| ObjectiveLigustri lucidi Fructus has pharmacological effects of nourishing yin and tonifying kidney,regulating immunity,anti-inflammatory and anti-aging.Numerous studies have found that the processed wine tasting of Ligustri lucidi Fructus can enhance the effect of nourishing liver and kidney.The drug pairs of Ligustri lucidi Fructus and Epimedium are also commonly used to study the conditioning of osteoporosis caused by deficiency of kidney Yin and Yang.In addition,the preparation Xianzhen Fuzheng granule with Ligustrum lucidum Fructus and Epimedium as the king drug is clinically used to improve the immunity of tumor patients.Based on this,the purpose of this study is to explore the protective effect and mechanism of Ligustri lucidi Fructus and its compound preparation on the nephrotoxicity induced by CPplatin in rats.Firstly,the protective effect of wine-processed Ligustri lucidi on liver and kidney injury induced by three different kinds of chemotherapeutic drugs was compared,and it was clear that wine-processed Ligustri lucidi had the significant protective effect on liver and kidney injury induced by chemotherapeutic drugs.Secondly,to explore the protective effect of wine-processed Ligustri lucidi on the liver and kidney injury model of rats established with equivalent dose of CPplatin at different administration frequencies.Finally,the protective effect of Ligustri lucidi Fructus and its compound preparation on renal injury induced by single injection of CPplatin with clinical equivalent dose was investigated,providing scientific basis for the clinical application of Ligustri lucidi Fructus and its compound preparations.Method1.Protective effect of wine-processed Ligustri lucidi on liver and kidney injury induced by three chemotherapeutic drugs: male rats were randomly divided into control group,model group,wine-processed Ligustri lucidi（high,medium and low dose）group and high dose single use group,with 8 rats in each group.1)Grouping and administration of rat liver and kidney injury model established by CPplatin: the control group was intragastric with distilled water 25 mg/kg for consecutive 7 days;the model group was intraperitoneally injected with CPplatin 8 mg/kg on the 5th day;the wineprocessed Ligustri lucidi（high,medium and low dose）groups were injected with CPplatin 8 mg/kg on the 5th day,and gavage 400 mg/kg,200 mg/kg and 100 mg/kg extract of wine-processed Ligustri lucidi daily for 7 days from the first day of modeling;the wine-processed Ligustri lucidi high-dose single use group was given 400 mg/kg intragastric administration daily for 7 consecutive days.2)Grouping and administration of rat liver and kidney injury model established by 5-fluorouracil: the control group was intragastric with distilled water 25 mg/kg for consecutive 14 days;the model group was intraperitoneally injected 20 mg/kg 5-fluorouracil from day 9 to day 14;the wineprocessed Ligustri lucidi（high,medium and low dose）groups were injected with 5-fluorouracil 20 mg/kg from day 9 to day 14,and gavage 400 mg/kg,200 mg/kg and 100mg/kg extract of wine-processed Ligustri lucidi daily for 14 days from the first day of modeling;the wine-processed Ligustri lucidi high-dose single use group was given 400mg/kg intragastric administration daily for 14 consecutive days.3)Grouping and administration of rat liver and kidney injury model established by cyclophosphamide:the control group was intragastric with distilled water 25 mg/kg for consecutive 21 days;the model group was intraperitoneally injected with cyclophosphamide 150 mg/kg on the 16 th day;the wine-processed Ligustri lucidi（high,medium and low dose）groups were injected with cyclophosphamide 150 mg/kg on the 16 th day,and gavage 400mg/kg,200 mg/kg and 100 mg/kg extract of wine-processed Ligustri lucidi daily for 21 days from the first day of modeling;the wine-processed Ligustri lucidi high-dose single use group was given 400 mg/kg intragastric administration daily for 21 consecutive days.After administration,the change rate of body weight and liver and kidney organ indexes were calculated,the biochemical indexes of serum liver and kidney function were detected,and the histopathological changes of liver and kidney were observed.2.Protective effect of wine-processed Ligustri lucidi on liver and kidney injury model induced by different injection times of equivalent dose CPplatin in rats: Wistar male rats were randomly divided into control group,model group and wine-processed Ligustri lucidi equivalent dose group,with 10 rats in each group.1)Liver and kidney injury model induced by single injection of clinical equivalent dose of CPplatin in rats:Except for the control group,rats in the other two groups were intraperitoneally injected with 3 mg/kg CPplatin on day 4,and wine-processed Ligustri lucidi equivalent dose group gavage 200 mg/kg extract on day 1 for 7 consecutive days.2)Liver and kidney injury model induced by twice injection of clinical equivalent dose of CPplatin in rats:except for the control group,rats in the other two groups were intraperitoneally injected with 3 mg/kg CPplatin on the 4th and 8th day,and rats in the wine-processed Ligustri lucidi equivalent dose group were intragastally with 200 mg/kg extract on the 1st day for 10 consecutive days.At the end of the experiment,the change rate of body weight and the indexes of liver and kidney organs were calculated,the biochemical indexes of serum liver