| Objective1.Prospective clinical randomized controlled trial was used to observe the clinical efficacy of fire needle on refractory chemotherapy-induced nausea and vomiting(CINV)in breast cancer,and serum 5-hydroxytryptamine(5HT)and Substance P(SP),plasma cholecystokinin(CCK)and ghrelin were evaluated to evaluate its efficacy,safety and impact on cerebral intestinal peptides and neurotransmitters.2.The molecular targets and pathway of pica rats against nausea and vomiting by fire needle were preliminary discussed through a model of pica rats by cyclophosphamide(CPA).Methods1.Clinical study:A prospective randomized controlled study included patients with breast cancer using highly vomiting chemotherapy.A total of 74 patients were included,randomly divided into treatment and control groups,and two follow-up chemotherapy cycles were observed.Control group used basic treatment with the combination of tropisetron hydrochloride and dexamethasone.Treatment group added fire needle treatment on basic treatment,used acupoints including Zusanli,Zhongwan,Neiguan,Gongsun,Yinbai and Lidui.Each point was treated Lingnan fire needle 7 stimulation,3 times;fire needle treatment was treated once after the first cycle(about 4-6 hours),and once before second cycles about 12-16 hours before chemotherapy.MAT observed at 24 and 120 hours after each chemotherapy;HADS before chemotherapy;SNAQ and TCM gastrointestinal symptoms at 72 hours after chemotherapy,and follow-up days of loss of appetite.Vomiting complete response(CR)rate,complete control(CC)rate,total control(TC)rate,none nausea(NN)rate as efficacy evaluation indicators,and recorded adverse reactions to safety and compliance analysis,in order to evaluate the efficacy of Lingnan fire needle for breast cancer CINV.ELISA was used to examine patient serum 5HT,SP and plasma CCK,ghrelin to observe effect of fire needle on CINV-related neurotransmitters and appetite-related brainintestinal peptides.2.Experimental research:First,CPA dose selected of pica rat model.Twelve female healthy SD rats were adaptive fed on kaolin diet for 3 days,and then selected which intake<1g were divided into control,low-dose 50mg/kg,medium-dose 100mg/kg and high-dose 200mg/kg groups.Control group received equal saline injection,and the other groups were injected with CPA intraperitoneally by respective dose to observe pica behavior by rat weight,feed intake and kaolin intake,detect the gastric tissue morphology and c-Fos protein expression at nucleus of solitary tract(NTS).Meanwhile measure temperature of fire needle to "red and white bright" in the fire and after leaving the fire to clarify the temperature of the fire needle stimulation.Secondly,explore the "brain-stomach" changes to fire needle prevent treatment.Healthy SD rats,each male and female half,adaptive fed with kaolin,intake<1g sixty rats were randomly divided into five groups:control,model,tropisetron,fire needle and electroacupuncture.The above superior dose of CPA was used for all groups expect control,which was injected with equal saline.Tropisetron was injected 0.8mg/kg by tail intravenous before CPA injection in Tropisetron.Fire needle and electroacupuncture group were treated 1 time 24 hours before CPA injection,and selected Zusanli and Zhongwan acupoints,fire needle at 7 stimulations for each time,3 times;electroacupuncture was 2/15Hz,2mA and 30 minutes at Zusanli,Zhongwan only acupuncture,and the current was gradually increased by 0.5mA to 2mA,on 2-3-3min.Rat pica behavioral observations were performed;temperature changes in rat acupoint region were detected by infrared thermal imaging instrument;gastric tissue was detected by HE staining;c-Fos expression in brain by immunohistochemistry;Western Blotting for 5HT3 receptor,Extracellular Signal Regulated Kinase(ERK1/2),Phosphorylated Protein Kinase A(pPKA),Heat Shock Protein(Hsp90),Phophatidylinositol Kinase(PIK3CA)expression in bulbus medullae,and ELISA for serum 5HT,SP and plasma CCK and ghrelin content.Finally,different genes and pathways involved in fire needle treatment were clarified.Thirty healthy SD rats were randomly divided into five groups,grouped and treated same as the previous section.Observe their pica behavior and gene expression of rat bulbus medulla was detected by transcriptomics,selecting significantly expressed genes by P<0.05 and |log2FC|>1.5,and related gene functions and pathway enrichment by GO and KEGG.Results1.Clinical study:(1)Fire needle treatment before chemotherapy,CR was higher than control group(P<0.001);CC was no difference between groups at any time points(P>0.05);TC of fire needle treatment before chemotherapy was significantly higher than that of control group(P<0.001);NN at 24 and 120 hours of fire needle treatment pre-chemotherapy intervention was significantly higher than control group(P<0.05).