight of the mice in the DDP-ONP groups was decreased slowly,and the survival rate was as high as 58%.The color of the kidneys of the DDP model group was grey which was absolutely different from the reddish-brown color of the kidneys in the control group.But the renal color of ONP treatment group was rather ruddy and the renal color of DDP-ONP-H group was similar to that of the control.The results of H&E staining showed that ONP could alleviate the necrosis of renal tubule cells induced by DDP.The contents of BUN,CRE and P were increased by 4 times,2times and 1 time,respectively,while the content of Ca was decreased by about 50%.Compared with the model group,the contents of CRE,BUN and P in the serum were significantly reduced,and the content of Ca was increased in the ONP treatment groups,among which the high dose ONP group had better inhibition effect on nephrotoxicity,and several serum biochemical parameters were almost similar to the normal.The above results proved that ONP treatment could indeed alleviate the kidney damage caused by DDP.3.The mechanism of ONP in alleviating the nephrotoxicity induced by cisplatinIn this study,the contents of MDA,SOD,and GSH-PX in renal tissues of mice were detected;LPS was used to induce inflammation in RAW264.7 cells in order to preliminarily study the anti-inflammatory activity of ONP.Real-time quantitative PCR was used to analyze the m RNA expression levels of TNF-α,IL-1β,and IL-10 related to inflammatory signal pathway in renal tissues.Western Blot was used to detect the expression levels of inflammation-related proteins NOX-4,TNF-α,IL-1β,and apoptosis-related proteins Bax,Bcl-2,JNK,p-JNK,p38,p-p38,ERK1/2,p-ERK1/2,Caspase 3,Cleaved Caspase-3,Caspase 8,Caspase 9.Moreover,the apoptosis of renal cells was observed by TUNEL staining.The results showed that ONP could reduce the level of oxidative stress in renal tissues induced by DDP,increase the levels of antioxidant enzymes(SOD and GSH-PX),reduce the expression levels of MDA and NOX-4.In vitro,ONP restrained LPS-induced inflammatory response in RAW264.7 cells by increasing the expression level of IL-10;in vivo,ONP also inhibited the expression levels of DDP-induced pro-inflammatory factors(TNF-αand IL-1β)in renal tissues by increasing the level of IL-10,thereby attenuating the inflammatory response in renal tissues;meanwhile,ONP suppressed the activation of MAPK and then decreased the expression levels of Bax and Caspase family proteins,increased the expression level of Bcl-2 protein.Ultimately,the apoptosis of renal cells caused by DDP was alleviated.