and kidney functions were detected,and the pathological changes of H＆E staining of liver and kidney tissues were observed.3.Protective effect of Ligustri lucidi Fructus and its compound preparation on equivalent dose CPplatin induced renal injury in rats: Wistar male rats were randomly divided into control group,model group,Ligustri lucidi Fructus group,drug pair（Ligustri lucidi Fructus-Epimedium）group and compound preparation（Xianzhen Fuzheng granule）group according to body weight,with 10 rats in each group.Except for the control group,the other four groups were intraperitoneally injected with CPplatin 3 mg/kg on day 4;Ligustri lucidi Fructus group gavage 200 mg/kg;drug control group gavage 350 mg/kg;compound preparation group gavage 120 mg/kg for7 days.After sampling,in addition to the changes of body weight,organ index,renal function index and pathological changes,immunohistochemical method was used to observe the expression and distribution of positive signals of KIM-1 and NOX4 in the kidney of rats;Western blot was used to detect the effects of Ligustri lucidi Fructus and its compound preparation on the expression of KIM-1,NOX4,Nrf-2,HO-1 and NFk B proteins in the kidney of CPplatin induced nephrotoxicity rats;The levels of MDA,SOD,CAT,GSH and NADP+/NADPH in rat kidney were detected by kit method.The effects of Ligustri lucidi Fructus and its compound preparation on IL-1b,IL-6 and TNFa in rat kidney tissue were detected by ELISA.In addition,the concentration of platinum atoms in rat kidney tissue was detected by graphite furnace atomic absorption spectrometry.Result1.The weight of rats was recorded on the first and last day of the experiment.After taking the materials,the weight of liver and kidney tissue were weighed respectively.It was found that the three chemotherapeutic drugs made the weight growth of rats slow,and 5-fluorouracil inhibited the growth of rats most significantly（P<0.05）,After administration of wine-processed Ligustri lucidi,the slow growth of body weight was improved in a dose-dependent manner.Compared with the control group,the indexes of liver and kidney were significantly increased by the three chemotherapy drugs（P<0.05）.After the intervention of wine chastity,the liver organ index did not change significantly,and the kidney organ index decreased,but there was no significant difference.The serum biochemical results of CPplatin induced liver and kidney injury in rats: compared with control group,liver function indexes ALT,AST,ALP,TP,ALB and GLO in model group were significantly decreased（P<0.05）,kidney function indexes BUN was increased 6 times,CREA was increased 7 times and UA was decreased 5 times,with statistical differences（P<0.05）;compared with model group,TP,ALB and GLO were significantly increased in a dose-dependent manner（P<0.05）,and high dose of wine-processed Ligustri lucidi can make the BUN and CREA significantly lower,UA significantly increased（P<0.05）.The serum biochemical results of 5-fluorouracil induced liver and kidney injury in rats: compared with the control group,liver function indexes except ALB in model group were significantly decreased（P<0.05）,renal function indexes CREA was significantly increased（P<0.05）,BUN was increased and UA was decreased,but there was no significant difference;after wine-processed Ligustri lucidi the intervention,compared with the model group,only the high dose of wine-processed Ligustri lucidi has a significant alleviating effect on AST,TP and GLO,significantly reduces CREA in a dose-dependent manner（P<0.05）,and has no significant alleviating effect on other indexes.The serum biochemical results of cyclophosphamide induced liver and kidney injury in rats:compared with the control group,ALT of liver function index in model group was significantly increased（P<0.05）,AST and ALP were significantly decreased（P<0.05）,but other related indexes had no significant changes,BUN and CREA of renal function were significantly increased（P<0.05）,UA was significantly decreased（P<0.05）;compared with the model group,there was no significant reversal of liver function index after the intervention of wine-processed Ligustri lucidi and only UA in renal function index was significantly increased（P<0.05）.H＆E staining of liver pathology showed that rats exposed to the three chemotherapeutic drugs had severe liver lesions,including inflammatory cell infiltration,hepatic cell swelling,necrosis and nuclear pyknosis.There was no significant reversal of pathological phenomena after the intervention of wine-processed Ligustri lucidi.According to the H ＆ E staining of renal pathology,the mechanism of renal lesions in rats exposed to three chemotherapeutic drugs is different.In CPplatin model group,the lumen of renal tubules is deformed,and the epithelial cells of renal tubules fall off and necrosis;neutrophil infiltration and interstitial hemorrhage were observed in kidney of rats in 5-fluorouracil model group.In the cyclophosphamide model group,the glomerular atrophy and inflammatory cell infiltration were significant.After the administration of wine-processed Ligustri lucidi,the pathological improvement of kidney injury induced by 5-fluorouracil and cyclophosphamide was not significant,but the pathological improvement of kidney injury induced by CPplatin was alleviated.2.In the experiment of liver and kidney injury induced by single injection of clinical equivalent dose of CPplatin in rats,compared