(2)The number of vomiting and nausea Visual Analogue Scale(VAS)scores at 24 and 120 hours of fire treatment before chemotherapy was significant less than ire treatment after chemotherapy(P<0.05);moreover,the number of vomiting and VAS score at 24h in 2nd cycle were significantly lower than in control(P<0.001);at 120h only VAS significantly lower than in control(P<0.001).No significant change in HADS(P>0.05);at 2nd cycle,compared with control group,SNAQ score of treatment group were significantly higher,and higher than 1st cycle(P<0.001)as well as,TCM gastrointestinal symptom score,anorexia scores and day of anorexia of treatment group were significantly decreased,and decreased than 1st cycle as well(P<0.05);however,compared with 1st cycle,in control group,SNAQ score decreased(P<0.05)and TCM gastrointestinal symptom score,anorexia scores and day of anorexia increased at 2nd cycle(P<0.05).(3)Only one case of skin herpes was reported after fire needle,and Wanhua oil covered.There was no difference on blood analysis and biochemical examination between groups(P>0.05);the compliance with fire needle treatment was up to 96.4%.(4)There was no difference in serum 5HT,SP and plasma CCK and ghrelin before treatment in the first cycle(P>0.05).Fire needle intervention before chemotherapy reduced serum content of 5HT,SP and plasma CCK(P<0.001),promoted ghrelin rise(P<0.001),and the difference in group of the four indicators was significant different(P<0.05).3.Experimental research:(1)High-dose group increased kaolin intake(P<0.05),increased expression of c-Fos protein in NTS(P<0.001)and showed significant inflammatory changes in gastric mucosa;the temperature of fire needle burning to "red and white bright" is(231.06±12.10)℃,and leaved fire temperature is(100.74±4.59)℃.(2)Fire needle can alleviate CPA induced with increased kaolin intake(P<0.05),which is consistent with tropisetron and electroacupuncture.The temperature of Zhongwan acupoint area was significantly reduced(P<0.05).There was no obvious difference in Zusanli acupoint(P>0.05).Only female rats produced significant gastric mucosal damage,and the fire needle and electroacupuncture promoted gastric mucosal recovery(P<0.05).All other group lower expression of c-Fos protein in NTS compared with the model(P<0.05).There were no differences in 5TH3 receptor,ERK1/2,pPKA,PIK3CA,and Hsp90 in bulbus medulla of brain stem(P>0.05).Fire needle inhibited serum 5HT,SP and plasma CCK,elevated plasma ghrelin content than model which is consistent with electroacupuncture and tropisetron(P<0.001).(3)Transcriptomic study found differences between female and male rats,analyzed the number of different genes in different groups of female rats,compared with the model group,there were 280 in control group,141 in fire needle group,185 in electroacupuncture group,and 258 in tropisetron group.Enrichment showed that fire needle is mainly related to bile secretion and fatty degeneration in female rats.Gene verification results that the expression of female RT1-Ba,Slcola6,PCK1,oas1a were different from model(P<0.05)and had no difference from control(P>0.05).Conclusion1.Lingnan fire needle therapy before chemotherapy combined with western medicine basic treatment can effectively control CINV symptoms,not only can inhibit vomiting,reduce nausea symptoms,but also can promote recovery of appetite,and good safety of high patient compliance,its effect is related reducing serum 5HT,SP and plasma CCK and increasing plasma ghrelin.2.Temperature of fire needle at stimulation at(100.74±4.59)℃,which for Zusanli and Zhongwan acupoint can reduce temperature of Zhongwan area,alleviate the pica behavior of CPA in rats,and relieve gastric mucosal damage,which may be related to regulation of CCK by bile secretion pathway,5HT/5TH3 receptor pathway regulation ghrelin in its female. |
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