with the control group,the weight of rats in model group increased slowly,liver organ index did not change significantly,but kidney organ index increased significantly（P<0.05）.After the administration of wine-processed Ligustri lucidi,the weight loss degree of rats was alleviated,and kidney organ index was decreased.Serum biochemical indexes showed that the levels of ALT,AST,GLO,BUN,UA and CREA of liver and kidney function were significantly increased（P<0.05）,after the treatment of wine-processed Ligustri lucidi,only BUN and UA levels of renal function indexes were significantly decreased（P<0.05）,and other indexes had no significant changes;the pathological manifestations of rat liver tissue are hepatocyte swelling,vacuolization and inflammatory cell infiltration,the pathological manifestations of renal tissue are tubular deformation and brush marginal membrane loss.After giving wine-processed Ligustri lucidi,it can significantly improve the pathological phenomenon of rat kidney injury,and has no significant reversal effect on rat liver injury.In the experiment of liver and kidney injury induced by twice injection of clinical equivalent dose of CPplatin in rats,compared with the control group,the weight of rats in model group was significantly decreased（P<0.05）,kidney organ index was significantly increased（P<0.05）,liver organ index had no significant change,after the intervention of wine-processed Ligustri lucidi,the decrease of body weight of the rats was reversed,and the renal organ index also decreased significantly（P<0.05）;serum biochemical results showed that the levels of ALT,TP,BUN and CREA were significantly increased in rats（P<0.05）,but there were no significant changes in other indexes.After treatment with wine-processed Ligustri lucidi,the levels of ALT and TP were significantly decreased（P<0.05）,and there were no significant changes in renal function indexes.The histopathological changes of rats showed obvious swelling of liver cells,local diffuse hemorrhage and inflammatory cell infiltration,severe dilatation of renal tubules and absence of brush-like border membrane.The histopathological films of liver and kidney were still observed after treatment with wine-processed Ligustri lucidi.3.In the experiment of renal injury induced by injection of clinical equivalent dose of CPplatin in rats,compared with the control group,the rate of change of body weight in model group was significantly decreased,renal organ index was significantly increased（P<0.05）,BUN and UA were significantly increased（P<0.05）.After the intervention of Ligustri lucidi Fructus and its compound preparation,there was no significant change in body weight,kidney organ index in the compound preparation group was significantly decreased（P<0.05）,BUN and UA in the drug pair group and the compound preparation group returned to normal value;H＆E staining of renal tissue showed that compared with the control group,the renal tubule lumen deformation,epithelial cell necrosis and brush border membrane loss were observed in the model group,and the pathological phenomena were improved after treatment with Ligustri lucidi Fructus and its compound preparation.Immunohistochemistry was used to investigate the protein expression and distribution of KIM-1 and NOX4,and it was found that positive signals of KIM-1 and NOX4 were strongly expressed in the kidney tissues of the model group,and the positive expressions of KIM-1 and NOX4 were decreased after the intervention of drug pairs and compound preparations respectively.In addition,drug pair and compound preparation can activate the Nrf-2/HO-1antioxidant pathway,up-regulate the expression of Nrf-2 and HO-1 and reduce the levels of antioxidant enzymes SOD,CAT and GSH,and inhibit the release of inflammatory factors TNF-a,IL-6 and IL-1b and the expression of NF-k B protein to play a protective role in kidney,thus exerting the renal protective effect of Ligustri lucidi Fructus and its compound preparation.ConclusionIn the rat model of liver and kidney injury induced by three chemotherapeutic drugs,wine-processed Ligustri lucidi had a significant protective effect on kidney injury induced by CPplatin,which was much higher than the clinical equivalent dose.However,the modeling dose of CPplatin injection was different from the clinical dose.In order to make this study more clinical and practical,the model induction scheme was changed to study the protective effect of wine-processed Ligustri lucidi on rat model of liver and kidney injury induced by equivalent dose of CPplatin for different times of administration.The results showed that f wine-processed Ligustri lucidi had significant renal protective effect on rat model of kidney injury induced by single intraperitoneal injection of CPplatin.At the same time,Traditional Chinese medicine plays a role in enhancing the curative effect under the combination of two or more drugs.Therefore,further investigate the protective effect and molecular mechanism of Ligustri lucidi Fructus and its compound preparation on CPplatin nephrotoxicity.It was found that the drug pair and compound preparation could increase the levels of SOD,CAT and GSH by activating Nrf-2/HO-1 antioxidant pathway,inhibit the expression of NF-k B protein and the release of inflammatory factors TNF-a,IL-6 and IL-1b,and play a protective role against CPplatin induced renal injury. |
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